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Drug-resistant tuberculosis (DR-TB) threatens progress in the control of TB. Mathematical models are increasingly being used to guide public health decisions on managing both antimicrobial resistance (AMR) and TB. It is important to consider bacterial heterogeneity in models as it can have consequences for predictions of resistance prevalence, which may affect decision-making. We conducted a systematic review of published mathematical models to determine the modelling landscape and to explore methods for including bacterial heterogeneity. Our first objective was to identify and analyse the general characteristics of mathematical models of DR-mycobacteria, including M. tuberculosis. The second objective was to analyse methods of including bacterial heterogeneity in these models. We had different definitions of heterogeneity depending on the model level. For between-host models of mycobacterium, heterogeneity was defined as any model where bacteria of the same resistance level were further differentiated. For bacterial population models, heterogeneity was defined as having multiple distinct resistant populations. The search was conducted following PRISMA guidelines in five databases, with studies included if they were mechanistic or simulation models of DR-mycobacteria. We identified 195 studies modelling DR-mycobacteria, with most being dynamic transmission models of non-treatment intervention impact in M. tuberculosis (n = 58). Studies were set in a limited number of specific countries, and 44% of models (n = 85) included only a single level of "multidrug-resistance (MDR)". Only 23 models (8 between-host) included any bacterial heterogeneity. Most of these also captured multiple antibiotic-resistant classes (n = 17), but six models included heterogeneity in bacterial populations resistant to a single antibiotic. Heterogeneity was usually represented by different fitness values for bacteria resistant to the same antibiotic (61%, n = 14). A large and growing body of mathematical models of DR-mycobacterium is being used to explore intervention impact to support policy as well as theoretical explorations of resistance dynamics. However, the majority lack bacterial heterogeneity, suggesting that important evolutionary effects may be missed. Author summary: The emergence of drug-resistant tuberculosis (DR-TB), where the causative bacterium Mycobacterium tuberculosis is resistant to key antibiotics such as rifampicin and isoniazid, poses a significant threat to TB control efforts. To gain a broader understanding of the challenges surrounding DR-TB, mathematical models are increasingly being employed to estimate the impact of interventions, effectiveness of treatment, and to predict the evolution of drug-resistance. However, pragmaticism surrounding model construction often means that important aspects, such as bacterial heterogeneity, are overlooked. We undertook a systematic review of the existing DR-mycobacterium modelling literature, with the specific aim of capturing methods for including bacterial heterogeneity. Our analysis revealed that most models of drug-resistance in mycobacteria primarily focus on intervention strategies and cost-effectiveness analyses, with minimal attention to bacterial heterogeneity. Where heterogeneity is included it mostly consisted of different fitness costs for resistance. [ABSTRACT FROM AUTHOR]

The human polyomavirus JCPyV is an opportunistic pathogen that infects greater than 60% of the world's population. The virus establishes a persistent and asymptomatic infection in the urogenital system but can cause a fatal demyelinating disease in immunosuppressed or immunomodulated patients following invasion of the CNS. The mechanisms responsible for JCPyV invasion into CNS tissues are not known but direct invasion from the blood to the cerebral spinal fluid via the choroid plexus has been hypothesized. To study the potential of the choroid plexus as a site of neuroinvasion, we used an adult human choroid plexus epithelial cell line to model the blood-cerebrospinal fluid (B-CSF) barrier in a transwell system. We found that these cells formed a highly restrictive barrier to virus penetration either as free virus or as virus associated with extracellular vesicles (EVJC+). The restriction was not absolute and small amounts of virus or EVJC+ penetrated and were able to establish foci of infection in primary astrocytes. Disruption of the barrier with capsaicin did not increase virus or EVJC+ penetration leading us to hypothesize that virus and EVJC+ were highly cell-associated and crossed the barrier by an active process. An inhibitor of macropinocytosis increased virus penetration from the basolateral (blood side) to the apical side (CSF side). In contrast, inhibitors of clathrin and raft dependent transcytosis reduced virus transport from the basolateral to the apical side of the barrier. None of the drugs inhibited apical to basolateral transport suggesting directionality. Pretreatment with cyclosporin A, an inhibitor of P-gp, MRP2 and BCRP multidrug resistance transporters, restored viral penetration in cells treated with raft and clathrin dependent transcytosis inhibitors. Because choroid plexus epithelial cells are known to be susceptible to JCPyV infection both in vitro and in vivo we also examined the release of infectious virus from the barrier. We found that virus was preferentially released from the cells into the apical (CSF) chamber. These data show clearly that there are two mechanisms of penetration, direct transcytosis which is capable of seeding the CSF with small amounts of virus, and infection followed by directional release of infectious virions into the CSF compartment. Author summary: In patients with compromised immune systems due to underlying immunosuppressive diseases or due to treatment with immunomodulatory drugs, JC virus (JCPyV) invades the brain and causes a rapidly progressing and often fatal demyelinating disease known as progressive multifocal leukoencephalopathy (PML). The brain is protected from invading pathogens by two major barriers, the blood-brain barrier (BBB), and the blood-cerebrospinal fluid barrier (BCSFB). The BCSFB is composed of choroid plexus epithelial cells that are directly exposed to the blood. The epithelial cells in the choroid plexus are held together by tight junctions that limit and regulate the movement of molecules and cells from the blood to the cerebral spinal fluid. In this paper we used an established adult choroid plexus epithelial cell line to model the blood-cerebrospinal fluid barrier and found that it was highly restrictive of virus penetration. The restriction was not absolute and small amounts of virus penetrated and established infection in underlying human glial cells. We also show a second mechanism of virus invasion involving direct infection of choroid plexus epithelial cells by JCPyV and directional release of infectious virions across the barrier. Both mechanisms likely contribute to the neuroinvasiveness of JCPyV. [ABSTRACT FROM AUTHOR]

The bloodstream form of Trypanosoma brucei expresses large poly-N-acetyllactosamine (pNAL) chains on complex N-glycans of a subset of glycoproteins. It has been hypothesised that pNAL may be required for receptor-mediated endocytosis. African trypanosomes contain a unique family of glycosyltransferases, the GT67 family. Two of these, TbGT10 and TbGT8, have been shown to be involved in pNAL biosynthesis in bloodstream form Trypanosoma brucei, raising the possibility that deleting both enzymes simultaneously might abolish pNAL biosynthesis and provide clues to pNAL function and/or essentiality. In this paper, we describe the creation of a TbGT10 null mutant containing a single TbGT8 allele that can be excised upon the addition of rapamycin and, from that, a TbGT10 and TbGT8 double null mutant. These mutants were analysed by lectin blotting, glycopeptide methylation linkage analysis and flow cytometry. The data show that the mutants are defective, but not abrogated, in pNAL synthesis, suggesting that other GT67 family members can compensate to some degree for loss of TbGT10 and TbGT8. Despite there being residual pNAL synthesis in these mutants, certain glycoproteins appear to be particularly affected. These include the lysosomal CBP1B serine carboxypeptidase, cell surface ESAG2 and the ESAG6 subunit of the essential parasite transferrin receptor (TfR). The pNAL deficient TfR in the mutants continued to function normally with respect to protein stability, transferrin binding, receptor mediated endocytosis of transferrin and subcellular localisation. Further the pNAL deficient mutants were as viable as wild type parasites in vitro and in in vivo mouse infection experiments. Although we were able to reproduce the inhibition of transferrin uptake with high concentrations of pNAL structural analogues (N-acetylchito-oligosaccharides), this effect disappeared at lower concentrations that still inhibited tomato lectin uptake, i.e., at concentrations able to outcompete lectin-pNAL binding. Based on these findings, we recommend revision of the pNAL-dependent receptor mediated endocytosis hypothesis. Author summary: Blood-stage trypanosome parasites have a specialised invagination on the cell surface named the flagellar pocket (FP), where invariant essential nutrient receptors are located. The pocket houses diverse proteins, including a transferrin receptor (TfR), which facilitates uptake of host transferrin-bound iron for survival. Several FP proteins, including TfR, are linked to complex sugar molecules (carbohydrates), the functions of which are not well understood. Complex carbohydrates are made by enzymes called glycosyltransferases (GTs) and previously we partially inhibited complex carbohydrate synthesis by deletion of either TbGT8 or TbGT10. However, mutant parasites lacking either one of these enzymes survived, suggesting functional redundancy. Here, we created a parasite mutant that lacks both TbGT8 and TbG10 to understand the combined effect of losing both enzymes. The mutant parasites showed a decreased ability to uptake tomato lectin, a protein that specifically binds to these sugar conjugates in the FP, indicating a reduction in carbohydrate complexity. Despite reduced complexity in the sugar structures attached to TfR, its critical function in transferrin/iron uptake remained effective. Furthermore, the mutants remained viable in culture and in animal models, challenging previous assumptions about the necessity and function of these carbohydrate conjugates. Our findings imply a greater flexibility and redundancy in the carbohydrate complex roles than previously appreciated. [ABSTRACT FROM AUTHOR]

Noroviruses (NoVs) are a leading cause of viral gastroenteritis. Despite global clinical relevance, our understanding of how host factors, such as antiviral cytokines interferons (IFNs), modulate NoV population dynamics is limited. Murine NoV (MNoV) is a tractable in vivo model for the study of host regulation of NoV. A persistent strain of MNoV, CR6, establishes a reservoir in intestinal tuft cells for chronic viral shedding in stool. However, the influence of host innate immunity and permissive cell numbers on viral population dynamics is an open question. We generated a pool of 20 different barcoded viruses (CR6BC) by inserting 6-nucleotide barcodes at the 3' position of the NS4 gene and used this pool as our viral inoculum for in vivo infections of different mouse lines. We found that over the course of persistent CR6 infection, shed virus was predominantly colon-derived, and viral barcode richness decreased over time irrespective of host immune status, suggesting that persistent infection involves a series of reinfection events. In mice lacking the IFN-λ receptor, intestinal barcode richness was enhanced, correlating with increased viral intestinal replication. IL-4 treatment, which increases tuft cell numbers, also increased barcode richness, indicating the abundance of permissive tuft cells to be a bottleneck during CR6 infection. In mice lacking type I IFN signaling (Ifnar1-/-) or all IFN signaling (Stat1-/-), barcode diversity at extraintestinal sites was dramatically increased, implicating different IFNs as critical bottlenecks at specific tissue sites. Of interest, extraintestinal barcodes were overlapping but distinct from intestinal barcodes, indicating that disseminated virus represents a distinct viral population than that replicating in the intestine. Barcoded viruses are a valuable tool to explore the influence of host factors on viral diversity in the context of establishment and maintenance of infection as well as dissemination and have provided important insights into how NoV infection proceeds in immunocompetent and immunocompromised hosts. Author summary: Defining the host factors responsible for controlling viral population dynamics during infection is critical for establishing a thorough understanding of viral transmission, dissemination, pathogenesis, and immune evasion. Here, we employed a barcoded virus strategy to interrogate how host factors modulate viral diversity of CR6, a persistent strain of murine norovirus. By evaluating barcode levels in tissues and stool of wild-type mice, mice lacking critical innate immune response genes, and mice treated with cytokine to enhance susceptible tuft cell levels, we found that both the availability of tuft cells and viral replication limitations imposed by interferon signaling serve as critical bottlenecks for CR6 diversity. Our studies also indicated that stool virus is likely predominantly derived from the colon, and that extraintestinal dissemination of CR6 in immunodeficient mouse strains likely occurs independently of intestinal infection. Our study thus revealed key constraints regulating norovirus population dynamics and provided additional insights into the mechanisms of viral shedding and dissemination. [ABSTRACT FROM AUTHOR]

Recent advances in the in vitro cultivation of Cryptosporidium parvum using hollow fiber bioreactor technology (HFB) have permitted continuous growth of parasites that complete all life cycle stages. The method provides access to all stages of the parasite and provides a method for non-animal production of oocysts for use in clinical trials. Here we examined the effect of long-term (>20 months) in vitro culture on virulence-factors, genome conservation, and in vivo pathogenicity of the host by in vitro cultured parasites. We find low-level sequence variation that is consistent with that observed in calf-passaged parasites. Further using a calf model infection, oocysts obtained from the HFB caused diarrhea of the same volume, duration and oocyst shedding intensity as in vivo passaged parasites. Author summary: Cryptosporidium parvum and C. hominis are waterborne parasites that are the second to third leading cause of diarrheal disease, and a major contributor to childhood deaths worldwide. Traditionally these intestinal parasites have proven difficult to culture for more than 2–3 days, which hampers long term in vitro studies. We reasoned that cultures of intestinal epithelial cells as monolayers in static plates results in the production of unpolarized epithelial cells. Utilizing hollow fiber technology, we have developed a method for producing intestinal epithelial cell growth that simulates the body resulting in polarized intestinal epithelial cells that have basal and apical surfaces, tight junctions, and develop functional villi. Using this system, we have maintained in vitro cultures of C. parvum that produce all life cycle stages for 20 months. Long-term in vitro culture of parasites often results in the development of a phenotype that is no longer pathogenic to the host; In this publication we show that using a calf model C. parvum BGF-T20HF after 20 months of in vitro culture was unchanged with respect to diarrhea output, parasite load, and clinical scores from the isolate used to initiate the culture (BGF-T0). In addition, we can show that the genome of the cultured parasites (BGF-T20HF) has undergone a similar genomic drift as the parent isolate (BGF-T0) used to start the inoculum that had been maintained by passage through fetal calves. Collectively the data supports the use of the in vitro cultured isolate, BGF-T20HF, for human trials, and provides a long-term model for the development of novel chemotherapeutic drugs to treat this disease. [ABSTRACT FROM AUTHOR]

SARS-CoV-2 spike protein (SARS-2-S) induced cell–cell fusion in uninfected cells may occur in long COVID-19 syndrome, as circulating SARS-2-S or extracellular vesicles containing SARS-2-S (S-EVs) were found to be prevalent in post-acute sequelae of COVID-19 (PASC) for up to 12 months after diagnosis. Although isolated recombinant SARS-2-S protein has been shown to increase the SASP in senescent ACE2-expressing cells, the direct linkage of SARS-2-S syncytia with senescence in the absence of virus infection and the degree to which SARS-2-S syncytia affect pathology in the setting of cardiac dysfunction are unknown. Here, we found that the senescent outcome of SARS-2-S induced syncytia exacerbated heart failure progression. We first demonstrated that syncytium formation in cells expressing SARS-2-S delivered by DNA plasmid or LNP-mRNA exhibits a senescence-like phenotype. Extracellular vesicles containing SARS-2-S (S-EVs) also confer a potent ability to form senescent syncytia without de novo synthesis of SARS-2-S. However, it is important to note that currently approved COVID-19 mRNA vaccines do not induce syncytium formation or cellular senescence. Mechanistically, SARS-2-S syncytia provoke the formation of functional MAVS aggregates, which regulate the senescence fate of SARS-2-S syncytia by TNFα. We further demonstrate that senescent SARS-2-S syncytia exhibit shrinked morphology, leading to the activation of WNK1 and impaired cardiac metabolism. In pre-existing heart failure mice, the WNK1 inhibitor WNK463, anti-syncytial drug niclosamide, and senolytic dasatinib protect the heart from exacerbated heart failure triggered by SARS-2-S. Our findings thus suggest a potential mechanism for COVID-19-mediated cardiac pathology and recommend the application of WNK1 inhibitor for therapy especially in individuals with post-acute sequelae of COVID-19. Author summary: In this paper, we directly linked SARS-2-S-triggered syncytium formation in the absence of infection with the ensuing induction of cellular senescence and its pathophysiological contribution to heart failure. We propose that both SARS-2-S expression and SARS-2-S protein internalization were sufficient to induce senescence in nonsenescent ACE2-expressing cells. This is important because of the persistent existence of SARS-2-S or extracellular vesicles containing SARS-2-S during the post-acute stages of SARS-CoV-2 infection in human subjects. In searching for the underlying molecular mechanisms determining syncytial fate, the formation of functional MAVS aggregates dependent on RIG-I was observed at an early stage during fusion and regulated the anti-death to senescence fate of SARS-2-S syncytia through the TNFα-TNFR2 axis. We also found impaired cardiac metabolism in SARS-2-S syncytia induced by condensed WNK1. However, syncytium formation or cellular senescence observed with the wild-type fusogenic S protein does not occur with the spike proteins produced by currently approved COVID-19 mRNA vaccines. Importantly, SARS-2-S-exacerbated heart failure could be largely rescued by WNK1 inhibitor, anti-syncytial drug or senolytic agent. Together, we suggest that rescuing metabolism dysfunction in senescent SARS-2-S syncytia should be taken into consideration in individuals with post-acute sequelae of COVID-19 (PASC). [ABSTRACT FROM AUTHOR]

NADPH oxidase 2 (NOX2) is an enzyme responsible for generating reactive oxygen species, primarily found in phagocytes. Chronic Granulomatous Disease (CGD), along with bacterial infections such as Mycobacterium tuberculosis (Mtb), is a representative NOX2-deficient X-linked disease characterized by uncontrolled inflammation. However, the precise roles of host-derived factors that induce infection-mediated hyperinflammation in NOX2-deficient condition remain incompletely understood. To address this, we compared Mtb-induced pathogenesis in Nox2-/- and wild type (WT) mice in a sex-dependent manner. Among age- and sex-matched mice subjected to Mtb infection, male Nox2-/- mice exhibited a notable increase in bacterial burden and lung inflammation. This was characterized by significantly elevated pro-inflammatory cytokines such as G-CSF, TNF-α, IL-1α, IL-1β, and IL-6, excessive neutrophil infiltration, and reduced pulmonary lymphocyte levels as tuberculosis (TB) progressed. Notably, lungs of male Nox2-/- mice were predominantly populated with CD11bintLy6GintCXCR2loCD62Llo immature neutrophils which featured mycobacterial permissiveness. By diminishing total lung neutrophils or reducing immature neutrophils, TB immunopathogenesis was notably abrogated in male Nox2-/- mice. Ultimately, we identified G-CSF as the pivotal trigger that exacerbates the generation of immature permissive neutrophils, leading to TB immunopathogenesis in male Nox2-/- mice. In contrast, neutralizing IL-1α and IL-1β, which are previously known factors responsible for TB pathogenesis in Nox2-/- mice, aggravated TB immunopathogenesis. Our study revealed that G-CSF-driven immature and permissive pulmonary neutrophils are the primary cause of TB immunopathogenesis and lung hyperinflammation in male Nox2-/- mice. This highlights the importance of quantitative and qualitative control of pulmonary neutrophils to alleviate TB progression in a phagocyte oxidase-deficient condition. Author summary: While tuberculosis (TB) remains a global threat to public health, the immunologic factors contributing to TB susceptibility are not yet fully defined. To enhance our understanding of TB immunopathogenesis, we utilized TB-susceptible male Nox2-/- mice to dissect the immunologic factors accelerating TB pathogenesis. In this study, we observed that male Nox2-/- mice infected with Mycobacterium tuberculosis (Mtb) exhibited increased lung hyperinflammation and mycobacterial burden compared to female Nox2-/- mice and WT mice. Exacerbated TB immunopathogenesis in male Nox2-/- mice was strongly correlated with an extreme infiltration of pulmonary neutrophils and the loss of pulmonary lymphocytes. The pulmonary neutrophils in male Nox2-/- mice displayed immature phenotypes and mycobacterial permissiveness. The depletion of total neutrophils or the AM80-induced maturation of neutrophils ameliorated TB pathogenesis in Nox2-/- mice, reducing pulmonary neutrophil counts, lung hyperinflammation, and mycobacterial load. Furthermore, neutralization of G-CSF mitigated TB pathogenesis in male Nox2-/- mice by decreasing immature neutrophil counts. Thus, we identified that G-CSF-mediated generation of permissive immature neutrophils contributes to TB susceptibility in male Nox2-/- mice. [ABSTRACT FROM AUTHOR]

Influenza infections result in a significant number of severe illnesses annually, many of which are complicated by secondary bacterial super-infection. Primary influenza infection has been shown to increase susceptibility to secondary methicillin-resistant Staphylococcus aureus (MRSA) infection by altering the host immune response, leading to significant immunopathology. Type III interferons (IFNs), or IFNλs, have gained traction as potential antiviral therapeutics due to their restriction of viral replication without damaging inflammation. The role of IFNλ in regulating epithelial biology in super-infection has recently been established; however, the impact of IFNλ on immune cells is less defined. In this study, we infected wild-type and IFNLR1-/- mice with influenza A/PR/8/34 followed by S. aureus USA300. We demonstrated that global IFNLR1-/- mice have enhanced bacterial clearance through increased uptake by phagocytes, which was shown to be cell-intrinsic specifically in myeloid cells in mixed bone marrow chimeras. We also showed that depletion of IFNLR1 on CX3CR1 expressing myeloid immune cells, but not neutrophils, was sufficient to significantly reduce bacterial burden compared to mice with intact IFNLR1. These findings provide insight into how IFNλ in an influenza-infected lung impedes bacterial clearance during super-infection and show a direct cell intrinsic role for IFNλ signaling on myeloid cells. Author summary: Influenza virus infects millions of people each year and is characterized by a mostly self-resolving mild to moderate illness. However, severe disease is observed in a subset of patients and is often characterized by bacterial super-infection, which occurs following influenza virus infection. In this setting, there can be a lethal synergy between the virus and bacteria that results in severe lung injury. In recent years, the field has determined that type III interferon (IFNλ), induced by influenza virus as part of the anti-viral response, may play a detrimental role during super-infection. This has been attributed to IFNλ impacting structural cells of the airways. In this study, we define a novel role for IFNλ impacting bacterial clearance during super-infection via directly altering lung phagocytes responsible for bacterial host-defense. We use three distinct mouse models to define this new role of IFNλ in regulating phagocyte function in the lung. This study is important because it defines a new mechanism by which anti-viral IFNλ leads to increased susceptibility to bacterial super-infection. This has implications for potential use of IFNλ as an anti-viral therapeutic and identifies a new biologic function of IFNλ in the lung. [ABSTRACT FROM AUTHOR]

There is a critical gap in knowledge about how Gram-negative bacterial pathogens, using survival strategies developed for other niches, cause lethal bacteremia. Facultative anaerobic species of the Enterobacterales order are the most common cause of Gram-negative bacteremia, including Escherichia coli, Klebsiella pneumoniae, Serratia marcescens, Citrobacter freundii, and Enterobacter hormaechei. Bacteremia often leads to sepsis, a life-threatening organ dysfunction resulting from unregulated immune responses to infection. Despite a lack of specialization for this host environment, Gram-negative pathogens cause nearly half of bacteremia cases annually. Based on our existing Tn-Seq fitness factor data from a murine model of bacteremia combined with comparative genomics of the five Enterobacterales species above, we prioritized 18 conserved fitness genes or operons for further characterization. Mutants were constructed for all genes in all five species. Each mutant was used to cochallenge C57BL/6 mice via tail vein injection along with each respective wild-type strain to determine competitive indices for each fitness gene. Five fitness factor genes, when mutated, attenuated mutants in four or five species in the spleen and liver (tatC, ruvA, gmhB, wzxE, arcA). Five additional fitness factor genes or operons were validated as outcompeted by wild-type in three, four, or five bacterial species in the spleen (xerC, prc, apaGH, atpG, aroC). Overall, 17 of 18 fitness factor mutants were attenuated in at least one species in the spleen or liver. Together, these findings allow for the development of a model of bacteremia pathogenesis that may include future targets of therapy against bloodstream infections. Author summary: Frequent cases of bacteremia plague our ICUs, bone marrow transplant units, and inpatient facilities. Nearly half of these infections are caused by Gram-negative bacteria. The Enterobacterales order including E. coli, K. pneumoniae, S. marcescens, C. freundii, and E. hormaechei are leading causes of bacteremia. An alarming proportion of these are due to antibiotic-resistant isolates, which are four times more likely to kill than antibiotic-susceptible isolates. Clearly, we need new therapeutic targets to treat cases of bacteremia and sepsis. Previously, it has been unclear what genes contribute to their ability to survive in this hostile host environment. We have previously undertaken unbiased genetic screens to identify genes shared by all five bacterial genera that are required for maintaining bacterial burden in the blood and blood-filtering organs. These include genes that encode proteins that maintain proton motive force, resist antimicrobial peptides and complement, mediate genome maintenance, transport key metabolites and proteins, avoid oxidative stress, acquire iron, and regulate key pathways. Mutants, constructed in these shared genes in the five species, were validated for a high proportion of genes as critical for infection in the mouse model of bacteremia. [ABSTRACT FROM AUTHOR]

Alveolar echinococcosis (AE) is a highly lethal helminth infection. Current chemotherapeutic strategies for AE primarily involve the use of benzimidazoles (BZs) such as mebendazole (MDZ) and albendazole (ABZ), which exhibit limited efficacy. In a previous study, the vaccine of recombinant Echinococcus granulosus P29 (rEgP29) showed significant immunoprotection against E. granulosus in both mice and sheep. In the current study, we utilized hybridoma technology to generate five monoclonal antibodies (mAbs) against P29, among which 4G10F4 mAb exhibited the highest antigen-specific binding capacity. This mAb was selected for further investigation of anti-AE therapy, both in vivo and in vitro. In vitro, 4G10F4 inhibited a noteworthy inhibition of E. multilocularis protoscoleces and primary cells viability through complement-dependent cytotoxicity (CDC) mechanism. In vivo, two experiments were conducted. In the first experiment, mice were intraperitoneally injected with Em protoscoleces, and subsequently treated with 4G10F4 mAb (2.5/5/10 mg/kg) at 12 weeks postinfection once per week for 8 times via tail vein injection. Mice that were treated with 4G10F4 mAb only in dosage of 5mg/kg exhibited a significant lower mean parasite burden (0.89±0.97 g) compared to isotype mAb treated control mice (2.21±1.30 g). In the second experiment, mice were infected through hepatic portal vein and treated with 4G10F4 mAb (5mg/kg) at one week after surgery once per week for 8 times. The numbers of hepatic metacestode lesions of the 4G10F4 treatment group were significantly lower in comparison to the isotype control group. Pathological analysis revealed severe disruption of the inner structure of the metacestode in both experiments, particularly affecting the germinal and laminated layers, resulting in the transformation into infertile vesicles after treatment with 4G10F4. In addition, the safety of 4G10F4 for AE treatment was confirmed through assessment of mouse weight and evaluation of liver and kidney function. This study presents antigen-specific monoclonal antibody immunotherapy as a promising therapeutic approach against E. multilocularis induced AE. Author summary: Echinococcosis encompasses two significant zoonotic tapeworm diseases, cystic echinococcosis (CE) and alveolar echinococcosis (AE), caused by Echinococcus granulosus and Echinococcus multilocularis, respectively. AE is widely acknowledged as the most fatal helminth infection, with a mortality rate exceeding 90% within 10–15 years of diagnosis if left untreated or inadequately treated. Two benzimidazole carbamates, albendazole and mebendazole, are the only anti-infective drugs that are clinically efficient in interrupting the larval growth of Echinococcus spp. However, due to their toxicity and limited efficacy, there is a pressing need to explore novel therapeutic strategies for AE. In this study, a therapeutic antibody named 4G10F4 was produced against hydatid P29 using hybridoma technology. This candidate antibody significantly inhibited E. multilocularis both in vitro and in vivo, representing a potentially efficacious and safe antigen-specific monoclonal antibody immunotherapy for the treatment of AE. Additionally, it has been identified as a promising antibody drug and molecular target for the development of anti-AE therapy. [ABSTRACT FROM AUTHOR]

R-loops are trimeric nucleic acid structures that form when an RNA molecule hybridizes with its complementary DNA strand, displacing the opposite strand. These structures regulate transcription as well as replication, but aberrant R-loops can form, leading to DNA breaks and genomic instability if unresolved. R-loop levels are elevated in many cancers as well as cells that maintain high-risk human papillomaviruses. We investigated how the distribution as well as function of R-loops changed between normal keratinocytes and HPV positive cells derived from a precancerous lesion of the cervix (CIN I). The levels of R-loops associated with cellular genes were found to be up to 10-fold higher in HPV positive cells than in normal keratinocytes while increases at ALU1 elements increased by up to 500-fold. The presence of enhanced R-loops resulted in altered levels of gene transcription, with equal numbers increased as decreased. While no uniform global effects on transcription due to the enhanced levels of R-loops were detected, genes in several pathways were coordinately increased or decreased in expression only in the HPV positive cells. This included the downregulation of genes in the innate immune pathway, such as DDX58, IL-6, STAT1, IFN-β, and NLRP3. All differentially expressed innate immune genes dependent on R-loops were also associated with H3K36me3 modified histones. Genes that were upregulated by the presence of R-loops in HPV positive cells included those in the DNA damage repair such as ATM, ATRX, and members of the Fanconi Anemia pathway. These genes exhibited a linkage between R-loops and H3K36me3 as well as γH2AX histone marks only in HPV positive cells. These studies identify a potential link in HPV positive cells between DNA damage repair as well as innate immune regulatory pathways with R-loops and γH2AX/H3K36me3 histone marks that may contribute to regulating important functions for HPV pathogenesis. Author summary: R-loops are trimeric RNA: DNA hybrids that regulate transcription but can also lead to DNA breaks. R-loop levels are elevated in many cancers as well in precancerous cells containing high-risk HPVs. How the distribution and function of R-loops changes due to the presence of viral genomes was examined by comparing cells derived from an HPV 31 positive precancer (CIN 612) to normal keratinocytes. Up to 10-fold higher R-loop levels were detected on cellular genes in CIN 612 cells, with over 500-fold increases at ALU elements. Analysis of the changes in R-loop landscape between normal and HPV positive cells identified a functional association between R-loop formation, gene expression, and chromatin states. R-loop formation in HPV postive cells was equally associated with negatively and positively regulated gene expression; however, coordinated expression of genes in specific pathways by R-loops was observed. This included repression of genes in the innate immune surveillance pathway, while those regulating DNA damage repair and metabolism were activated. [ABSTRACT FROM AUTHOR]

Entamoeba histolytica is a protozoan parasite belonging to the phylum Amoebozoa that causes amebiasis, a global public health problem. E. histolytica alternates its form between a proliferative trophozoite and a dormant cyst. Trophozoite proliferation is closely associated with amebiasis symptoms and pathogenesis whereas cysts transmit the disease. Drugs are available for clinical use; however, they have issues of adverse effects and dual targeting of disease symptoms and transmission remains to be improved. Development of new drugs is therefore urgently needed. An untargeted lipidomics analysis recently revealed structural uniqueness of the Entamoeba lipidome at different stages of the parasite's life cycle involving very long (26–30 carbons) and/or medium (8–12 carbons) acyl chains linked to glycerophospholipids and sphingolipids. Here, we investigated the physiology of this unique acyl chain diversity in Entamoeba, a non-photosynthetic protist. We characterized E. histolytica fatty acid elongases (EhFAEs), which are typically components of the fatty acid elongation cycle of photosynthetic protists and plants. An approach combining genetics and lipidomics revealed that EhFAEs are involved in the production of medium and very long acyl chains in E. histolytica. This approach also showed that the K3 group herbicides, flufenacet, cafenstrole, and fenoxasulfone, inhibited the production of very long acyl chains, thereby impairing Entamoeba trophozoite proliferation and cyst formation. Importantly, none of these three compounds showed toxicity to a human cell line; therefore, EhFAEs are reasonable targets for developing new anti-amebiasis drugs and these compounds are promising leads for such drugs. Interestingly, in the Amoebazoan lineage, gain and loss of the genes encoding two different types of fatty acid elongase have occurred during evolution, which may be relevant to parasite adaptation. Acyl chain diversity in lipids is therefore a unique and indispensable feature for parasitic adaptation of Entamoeba. Author summary: Entamoeba, a protozoan parasite, thrives in its niche by alternating its form between a proliferative trophozoite and a dormant cyst. Entamoeba parasites were recently shown to generate lipid acyl chain diversity throughout their life cycle. Here, we provide insight into the mechanism generating this acyl chain diversity and its importance in maintaining the parasite's life cycle. We also show that the E. histolytica isozymes responsible for generating this diversity are reasonable targets for developing new anti-amebiasis drugs, and that the herbicides, flufenacet, cafenstrole, and fenoxasulfone, are promising leads for such drugs. Interestingly, gain and loss of the two genes encoding the isozymes controlling acyl chain diversity is plausibly related to E. histolytica host adaptation. [ABSTRACT FROM AUTHOR]

Persistence of the rebound-competent viral reservoir (RCVR) within the CD4+ T cell compartment of people living with HIV remains a major barrier to HIV cure. Here, we determined the effects of the pan-lymphocyte-depleting monoclonal antibody (mAb) alemtuzumab on the RCVR in SIVmac239-infected rhesus macaques (RM) receiving antiretroviral therapy (ART). Alemtuzumab administered during chronic ART or at the time of ART initiation induced >95% depletion of circulating CD4+ T cells in peripheral blood and substantial CD4+ T cell depletion in lymph nodes. However, treatment was followed by proliferation and reconstitution of CD4+ T cells in blood, and despite ongoing ART, levels of cell-associated SIV DNA in blood and lymphoid tissues were not substantially different between alemtuzumab-treated and control RM after immune cell reconstitution, irrespective of the time of alemtuzumab treatment. Upon ART cessation, 19 of 22 alemtuzumab-treated RM and 13 of 13 controls rebounded in <28 days with no difference in the time to rebound between treatment groups. Time to rebound and reactivation rate was associated with plasma viral loads (pVLs) at time of ART initiation, suggesting lymphocyte depletion had no durable impact on the RCVR. However, 3 alemtuzumab-treated RM that had lowest levels of pre-ART viremia, failed to rebound after ART withdrawal, in contrast to controls with similar levels of SIV replication. These observations suggest that alemtuzumab therapy has little to no ability to reduce well-established RCVRs but may facilitate RCVR destabilization when pre-ART virus levels are particularly low. Author summary: A major challenge to curing HIV is the presence of cells with rebound-competent viral reservoirs (RCVR) capable of re-establishing systemic infection when ART is interrupted. CD4+ T cells harboring RCVR persist for life due to homeostatic mechanisms that allow them to proliferate and expand during ART. In this study, we explored whether pan-lymphocyte depletion with a licensed CD52-depleting monoclonal antibody called alemtuzumab, can eliminate or reduce RCVR in SIV-infected rhesus macaques (RM) when administered either at the time of ART initiation or during chronic ART administration. While alemtuzumab induced massive depletion of lymphocytes in blood, including >95% depletion of CD4+ T cells, there were no measurable effects of alemtuzumab on cell-associated viral loads during ART or SIV rebound dynamics following ART interruption in most RM. Three alemtuzumab-treated RM failed to manifest rebound viremia during extended follow up after ART withdrawal. However, these animals had the lowest levels of pre-ART SIV replication, suggesting that alemtuzumab may have been effective in RM with early and presumably smaller RCVR. Overall, our study highlights the current limitations of lymphocyte depletion strategies in facilitating the permanent disruption of RCVR, particularly once the RCVR is established above levels that induce consistent post-ART viremia. [ABSTRACT FROM AUTHOR]

The severity of COVID-19 is linked to excessive inflammation. Neutrophils represent a critical arm of the innate immune response and are major mediators of inflammation, but their role in COVID-19 pathophysiology remains poorly understood. We conducted transcriptomic profiling of neutrophils obtained from patients with mild and severe COVID-19, as well as from SARS-CoV-2 infected mice, in comparison to non-infected healthy controls. In addition, we investigated the inflammasome formation potential in neutrophils from patients and mice upon SARS-CoV-2 infection. Transcriptomic analysis of polymorphonuclear cells (PMNs), consisting mainly of mature neutrophils, revealed a striking type I interferon (IFN-I) gene signature in severe COVID-19 patients, contrasting with mild COVID-19 and healthy controls. Notably, low-density granulocytes (LDGs) from severe COVID-19 patients exhibited an immature neutrophil phenotype and lacked this IFN-I signature. Moreover, PMNs from severe COVID-19 patients showed heightened nigericin-induced caspase1 activation, but reduced responsiveness to exogenous inflammasome priming. Furthermore, IFN-I emerged as a priming stimulus for neutrophil inflammasomes. These findings suggest a potential role for neutrophil inflammasomes in driving inflammation during severe COVID-19. Altogether, these findings open promising avenues for targeted therapeutic interventions to mitigate the pathological processes associated with the disease. Author summary: COVID-19, caused by the SARS-CoV-2, ranges from mild "flu"-like symptoms to severe respiratory distress or even death. Neutrophils are important cells of our immune system which are strongly involved in inflammatory responses, including those occurring in COVID-19. However, despite extensive research, the precise contribution of neutrophils to the pathogenesis of COVID-19 remains elusive, and further clarification on their role is still needed. In this study, we isolated neutrophils from COVID-19 patients and healthy controls to analyze changes in their gene expression profile and inflammatory functions. These analyses revealed a distinct type I interferon (IFN-I) gene signature expressed by mature, but not immature, neutrophils from severe COVID-19 patients, which was absent in mild cases and healthy controls. Additionally, neutrophils from severe COVID-19 showed signs of increased inflammasome activation, a protein complex that contributes to inflammation by releasing inflammatory cytokines. Notably, IFN-I alone was able to promote neutrophil inflammasome formation in vitro suggesting a direct link between IFN-I response and inflammasome formation during COVID-19. Furthermore, increased neutrophil inflammasome activity was detected also in a mouse model of COVID-19. These findings suggest a potential role for neutrophils in driving excessive inflammation during severe COVID-19, and a role for IFN-I in priming the assembly of inflammasomes in these cells. [ABSTRACT FROM AUTHOR]

Identifying new molecular therapies targeted at the severe hepatic fibrosis associated with the granulomatous immune response to Schistosoma mansoni infection is essential to reduce fibrosis-related morbidity/mortality in schistosomiasis. In vitro cell activation studies suggested the lipid molecule prostaglandin D2 (PGD2) as a potential pro-fibrotic candidate in schistosomal context, although corroboratory in vivo evidence is still lacking. Here, to investigate the role of PGD2 and its cognate receptor DP2 in vivo, impairment of PGD2 synthesis by HQL-79 (an inhibitor of the H-PGD synthase) or DP2 receptor inhibition by CAY10471 (a selective DP2 antagonist) were used against the fibrotic response of hepatic eosinophilic granulomas of S. mansoni infection in mice. Although studies have postulated PGD2 as a fibrogenic molecule, HQL-79 and CAY10471 amplified, rather than attenuated, the fibrotic response within schistosome hepatic granulomas. Both pharmacological strategies increased hepatic deposition of collagen fibers–an unexpected outcome accompanied by further elevation of hepatic levels of the pro-fibrotic cytokines TGF-β and IL-13 in infected animals. In contrast, infection-induced enhanced LTC4 synthesis in the schistosomal liver was reduced after HQL-79 and CAY10471 treatments, and therefore, inversely correlated with collagen production in granulomatous livers. Like PGD2-directed maneuvers, antagonism of cysteinyl leukotriene receptors CysLT1 by MK571 also promoted enhancement of TGF-β and IL-13, indicating a key down-regulatory role for endogenous LTC4 in schistosomiasis-induced liver fibrosis. An ample body of data supports the role of S. mansoni-driven DP2-mediated activation of eosinophils as the source of LTC4 during infection, including: (i) HQL-79 and CAY10471 impaired systemic eosinophilia, drastically decreasing eosinophils within peritoneum and hepatic granulomas of infected animals in parallel to a reduction in cysteinyl leukotrienes levels; (ii) peritoneal eosinophils were identified as the only cells producing LTC4in PGD2-mediated S. mansoni-induced infection; (iii) the magnitude of hepatic granulomatous eosinophilia positively correlates with S. mansoni-elicited hepatic content of cysteinyl leukotrienes, and (iv) isolated eosinophils from S. mansoni-induced hepatic granuloma synthesize LTC4in vitro in a PGD2/DP2 dependent manner. So, our findings uncover that granulomatous stellate cells-derived PGD2 by activating DP2 receptors on eosinophils does stimulate production of anti-fibrogenic cysLTs, which endogenously down-regulates the hepatic fibrogenic process of S. mansoni granulomatous reaction–an in vivo protective function which demands caution in the future therapeutic attempts in targeting PGD2/DP2 in schistosomiasis. Author summary: Accumulation of scar tissue (fibrosis) in the liver is the main cause of health problems associated with schistosomiasis even after the parasite is eliminated by treatment with anthelmintics. Previous experiments with isolated cells in culture have identified a potential role for a lipid mediator, PGD2, in promoting liver fibrosis leading to suggestions that PGD2 inhibition may be beneficial for the people infected with Schistosoma parasite. However, there was no direct evidence in an infection model to support these claims. Here, we described the effect of inhibiting the production or action of PGD2 in a mouse model of schistosomiasis. We identified the cell target and mechanism of action of PGD2's participation in schistosomiasis. However, our data indicates that PGD2 protects the liver from fibrosis. Thus, inhibition of PGD2 action in patients infected with the Schistosoma parasite may aggravate the condition and promote faster liver failure and should not be pursued as a treatment option. [ABSTRACT FROM AUTHOR]

Multiple peptide resistance factor (MprF) confers resistance to cationic antimicrobial peptides (AMPs) in several pathogens, thereby enabling evasion of the host immune response. The role of MprF in commensals remains, however, uncharacterized. To close this knowledge gap, we used a common gut commensal of animals, Lactiplantibacillus plantarum, and its natural host, the fruit fly Drosophila melanogaster, as an experimental model to investigate the role of MprF in commensal-host interactions. The L. plantarum ΔmprF mutant that we generated exhibited deficiency in the synthesis of lysyl-phosphatidylglycerol (Lys-PG), resulting in increased negative cell surface charge and increased susceptibility to AMPs. Susceptibility to AMPs had no effect on ΔmprF mutant's ability to colonize guts of uninfected flies. However, we observed significantly reduced abundance of the ΔmprF mutant after infection-induced inflammation in the guts of wild-type flies but not flies lacking AMPs. Additionally, we found that the ΔmprF mutant compared to wild-type L. plantarum induces a stronger intestinal immune response in flies due to the increased release of immunostimulatory peptidoglycan fragments, indicating an important role of MprF in promoting host tolerance to commensals. Our further analysis suggests that MprF-mediated lipoteichoic acid modifications are involved in host immunomodulation. Overall, our results demonstrate that MprF, besides its well-characterized role in pathogen immune evasion and virulence, is also an important commensal resilience factor. Author summary: Commensal microbial communities residing in the host intestine often experience the exposure to host immune effectors triggered by the pathogens during infection. Despite these perturbations, intestinal commensals stably colonize the host, thus exhibiting resilience to inflammation. Here, we used the fruit fly Drosophila melanogaster and its gut commensal, Lactiplantibacillus plantarum, as a model to investigate the role of a specific bacterial gene, multiple peptide resistance factor (MprF), in commensal-host interactions during infection. We generated an L. plantarum ΔmprF mutant and demonstrated susceptibility of this mutant to AMPs and deficiency in the colonization of flies after infection. Importantly, ΔmprF mutant efficiently colonized flies that lack AMPs, demonstrating that MprF-mediated resistance to host AMPs is crucial for commensal persistence during infection. Additionally, we discovered that the ΔmprF mutant induced due to an increased release of cell-wall fragments enhanced immune response in flies as compared to wild-type bacteria. Hence, MprF confers two highly-relevant functions for commensal-host association: AMP resistance and immune evasion. Finally, we showed a role of MprF in regulating LTA size, implying a broader role of MprF in the bacterial physiology then previously appreciated. Collectively, our work shows that AMP resistance via MprF-mediated lipid and LTA modifications is essential for commensal resilience during inflammation. [ABSTRACT FROM AUTHOR]

Most viral diseases display a variable clinical outcome due to differences in virus strain virulence and/or individual host susceptibility to infection. Understanding the biological mechanisms differentiating a viral infection displaying severe clinical manifestations from its milder forms can provide the intellectual framework toward therapies and early prognostic markers. This is especially true in arbovirus infections, where most clinical cases are present as mild febrile illness. Here, we used a naturally occurring vector-borne viral disease of ruminants, bluetongue, as an experimental system to uncover the fundamental mechanisms of virus-host interactions resulting in distinct clinical outcomes. As with most viral diseases, clinical symptoms in bluetongue can vary dramatically. We reproduced experimentally distinct clinical forms of bluetongue infection in sheep using three bluetongue virus (BTV) strains (BTV-1IT2006, BTV-1IT2013 and BTV-8FRA2017). Infected animals displayed clinical signs varying from clinically unapparent, to mild and severe disease. We collected and integrated clinical, haematological, virological, and histopathological data resulting in the analyses of 332 individual parameters from each infected and uninfected control animal. We subsequently used machine learning to select the key viral and host processes associated with disease pathogenesis. We identified and experimentally validated five different fundamental processes affecting the severity of bluetongue: (i) virus load and replication in target organs, (ii) modulation of the host type-I IFN response, (iii) pro-inflammatory responses, (iv) vascular damage, and (v) immunosuppression. Overall, we showed that an agnostic machine learning approach can be used to prioritise the different pathogenetic mechanisms affecting the disease outcome of an arbovirus infection. Author summary: In this study we comprehensively investigated the pathogenetic mechanisms underlying the clinical outcomes of bluetongue, a viral disease of ruminants used as an experimental model for vector-borne infections. Arboviruses are the cause of major global health and economic burden. Each arbovirus infection induces its own distinctive clinical features. However, many of these vector-borne diseases are typically characterised by general symptoms such as mild febrile flu-like illness and rash, with only a minor proportion of cases exhibiting severe clinical manifestations. It is therefore critical to understand those biological processes distinguishing arbovirus infections resulting in mild or severe clinical diseases. Here, we used in vivo experiments in sheep, artificial intelligence and in vitro experiments to identify the key processes affecting the severity of bluetongue. We established that the clinical outcome of bluetongue in the infected animals is influenced by (i) levels of virus replication in target organs, (ii) modulation of the host innate immunity and (iii) pro-inflammatory responses, (iv) damage to the blood vessels, and (v) immunosuppression. This study provides an intellectual framework on how to prioritise experimentally the variety of biological processes determining the clinical outcome to a virus infection. [ABSTRACT FROM AUTHOR]

Genetic editing of the germline using CRISPR/Cas9 technology has made it possible to alter livestock traits, including the creation of resistance to viral diseases. However, virus adaptability could present a major obstacle in this effort. Recently, chickens resistant to avian leukosis virus subgroup J (ALV-J) were developed by deleting a single amino acid, W38, within the ALV-J receptor NHE1 using CRISPR/Cas9 genome editing. This resistance was confirmed both in vitro and in vivo. In vitro resistance of W38-/- chicken embryonic fibroblasts to all tested ALV-J strains was shown. To investigate the capacity of ALV-J for further adaptation, we used a retrovirus reporter-based assay to select adapted ALV-J variants. We assumed that adaptive mutations overcoming the cellular resistance would occur within the envelope protein. In accordance with this assumption, we isolated and sequenced numerous adapted virus variants and found within their envelope genes eight independent single nucleotide substitutions. To confirm the adaptive capacity of these substitutions, we introduced them into the original retrovirus reporter. All eight variants replicated effectively in W38-/- chicken embryonic fibroblasts in vitro while in vivo, W38-/- chickens were sensitive to tumor induction by two of the variants. Importantly, receptor alleles with more extensive modifications have remained resistant to the virus. These results demonstrate an important strategy in livestock genome engineering towards antivirus resistance and illustrate that cellular resistance induced by minor receptor modifications can be overcome by adapted virus variants. We conclude that more complex editing will be necessary to attain robust resistance. Author summary: The CRISPR/CAS9 technology accelerates genetic modification studies. Induction of the farm animals' virus resistance is particularly important. However, the high adaptability of viruses could present a serious drawback to this effort. Their adaptive evolution in hosts with biotechnologically induced resistance has received minimal attention so far. Using a single amino acid deletion in the specific receptor we previously created a chicken line resistant to avian leukosis virus subgroup J. As little change as possible is desirable to preserve gene function and minimize any side effects on the fitness of the chickens. Here we however show that the virus rapidly adapts to the single amino acid deletion in the receptor while the cells with extensive receptor changes remain resistant. Our study highlights the need for complex gene editing and validating the resistance to minimize the risk of virus evolution and adaptation to the genetically modified host. [ABSTRACT FROM AUTHOR]

The chemokine co-receptors CXCR4 and CCR5 mediate HIV entry and signal transduction necessary for viral infection. However, to date only the CCR5 antagonist maraviroc is approved for treating HIV-1 infection. Given that approximately 50% of late-stage HIV patients also develop CXCR4-tropic virus, clinical anti-HIV CXCR4 antagonists are needed. Here, we describe a novel allosteric CXCR4 antagonist TIQ-15 which inhibits CXCR4-tropic HIV-1 infection of primary and transformed CD4 T cells. TIQ-15 blocks HIV entry with an IC50 of 13 nM. TIQ-15 also inhibits SDF-1α/CXCR4-mediated cAMP production, cofilin activation, and chemotactic signaling. In addition, TIQ-15 induces CXCR4 receptor internalization without affecting the levels of the CD4 receptor, suggesting that TIQ-15 may act through a novel allosteric site on CXCR4 for blocking HIV entry. Furthermore, TIQ-15 did not inhibit VSV-G pseudotyped HIV-1 infection, demonstrating its specificity in blocking CXCR4-tropic virus entry, but not CXCR4-independent endocytosis or post-entry steps. When tested against a panel of clinical isolates, TIQ-15 showed potent inhibition against CXCR4-tropic and dual-tropic viruses, and moderate inhibition against CCR5-tropic isolates. This observation was followed by a co-dosing study with maraviroc, and TIQ-15 demonstrated synergistic activity. In summary, here we describe a novel HIV-1 entry inhibitor, TIQ-15, which potently inhibits CXCR4-tropic viruses while possessing low-level synergistic activities against CCR5-tropic viruses. TIQ-15 could potentially be co-dosed with the CCR5 inhibitor maraviroc to block viruses of mixed tropisms. Author summary: HIV uses the chemokine co-receptors CXCR4 or CCR5 for cell fusion and entry. While the CCR5-tropic viruses predominate early in HIV infection, the emergence of the CXCR4-tropic viruses at later stages in 50% of patients is associated with rapid disease progression. The CCR5 antagonist maraviroc has been approved for treating HIV infection. However, currently, there is no clinical anti-HIV CXCR4 inhibitor. Here we report a novel CXCR4 antagonist TIQ-15 that potently blocked CXCR4-tropic HIV infection of human CD4 T cells. TIQ-15 in combination with maraviroc also demonstrated synergistic activities against CCR5-tropic viruses and could be potentially used with maraviroc to block viruses of mixed tropisms. [ABSTRACT FROM AUTHOR]

CD8 T cells are the predominant effector cells of adaptive immunity in preventing cytomegalovirus (CMV) multiple-organ disease caused by cytopathogenic tissue infection. The mechanism by which CMV-specific, naïve CD8 T cells become primed and clonally expand is of fundamental importance for our understanding of CMV immune control. For CD8 T-cell priming, two pathways have been identified: direct antigen presentation by infected professional antigen-presenting cells (pAPCs) and antigen cross-presentation by uninfected pAPCs that take up antigenic material derived from infected tissue cells. Studies in mouse models using murine CMV (mCMV) and precluding either pathway genetically or experimentally have shown that, in principle, both pathways can congruently generate the mouse MHC/H-2 class-I-determined epitope-specificity repertoire of the CD8 T-cell response. Recent studies, however, have shown that direct antigen presentation is the canonical pathway when both are accessible. This raised the question of why antigen cross-presentation is ineffective even under conditions of high virus replication thought to provide high amounts of antigenic material for feeding cross-presenting pAPCs. As delivery of antigenic material for cross-presentation is associated with programmed cell death, and as CMVs encode inhibitors of different cell death pathways, we pursued the idea that these inhibitors restrict antigen delivery and thus CD8 T-cell priming by cross-presentation. To test this hypothesis, we compared the CD8 T-cell responses to recombinant mCMVs lacking expression of the apoptosis-inhibiting protein M36 or the necroptosis-inhibiting protein M45 with responses to wild-type mCMV and revertant viruses expressing the respective cell death inhibitors. The data reveal that increased programmed cell death improves CD8 T-cell priming in mice capable of antigen cross-presentation but not in a mutant mouse strain unable to cross-present. These findings strongly support the conclusion that CMV cell death inhibitors restrict the priming of CD8 T cells by antigen cross-presentation. Author summary: In patients as well as in experimental mouse models, CD8 T cells represent the most potent antiviral effector cells in preventing CMV disease in immunocompromised recipients of hematopoietic cell transplantation. Despite the clinical relevance of mounting a protective response, the mode of CMV antigen presentation to naïve CD8 T cells was long debated. In principle, naïve CD8 T cells can be sensitized through "direct antigen presentation" on the surface of infected professional antigen-presenting cells (pAPCs) or through "antigen cross-presentation" by uninfected pAPCs that take up antigenic material derived from infected cells following cell death. As CMVs are cytopathogenic, eventually killing host cells, and as they encode immune evasion proteins interfering with the MHC/HLA class-I pathway of direct antigen presentation, it seemed reasonable to propose a dominant role for antigen cross-presentation. Mouse models precluding either mode of antigen presentation, however, revealed that both can raise an equivalent CD8 T-cell response, and recent work has identified direct antigen presentation as the canonical pathway taken when both are accessible. Here we show that virus-encoded inhibitors of two programmed cell death modalities, apoptosis and necroptosis, reduce antigen release to below a level required for efficient cross-presentation. This solves an open question in CMV immunology. [ABSTRACT FROM AUTHOR]

The Middle East Respiratory Syndrome Coronavirus (MERS-CoV) causes severe and fatal acute respiratory disease in humans. High fatality rates and continued infectiousness remain a pressing concern for global health preparedness. Antibodies targeted at the receptor-binding domain (RBD) are major countermeasures against human viral infection. Here, we report four potent nanobodies against MERS-CoV, which are isolated from alpaca, and especially the potency of Nb14 is highest in the pseudotyped virus assay. Structural studies show that Nb14 framework regions (FRs) are mainly involved in interactions targeting a novel epitope, which is entirely distinct from all previously reported antibodies, and disrupt the protein-carbohydrate interaction between residue W535 of RBD and hDPP4 N229-linked carbohydrate moiety (hDPP4-N229-glycan). Different from Nb14, Nb9 targets the cryptic face of RBD, which is distinctive from the hDPP4 binding site and the Nb14 epitope, and it induces the β5-β6 loop to inflect towards a shallow groove of the RBD and dampens the accommodation of a short helix of hDPP4. The particularly striking epitopes endow the two Nbs administrate synergistically in the pseudotyped MERS-CoV assays. These results not only character unprecedented epitopes for antibody recognition but also provide promising agents for prophylaxis and therapy of MERS-CoV infection. Author summary: MERS-CoV is one of the most prevalent zoonotic virus that has spread through 27 countries and infected more than 2,605 people since its first outbreak in Saudi Arabia in 2012. The high fatality rate and its persistent wide spread infectiousness in animal reservoirs have generated tremendous global public health concern. However, no licensed therapeutic agents or vaccines against MERS-CoV are currently available. To address the risk of MERS-CoV and its variants re-emergence, we developed four highly effective Nbs from alpaca and solved two crystal complexes of Nb9 and Nb14 with RBD. Remarkably, the neutralizing activity of Nb14 represents the highest of MERS-CoV antibodies reported to date. The novel epitopes of Nb14 and Nb9 locate outside the RBD and hDPP4 interface, and the unique different epitopes and mechanisms of Nb14 and Nb9 provide a better neutralizing for synergistically against MERS-CoV infection. Our findings provide insights into the cryptic epitopes on RBD and the development of antibodies against MERS-CoV infection. [ABSTRACT FROM AUTHOR]

Intestinal helminth infection triggers a type 2 immune response that promotes a 'weep-and sweep' response characterised by increased mucus secretion and intestinal hypermotility, which function to dislodge the worm from its intestinal habitat. Recent studies have discovered that several other pathogens cause intestinal dysmotility through major alterations to the immune and enteric nervous systems (ENS), and their interactions, within the gastrointestinal tract. However, the involvement of these systems has not been investigated for helminth infections. Eosinophils represent a key cell type recruited by the type 2 immune response and alter intestinal motility under steady-state conditions. Our study aimed to investigate whether altered intestinal motility driven by the murine hookworm, Nippostrongylus brasiliensis, infection involves eosinophils and how the ENS and smooth muscles of the gut are impacted. Eosinophil deficiency did not influence helminth-induced intestinal hypermotility and hypermotility did not involve gross structural or functional changes to the ENS. Hypermotility was instead associated with a dramatic increase in smooth muscle thickness and contractility, an observation that extended to another rodent nematode, Heligmosomoides polygyrus. In summary our data indicate that, in contrast to other pathogens, helminth-induced intestinal hypermotility is driven by largely by myogenic, rather than neurogenic, alterations with such changes occurring independently of eosinophils. (<300 words) Author summary: Intestinal helminth infection is a global threat to those living in poverty without adequate sanitation. Expulsion of intestinal worms is driven by a host type 2 immune response, characterised by increased eosinophils, that results in the intestinal hypermotility and mucus secretion that dislodge the worm from its luminal habitat. Intestinal motility is largely controlled by the local enteric nervous system (ENS) and can be regulated by close interactions between neurons and intestinal immune cells. Using Nippostrongylus brasiliensis as a model of murine hookworm infection, we investigated the contribution of the ENS and eosinophils to intestinal hypermotility and worm expulsion. Despite the critical role of the ENS in regulating typical intestinal function, very little alteration to ENS structure or function was observed following Nb infection. Instead, Nb infected animals displayed dramatically increased smooth muscle thickness and contractile strength. Alterations of smooth muscle were also observed in response to infection with Heligmosomoides polygyrus. Neither Nippostrongylus brasiliensis -induced intestinal hypermotility nor altered smooth muscle morphology required eosinophils. Our findings reveal that, in contrast to other intestinal pathogens, myogenic rather than neurogenic alterations drive small intestinal hypermotility and pathogen expulsion following intestinal helminth infection. (<200 words) [ABSTRACT FROM AUTHOR]

Overnutrition with dietary sugar can worsen infection outcomes in diverse organisms including insects and humans, through generally unknown mechanisms. In the present study, we show that adult Drosophila melanogaster fed high-sugar diets became more susceptible to infection by the Gram-negative bacteria Providencia rettgeri and Serratia marcescens. We found that P. rettgeri and S. marcescens proliferate more rapidly in D. melanogaster fed a high-sugar diet, resulting in increased probability of host death. D. melanogaster become hyperglycemic on the high-sugar diet, and we find evidence that the extra carbon availability may promote S. marcescens growth within the host. However, we found no evidence that increased carbon availability directly supports greater P. rettgeri growth. D. melanogaster on both diets fully induce transcription of antimicrobial peptide (AMP) genes in response to infection, but D. melanogaster provided with high-sugar diets show reduced production of AMP protein. Thus, overnutrition with dietary sugar may impair host immunity at the level of AMP translation. Our results demonstrate that dietary sugar can shape infection dynamics by impacting both host and pathogen, depending on the nutritional requirements of the pathogen and by altering the physiological capacity of the host to sustain an immune response. Author summary: Diet has a critical impact on the quality of immune defense and high-sugar diets increase susceptibility to bacterial infection in many animals. Yet it is unknown which aspects of host and pathogen physiology are impacted by diet to influence infection dynamics. Here we show that high-sugar diets increase susceptibility to some bacterial infections in Drosophila. We find that feeding on high-sugar diet impairs the host immune response by reducing the level of antimicrobial peptides produced. The expression of genes encoding these peptides is not affected, so we infer that protein translation is impaired. We further show that flies on high-sugar diets are hyperglycemic, and that some pathogens may use the excess sugar in the host to promote growth during the infection. Thus, our study demonstrates that dietary impacts on infection outcome arise through physiological effects on both the host and pathogen. [ABSTRACT FROM AUTHOR]

Natural killer (NK) cells play a critical role in virus control. However, it has remained largely unclear whether NK cell mobilization in SARS-CoV-2 infections is beneficial or pathologic. To address this deficit, we employed a validated experimental NK cell depletion non-human primate (NHP) model with SARS-CoV-2 Delta variant B.1.617.2 challenge. Viral loads (VL), NK cell numbers, activation, proliferation, and functional measures were evaluated in blood and tissues. In non-depleted (control) animals, infection rapidly induced NK cell expansion, activation, and increased tissue trafficking associated with VL. Strikingly, we report that experimental NK cell depletion leads to higher VL, longer duration of viral shedding, significantly increased levels of pro-inflammatory cytokines in the lungs, and overt lung damage. Overall, we find the first significant and conclusive evidence for NK cell-mediated control of SARS-CoV-2 virus replication and disease pathology. These data indicate that adjunct therapies for infection could largely benefit from NK cell-targeted approaches. Author summary: Natural killer (NK) cells play a critically understudied role in controlling SARS-CoV-2 viral replication, clearance, and disease sequelae. In this manuscript, we investigated the protective role of NK cells in acute infection using a well-established NK cell depletion strategy in cynomolgus macaques (CM) and a SARS-CoV-2 delta variant infection model. Circulating NK cells exhibited an increased proliferative and activated phenotype following infection, concomitant with peak NK cell expansion at 10 days post-infection (DPI). Importantly, following experimental NK cell depletion, CM exhibited increased viral shedding and delayed viral clearance compared to controls. NK cell-depleted animals also exhibited significantly increased lung pathology and Luminex cytokine analyses of broncho-alveolar lavage (BAL) fluid showed a 5-fold increase in interferon-alpha (IFNα) compared to controls during peak infection. Collectively, our findings suggest that NK cells play a crucial role in controlling SARS-CoV-2 replication and reducing lung damage. These results underscore the potential of NK cell-based vaccines and therapies for COVID-19 and other infectious diseases, warranting further investigation in this area. [ABSTRACT FROM AUTHOR]

Group B Streptococcus (GBS) is a major human and animal pathogen that threatens public health and food security. Spill-over and spill-back between host species is possible due to adaptation and amplification of GBS in new niches but the evolutionary and functional mechanisms underpinning those phenomena are poorly known. Based on analysis of 1,254 curated genomes from all major GBS host species and six continents, we found that the global GBS population comprises host-generalist, host-adapted and host-restricted sublineages, which are found across host groups, preferentially within one host group, or exclusively within one host group, respectively, and show distinct levels of recombination. Strikingly, the association of GBS genomes with the three major host groups (humans, cattle, fish) is driven by a single accessory gene cluster per host, regardless of sublineage or the breadth of host spectrum. Moreover, those gene clusters are shared with other streptococcal species occupying the same niche and are functionally relevant for host tropism. Our findings demonstrate (1) the heterogeneity of genome plasticity within a bacterial species of public health importance, enabling the identification of high-risk clones; (2) the contribution of inter-species gene transmission to the evolution of GBS; and (3) the importance of considering the role of animal hosts, and the accessory gene pool associated with their microbiota, in the evolution of multi-host bacterial pathogens. Collectively, these phenomena may explain the adaptation and clonal expansion of GBS in animal reservoirs and the risk of spill-over and spill-back between animals and humans. Author summary: Group B Streptococcus (GBS) is a bacterium that represents a health and food security threat in three major host groups: humans (in particular neonates), bovines, and fishes. However, the genomic mechanisms driving adaptation to these hosts remain unclear. Here, we use powerful statistical approaches to compare genomes of GBS from around the world. We found that GBS' ability to exchange genes is a good indicator of how well it can adapt to different hosts. Additionally, three groups of genes appear crucial for GBS to infect either humans, cows, or fishes, regardless of the bacterial strain. These gene groups are also found in other similar bacteria that live in the same hosts or environments. Where a functional role is already known for two gene groups in humans and bovines, respectively, it is shown here for the first time for the third gene group in fishes. Our findings demonstrate the importance of considering the role of animals in the evolution of multi-host bacterial pathogens like GBS. Gene exchange between bacteria infecting multiple hosts represents a high threat to human health, as high-risk types of GBS could adapt and expand in animals, which could then act as reservoirs for highly pathogenic human infections. [ABSTRACT FROM AUTHOR]

The pathogenesis of HIV-1 infection is governed by a highly dynamic, time-dependent interaction between the host and the viral genome. In this study, we developed a novel systematic approach to assess the host-virus interaction, using average pairwise viral diversity as a proxy for time since infection, and applied this method to nearly whole viral genome sequences (n = 4,464), human leukocyte antigen (HLA) genotyping data (n = 1,044), and viral RNA load (VL) measurements during the untreated chronic phase (n = 829) of Swiss HIV Cohort Study participants. Our systematic genome-wide screen revealed for 98 HLA/viral-variant pairs a signature of immune-driven selection in the form of an HLA-dependent effect of infection time on the presence of HIV amino acid variants. Of these pairs, 12 were found to have an effect on VL. Furthermore, 28/58 pairs were validated by time-to-event analyses and 48/92 by computational HLA-epitope predictions. Our diversity-based approach allows a powerful and systematic investigation of the interaction between the virus and cellular immunity, revealing a notable subset of such interaction effects. From an evolutionary perspective, these observations underscore the complexity of HLA-mediated selection pressures on the virus that shape viral evolution and pathogenesis. Author summary: The intricate interplay between viruses and the human immune system is reflected in dynamic associations between the viral and the human genomes. These often take the form of escape dynamics, in which the virus acquires mutations that allow it to evade immune recognition. We developed a novel viral diversity-based method to screen for such interactions across the viral genome systematically and applied it to a unique dataset of HIV-1 sequences and human leukocyte antigen (HLA) variants. We could identify time-dependent interactions between 98 pairs of HLA and viral variants. Among these pairs, 12 were associated with the concentration of viral RNA, longitudinal time-to-event analyses confirmed 28, and 48 were consistent with computational predictions of viral peptide binding to HLA molecules. Our results highlight how the highly dynamic interaction between the viral genome and the immune system shapes viral evolution, and our approach offers new opportunities to systematically study such interactions from real-world cross-sectional data. [ABSTRACT FROM AUTHOR]

Manipulating evolutionary forces imposed by hosts on pathogens like genetic drift and selection could avoid the emergence of virulent pathogens. For instance, increasing genetic drift could decrease the risk of pathogen adaptation through the random fixation of deleterious mutations or the elimination of favorable ones in the pathogen population. However, no experimental proof of this approach is available for a plant-pathogen system. We studied the impact of pepper (Capsicum annuum) lines carrying the same major resistance gene but contrasted genetic backgrounds on the evolution of Potato virus Y (PVY). The pepper lines were chosen for the contrasted levels of genetic drift (inversely related to Ne, the effective population size) they exert on PVY populations, as well as for their contrasted resistance efficiency (inversely related to the initial replicative fitness, Wi, of PVY in these lines). Experimental evolution was performed by serially passaging 64 PVY populations every month on six contrasted pepper lines during seven months. These lines exhibited highly divergent evolutionary trajectories, ranging from viral extinctions to replicative fitness gains. The sequencing of the PVY VPg cistron, where adaptive mutations are likely to occur, allowed linking these replicative fitness gains to parallel adaptive nonsynonymous mutations. Evolutionary trajectories were well explained by the genetic drift imposed by the host. More specifically, Ne, Wi and their synergistic interaction played a major role in the fate of PVY populations. When Ne was low (i.e. strong genetic drift), the final PVY replicative fitness remained close to the initial replicative fitness, whereas when Ne was high (i.e. low genetic drift), the final PVY replicative fitness was high independently of the replicative fitness of the initially inoculated virus. We show that combining a high resistance efficiency (low Wi) and a strong genetic drift (low Ne) is the best solution to increase resistance durability, that is, to avoid virus adaptation on the long term. Author summary: Given their high evolutionary potential, viruses are notoriously difficult to control by drug treatments or by breeding resistant hosts. In plants, resistance genes targeting viruses become often rapidly inefficient due to virus mutational escape. Manipulating the evolutionary forces imposed by the plant on the virus population, like selection and genetic drift, could help control virus adaptation and prevent resistance breakdown. Theoretically, a plant genotype imposing low selective pressures could slow down the fixation of adaptive viral mutations, while a plant genotype imposing strong genetic drift could decrease virus fitness by favoring the random fixation of deleterious viral mutations and/or the elimination of favorable ones. Using experimental evolution, we studied the effect of plants imposing contrasted levels of genetic drift on the emergence of resistance-adapted virus mutants. Results indicate that the level of genetic drift during plant colonization, in part controlled by the genetic background of the plant, and the plant resistance level are the main drivers of the virus evolutionary trajectories, which varied from fast adaptation to extinction. Consequently, it is possible to achieve high resistance efficiency and durability by breeding plant genotypes through the introduction of major-effect resistance genes exerting a strong selection on virus populations into a genetic background increasing genetic drift. [ABSTRACT FROM AUTHOR]

Enteroviruses are a vast genus of positive-sense RNA viruses that cause diseases ranging from common cold to poliomyelitis and viral myocarditis. They encode a membrane-bound AAA+ ATPase, 2C, that has been suggested to serve several roles in virus replication, e.g. as an RNA helicase and capsid assembly factor. Here, we report the reconstitution of full-length, poliovirus 2C's association with membranes. We show that the N-terminal membrane-binding domain of 2C contains a conserved glycine, which is suggested by structure predictions to divides the domain into two amphipathic helix regions, which we name AH1 and AH2. AH2 is the main mediator of 2C oligomerization, and is necessary and sufficient for its membrane binding. AH1 is the main mediator of a novel function of 2C: clustering of membranes. Cryo-electron tomography reveal that several 2C copies mediate this function by localizing to vesicle-vesicle interfaces. 2C-mediated clustering is partially outcompeted by RNA, suggesting a way by which 2C can switch from an early role in coalescing replication organelles and lipid droplets, to a later role where 2C assists RNA replication and particle assembly. 2C is sufficient to recruit RNA to membranes, with a preference for double-stranded RNA (the replicating form of the viral genome). Finally, the in vitro reconstitution revealed that full-length, membrane-bound 2C has ATPase activity and ATP-independent, single-strand ribonuclease activity, but no detectable helicase activity. Together, this study suggests novel roles for 2C in membrane clustering, RNA membrane recruitment and cleavage, and calls into question a role of 2C as an RNA helicase. The reconstitution of functional, 2C-decorated vesicles provides a platform for further biochemical studies into this protein and its roles in enterovirus replication. Author summary: Enteroviruses are frequent causes of diseases that range from mild common colds to severe conditions such as poliomyelitis, acute flaccid myelitis and viral myocarditis. Enterovirus particles are tiny, even by virus standards, and they can only package a small amount of viral genetic material. With such a small genetic 'toolbox', how can these viruses so vigorously hijack the interior of a cell and turn it into a viral factory? One reason is that the viral genes often encode multifunctional proteins. Here, we studied one such multifunctional viral protein, called 2C. This protein is found in all enteroviruses and in related viruses, and the virus cannot replicate without it. We developed a new way of studying 2C that allowed us to work with the complete 2C protein 'in its element', namely bound to a membrane. We found that 2C not only binds one membrane, but can gather several membranes together–a function that may be important to building the viral factory. We also found that 2C can bind RNA (the chemical form of the virus' genetic material) to membranes and can cleave RNA. Our findings reinforce the view of 2C as an organizer of the viral factory. [ABSTRACT FROM AUTHOR]

Avian malaria is expanding upslope with warmer temperatures and driving multiple species of Hawaiian birds towards extinction. Methods to reduce malaria transmission are urgently needed to prevent further declines. Releasing Wolbachia-infected incompatible male mosquitoes could suppress mosquito populations and releasing Wolbachia-infected female mosquitoes (or both sexes) could reduce pathogen transmission if the Wolbachia strain reduced vector competence. We cleared Culex quinquefasciatus of their natural Wolbachia pipientis wPip infection and transinfected them with Wolbachia wAlbB isolated from Aedes albopictus. We show that wAlbB infection was transmitted transovarially, and demonstrate cytoplasmic incompatibility with wild-type mosquitoes infected with wPip from Oahu and Maui, Hawaii. We measured vector competence for avian malaria, Plasmodium relictum, lineage GRW4, of seven mosquito lines (two with wAlbB; three with natural wPip infection, and two cleared of Wolbachia infection) by allowing them to feed on canaries infected with recently collected field isolates of Hawaiian P. relictum. We tested 73 groups (Ntotal = 1176) of mosquitoes for P. relictum infection in abdomens and thoraxes 6–14 days after feeding on a range of parasitemias from 0.028% to 2.49%, as well as a smaller subset of salivary glands. We found no measurable effect of Wolbachia on any endpoint, but strong effects of parasitemia, days post feeding, and mosquito strain on both abdomen and thorax infection prevalence. These results suggest that releasing male wAlbB-infected C. quinquefasciatus mosquitoes could suppress wPip-infected mosquito populations, but would have little positive or negative impact on mosquito vector competence for P. relictum if wAlbB became established in local mosquito populations. More broadly, the lack of Wolbachia effects on vector competence we observed highlights the variable impacts of both native and transinfected Wolbachia infections in mosquitoes. Author summary: Avian malaria is expanding upslope in Hawaii with warmer temperatures and driving multiple species of birds towards extinction. Methods to reduce malaria transmission are urgently needed to prevent further declines. Releasing male mosquitoes infected with a type of bacteria (Wolbachia) could suppress mosquito populations by reducing offspring viability and releasing Wolbachia-infected female mosquitoes (or both sexes) could reduce pathogen transmission if the Wolbachia strain reduced vector competence. We cleared Culex quinquefasciatus of their natural Wolbachia pipientis wPip infection and transinfected them with Wolbachia wAlbB isolated from Aedes albopictus. We show that wAlbB infection was transmitted to offspring, and mating wAlbB males with wild-type mosquitoes produced mostly inviable offspring. We measured susceptibility to avian malaria infection of mosquitoes with native or transinfected Wolbachia (wPip or wAlbB). We found no measurable effect of Wolbachia on susceptibility. These results suggest that releasing male wAlbB-infected C. quinquefasciatus mosquitoes could suppress Hawaiian mosquito populations, but would have little positive or negative impact on mosquito vector competence for avian malaria. More broadly, the lack of Wolbachia effects on susceptibility to avian malaria we observed highlights the variable impacts of both native and transinfected Wolbachia infections in mosquitoes. [ABSTRACT FROM AUTHOR]

Epstein-Barr Virus (EBV) is associated with numerous cancers including B cell lymphomas. In vitro, EBV transforms primary B cells into immortalized Lymphoblastoid Cell Lines (LCLs) which serves as a model to study the role of viral proteins in EBV malignancies. EBV induced cellular transformation is driven by viral proteins including EBV-Nuclear Antigens (EBNAs). EBNA-LP is important for the transformation of naïve but not memory B cells. While EBNA-LP was thought to promote gene activation by EBNA2, EBNA-LP Knockout (LPKO) virus-infected cells express EBNA2-activated cellular genes efficiently. Therefore, a gap in knowledge exists as to what roles EBNA-LP plays in naïve B cell transformation. We developed a trans-complementation assay wherein transfection with wild-type EBNA-LP rescues the transformation of peripheral blood- and cord blood-derived naïve B cells by LPKO virus. Despite EBNA-LP phosphorylation sites being important in EBNA2 co-activation; neither phospho-mutant nor phospho-mimetic EBNA-LP was defective in rescuing naïve B cell outgrowth. However, we identified conserved leucine-rich motifs in EBNA-LP that were required for transformation of adult naïve and cord blood B cells. Because cellular PPAR-g coactivator (PGC) proteins use leucine-rich motifs to engage transcription factors including YY1, a key regulator of DNA looping and metabolism, we examined the role of EBNA-LP in engaging transcription factors. We found a significant overlap between EBNA-LP and YY1 in ChIP-Seq data. By Cut&Run, YY1 peaks unique to WT compared to LPKO LCLs occur at more highly expressed genes. Moreover, Cas9 knockout of YY1 in primary B cells prior to EBV infection indicted YY1 to be important for EBV-mediated transformation. We confirmed EBNA-LP and YY1 biochemical association in LCLs by endogenous co-immunoprecipitation and found that the EBNA-LP leucine-rich motifs were required for YY1 interaction in LCLs. We propose that EBNA-LP engages YY1 through conserved leucine-rich motifs to promote EBV transformation of naïve B cells. Author summary: Epstein-Barr Virus (EBV) is associated with various B cell lymphomas, particularly in immunosuppressed individuals. In the absence of a functional immune system, viral latency proteins, including EBV Nuclear Antigens (EBNAs) act as oncoproteins to promote tumorigenesis. EBNA-LP is one of the first viral proteins produced after infection and is important for the transformation of naïve B cells. However, the roles of EBNA-LP during infection are largely undefined. In this study, developed an assay in which the role of wild type and mutant EBNA-LP could be investigated in the context of primary naïve B cells infected with an EBNA-LP Knockout virus. Using this assay, we identified highly conserved leucine-rich motifs within EBNA-LP that are important for transformation of EBV-infected naïve B cells. These conserved motifs associate with the cellular transcription factor YY1, an important transcriptional regulator in B cell development and in many cancers, that we now show is essential for outgrowth of EBV infected B cells. Our study provides further insights into the mechanisms by which EBV transforms naïve B cells. [ABSTRACT FROM AUTHOR]

Recognizing that enteric tuft cells can signal the presence of nematode parasites, we investigated whether tuft cells are required for the expulsion of the cestode, Hymenolepis diminuta, from the non-permissive mouse host, and in concomitant anti-helminthic responses. BALB/c and C57BL/6 mice infected with H. diminuta expelled the worms by 11 days post-infection (dpi) and displayed DCLK1+ (doublecortin-like kinase 1) tuft cell hyperplasia in the small intestine (not the colon) at 11 dpi. This tuft cell hyperplasia was dependent on IL-4Rα signalling and adaptive immunity, but not the microbiota. Expulsion of H. diminuta was slowed until at least 14 dpi, but not negated, in tuft cell-deficient Pou2f3-/- mice and was accompanied by delayed goblet cell hyperplasia and slowed small bowel transit. Worm antigen and mitogen evoked production of IL-4 and IL-10 by splenocytes from wild-type and Pou2f3-/- mice was not appreciably different, suggesting similar systemic immune reactivity to infection with H. diminuta. Wild-type and Pou2f3-/- mice infected with H. diminuta displayed partial protection against subsequent infection with the nematode Heligmosomoides bakeri. We speculate that, with respect to H. diminuta, enteric tuft cells are important for local immune events driving the rapidity of H. diminuta expulsion but are not critical in initiating or sustaining systemic Th2 responses that provide concomitant immunity against secondary infection with H. bakeri. Author summary: The small intestinal tuft cell is emerging as a versatile sentinel cell type in a variety of infections involving protozoans, nematodes, and certain bacteria. Whether the tuft cell responds to enteric cestode infections and the extent of its involvement in mediating the anti-helminth response is not yet characterized. Using mice infected with Hymenolepis diminuta, we show that tuft cells help accelerate expulsion of this lumen-dwelling cestode, by coordinating local events that form an integral part of the classical "weep and sweep" anti-helminthic response. We further show that although infection with H. diminuta induces both tuft cell hyperplasia as well as protection from subsequent infection with the nematode Heligmosmoides bakeri, the tuft cell is not solely responsible for mediating systemic and other local Th2 responses against H. diminuta. Our work reveals subtle roles for the tuft cell in initiating and coordinating host anti-parasitic activity in cestode infections. At the same time, we show that redundancies exist in the host's mucosal arsenal that ultimately mediate worm expulsion in tuft cell deficient mice. [ABSTRACT FROM AUTHOR]

Reactive carbonyl and oxygen species (RCS/ROS), often generated as metabolic byproducts, particularly under conditions of pathology, can cause direct damage to proteins, lipids, and nucleic acids. Glyoxal oxidases (Gloxs) oxidize aldehydes to carboxylic acids, generating hydrogen peroxide (H2O2). Although best characterized for their roles in lignin degradation, Glox in plant fungal pathogens are known to contribute to virulence, however, the mechanism underlying such effects are unclear. Here, we show that Glox in the insect pathogenic fungus, Metarhizium acridum, is highly expressed in mycelia and during formation of infection structures (appressoria), with the enzyme localizing to the cell membrane. MaGlox targeted gene disruption mutants showed RCS and ROS accumulation, resulting in cell toxicity, induction of apoptosis and increased autophagy, inhibiting normal fungal growth and development. The ability of the MaGlox mutant to scavenge RCS was significantly reduced, and the mutant exhibited increased susceptibility to aldehydes, oxidative and cell wall perturbing agents but not toward osmotic stress, with altered cell wall contents. The ΔMaGlox mutant was impaired in its ability to penetrate the host cuticle and evade host immune defense resulting in attenuated pathogenicity. Overexpression of MaGlox promoted fungal growth and conidial germination, increased tolerance to H2O2, but had little to other phenotypic effects. Transcriptomic analyses revealed downregulation of genes related to cell wall synthesis, conidiation, stress tolerance, and host cuticle penetration in the ΔMaGlox mutant. These findings demonstrate that MaGlox-mediated scavenging of RCS is required for virulence, and contributes to normal fungal growth and development, stress resistance. Author summary: The ability of organisms to detoxify reactive metabolic byproducts is crucial for normal development, stress resistance, and where applicable, pathogenesis. Here, using the insect pathogenic fungus, Metarhizium acridum, we show that a glyoxal oxidase acts to detoxify cellular reactive carbonyl species (RCS) as well as leading to decreased reactive oxygen species (ROS) levels by alleviating aldehydes accumulation which can trigger ROS production. Mutants deficient in MaGlox showed increased levels of RCS/ROS leading to increased autophagy and apoptosis, resulting in impaired fungal growth, development, and reduced virulence. These findings link Glox mediated RCS/ROS detoxification to cell degradation/turnover processes and cell death, impacting fungal pathogenicity and development. [ABSTRACT FROM AUTHOR]

Pathogenic bacteria's metabolic adaptation for survival and proliferation within hosts is a crucial aspect of bacterial pathogenesis. Here, we demonstrate that citrate, the first intermediate of the tricarboxylic acid (TCA) cycle, plays a key role as a regulator of gene expression in Staphylococcus aureus. We show that citrate activates the transcriptional regulator CcpE and thus modulates the expression of numerous genes involved in key cellular pathways such as central carbon metabolism, iron uptake and the synthesis and export of virulence factors. Citrate can also suppress the transcriptional regulatory activity of ferric uptake regulator. Moreover, we determined that accumulated intracellular citrate, partly through the activation of CcpE, decreases the pathogenic potential of S. aureus in animal infection models. Therefore, citrate plays a pivotal role in coordinating carbon metabolism, iron homeostasis, and bacterial pathogenicity at the transcriptional level in S. aureus, going beyond its established role as a TCA cycle intermediate. Author summary: Citrate is the first intermediate of the TCA cycle, and its classic role is as a participant in metabolic reactions. In this study, we explored the non-metabolic role of citrate in Staphylococcus aureus. The results suggested that citrate can influence central carbon metabolism, iron homeostasis and the expression of virulence factors by modulating the regulatory activity of the catabolite control protein E (CcpE) and ferric uptake regulator (Fur). For instance, we revealed a new axis, named citrate-CcpE-pycA, was responsible for controlling the endogenous biosynthesis of aspartate, which likely resulted in changes in metabolic adaptation in the host and influenced the pathogenesis of this important human pathogen. Thus, citrate serves as a key signaling molecule for the modulation of gene expression in S. aureus. [ABSTRACT FROM AUTHOR]

Ergosterol is essential for fungal cell membrane integrity and growth, and numerous antifungal drugs target ergosterol. Inactivation or modification of ergosterol biosynthetic genes can lead to changes in antifungal drug susceptibility, filamentation and stress response. Here, we found that the ergosterol biosynthesis gene ERG251 is a hotspot for point mutations during adaptation to antifungal drug stress within two distinct genetic backgrounds of Candida albicans. Heterozygous point mutations led to single allele dysfunction of ERG251 and resulted in azole tolerance in both genetic backgrounds. This is the first known example of point mutations causing azole tolerance in C. albicans. Importantly, single allele dysfunction of ERG251 in combination with recurrent chromosome aneuploidies resulted in bona fide azole resistance. Homozygous deletions of ERG251 caused increased fitness in low concentrations of fluconazole and decreased fitness in rich medium, especially at low initial cell density. Homozygous deletions of ERG251 resulted in accumulation of ergosterol intermediates consistent with the fitness defect in rich medium. Dysfunction of ERG251, together with FLC exposure, resulted in decreased accumulation of the toxic sterol (14-ɑ-methylergosta-8,24(28)-dien-3β,6α-diol) and increased accumulation of non-toxic alternative sterols. The altered sterol composition of the ERG251 mutants had pleiotropic effects on transcription, filamentation, and stress responses including cell membrane, osmotic and oxidative stress. Interestingly, while dysfunction of ERG251 resulted in azole tolerance, it also led to transcriptional upregulation of ZRT2, a membrane-bound Zinc transporter, in the presence of FLC, and overexpression of ZRT2 is sufficient to increase azole tolerance in wild-type C. albicans. Finally, in a murine model of systemic infection, homozygous deletion of ERG251 resulted in decreased virulence while the heterozygous deletion mutants maintain their pathogenicity. Overall, this study demonstrates that single allele dysfunction of ERG251 is a recurrent and effective mechanism of acquired azole tolerance. We propose that altered sterol composition resulting from ERG251 dysfunction mediates azole tolerance as well as pleiotropic effects on stress response, filamentation and virulence. Author summary: Invasive infections caused by the fungal pathogen Candida albicans have high mortality rates (20–60%), even with antifungal drug treatment. Numerous mechanisms contributing to drug resistance have been characterized, but treatment failure remains a problem indicating that there are many facets that are not yet understood. The azole class of antifungals target production of ergosterol, an essential component of fungal cell membranes. Here, we provide insights into how ERG251, a component of the ergosterol biosynthesis pathway, contributes to enhanced growth in azoles, along with broader impacts on stress responses, filamentation, and pathogenicity. One of the most striking results from our study is that even a single nucleotide change in one allele of ERG251 in the diploid C. albicans can lead to azole tolerance. Tolerance, a distinct phenotype from resistance, is the ability of fungal cells to grow above the minimum inhibitory concentration in a drug concentration-independent manner. Tolerance frequently goes undetected in the clinic because it is not observable in standard assays. Strikingly, azole tolerant strains lacking one allele of ERG251 remained virulent in a mouse model of infection highlighting the potential for mutations in ERG251 to arise and contribute to treatment failure in patients. [ABSTRACT FROM AUTHOR]

Plant-parasitic nematodes constrain global food security. During parasitism, they secrete effectors into the host plant from two types of pharyngeal gland cells. These effectors elicit profound changes in host biology to suppress immunity and establish a unique feeding organ from which the nematode draws nutrition. Despite the importance of effectors in nematode parasitism, there has been no comprehensive identification and characterisation of the effector repertoire of any plant-parasitic nematode. To address this, we advance techniques for gland cell isolation and transcriptional analysis to define a stringent annotation of putative effectors for the cyst nematode Heterodera schachtii at three key life-stages. We define 717 effector gene loci: 269 "known" high-confidence homologs of plant-parasitic nematode effectors, and 448 "novel" effectors with high gland cell expression. In doing so we define the most comprehensive "effectorome" of a plant-parasitic nematode to date. Using this effector definition, we provide the first systems-level understanding of the origin, deployment and evolution of a plant-parasitic nematode effectorome. The robust identification of the effector repertoire of a plant-parasitic nematode will underpin our understanding of nematode pathology, and hence, inform strategies for crop protection. Author summary: Pathogenic organisms deliver so called "effectors", typically proteins, into their host to alter cell structure and function. Identifying and characterising the repertoire of effector-encoding genes is requisite to understanding infection. Here we advance methods and understanding, in combination with an extensive literature survey, to define a stringent annotation of putative effectors for the plant-parasitic cyst nematode, Heterodera schachtii. Using this effector definition, we provide the first systems-level understanding of the origin, deployment, and evolution of a plant-parasitic nematode effectorome. Given that plant-parasitic nematodes are major, and in some cases dominant, threats to global food security, it is widely cited that understanding how they infect their host (i.e. effectors) will deliver routes to alleviate their threat to agriculture. [ABSTRACT FROM AUTHOR]

Membrane-associated RING-CH (MARCH) 2 is a member of the MARCH protein family of RING-CH finger E3 ubiquitin ligases that play important roles in regulating the levels of proteins found on the cell surface. MARCH1, 2 and 8 inhibit HIV-1 infection by preventing the incorporation of the envelope glycoproteins into nascent virions. However, a better understanding on the mechanism utilized by MARCH proteins to restrict HIV-1 infection is needed. In this report, we identify an amino acid in human MARCH2, absent in mouse MARCH2, critical for its antiretroviral function. Moreover, we map the domains of human MARCH2 critical for restricting as well as binding to the HIV-1 envelope glycoproteins. In addition, we demonstrate that MARCH2 is present inside nascent virions and reduces particle infectivity by blocking virus entry in a RING-CH-independent manner. Finally, we show that MARCH2 acts as an HIV-1 restriction factor only in primary CD4+ T cells and can prevent cell-to-cell transmission of HIV-1. Our findings reveal important new aspects of the antiviral mechanism utilized by human MARCH2 to restrict HIV-1 that have potential implications to all MARCH proteins with antiviral functions and their viral targets. Author summary: Recently, Membrane-associated RING-CH (MARCH) proteins have emerged as important antiviral factors that block virus infection by preventing viral envelope glycoprotein incorporation into newly formed virions in an ill-defined mechanism. Here, we examine in depth the role of MARCH2 during Human Immunodeficiency Virus type 1 (HIV-1) infection and show that it utilizes two mechanisms to inhibit HIV-1: 1) prevents the envelope glycoprotein incorporation into nascent virions in a RING-CH-dependent manner, 2) when incorporated in virions, it blocks virus-cell membrane fusion in a RING-CH independent manner. We also demonstrate that MARCH2 restricts HIV-1 only in CD4+ T cells and can prevent cell-to-cell transmission. In conclusion, our findings reveal new mechanistic aspects regarding the anti-HIV-1 function of MARCH2. [ABSTRACT FROM AUTHOR]

Most people living with HIV-1 experience rapid viral rebound once antiretroviral therapy is interrupted; however, a small fraction remain in viral remission for an extended duration. Understanding the factors that determine whether viral rebound is likely after treatment interruption can enable the development of optimal treatment regimens and therapeutic interventions to potentially achieve a functional cure for HIV-1. We built upon the theoretical framework proposed by Conway and Perelson to construct dynamic models of virus-immune interactions to study factors that influence viral rebound dynamics. We evaluated these models using viral load data from 24 individuals following antiretroviral therapy interruption. The best-performing model accurately captures the heterogeneity of viral dynamics and highlights the importance of the effector cell expansion rate. Our results show that post-treatment controllers and non-controllers can be distinguished based on the effector cell expansion rate in our models. Furthermore, these results demonstrate the potential of using dynamic models incorporating an effector cell response to understand early viral rebound dynamics post-antiretroviral therapy interruption. Author summary: Most people living with HIV-1 experience rapid viral rebound once antiretroviral therapy is interrupted; however, a small fraction remain in viral remission for an extended duration. The factors that determine viral rebound dynamics after treatment interruption are not well understood. In this study, we built upon a previous theoretical framework to construct dynamic models of virus-immune interactions to study factors that influence viral rebound dynamics. We evaluated these models using viral load data from 24 individuals following antiretroviral therapy interruption. The best-performing model accurately captures the heterogeneity of viral dynamics and robustly shows that people who remain in viral remission for an extended duration after treatment interruption have a higher effector cell expansion rate compared to those who rapidly rebound. These results demonstrate the potential of using viral dynamic models incorporating an effector cell response to understand early viral rebound dynamics post-antiretroviral therapy interruption. [ABSTRACT FROM AUTHOR]

While Merkel cell polyomavirus (MCPyV or MCV) is an abundant virus frequently shed from healthy skin, it is one of the most lethal tumor viruses in immunocompromised individuals, highlighting the crucial role of host immunity in controlling MCPyV oncogenic potential. Despite its prevalence, very little is known about how MCPyV interfaces with the host immune response to maintain asymptomatic persistent infection and how inadequate control of MCPyV infection triggers MCC tumorigenesis. In this study, we discovered that the MCPyV protein, known as the Alternative Large Tumor Open Reading Frame (ALTO), also referred to as middle T, effectively primes and activates the STING signaling pathway. It recruits Src kinase into the complex of STING downstream kinase TBK1 to trigger its autophosphorylation, which ultimately activates the subsequent antiviral immune response. Combining single-cell analysis with both loss- and gain-of-function studies of MCPyV infection, we demonstrated that the activity of ALTO leads to a decrease in MCPyV replication. Thus, we have identified ALTO as a crucial viral factor that modulates the STING-TBK1 pathway, creating a negative feedback loop that limits viral infection and maintains a delicate balance with the host immune system. Our study reveals a novel mechanism by which a tumorigenic virus-encoded protein can link Src function in cell proliferation to the activation of innate immune signaling, thereby controlling viral spread, and sustaining persistent infection. Our previous findings suggest that STING also functions as a tumor suppressor in MCPyV-driven oncogenesis. This research provides a foundation for investigating how disruptions in the finely tuned virus-host balance, maintained by STING, could alter the fate of MCPyV infection, potentially encouraging malignancy. Author summary: Merkel cell polyomavirus (MCPyV) is a small DNA virus responsible for the majority cases of Merkel Cell Carcinoma (MCC), a rare yet highly aggressive form of cancer. While MCPyV latently infects a vast majority of individuals, the mechanisms governing its control, persistence, and oncogenic triggers remain obscure. Our research reveals that the MCPyV-derived Alternative Large Tumor Open Reading Frame (ALTO) protein primes and activates the cGAS-STING-TBK1 innate immune pathway within cells. Such activation elicits an antiviral response, effectively curbing MCPyV replication. This phenomenon illustrates the virus's cunning strategy to exploit cellular mechanisms, ensuring low viral presence while facilitating long-term infection. Consequently, our study sheds light on a novel tactic utilized by MCPyV to maintain a persistent infection in its host. [ABSTRACT FROM AUTHOR]

The intracellular symbiont Wolbachia pipientis evolved after the divergence of arthropods and nematodes, but it reached high prevalence in many of these taxa through its abilities to infect new hosts and their germlines. Some strains exhibit long-term patterns of co-evolution with their hosts, while other strains are capable of switching hosts. This makes strain selection an important factor in symbiont-based biological control. However, little is known about the ecological and evolutionary interactions that occur when a promiscuous strain colonizes an infected host. Here, we study what occurs when two strains come into contact in host cells following horizontal transmission and infection. We focus on the faithful wMel strain from Drosophila melanogaster and the promiscuous wRi strain from Drosophila simulans using an in vitro cell culture system with multiple host cell types and combinatorial infection states. Mixing D. melanogaster cell lines stably infected with wMel and wRi revealed that wMel outcompetes wRi quickly and reproducibly. Furthermore, wMel was able to competitively exclude wRi even from minuscule starting quantities, indicating that this is a nearly deterministic outcome, independent of the starting infection frequency. This competitive advantage was not exclusive to wMel's native D. melanogaster cell background, as wMel also outgrew wRi in D. simulans cells. Overall, wRi is less adept at in vitro growth and survival than wMel and its in vivo state, revealing differences between the two strains in cellular and humoral regulation. These attributes may underlie the observed low rate of mixed infections in nature and the relatively rare rate of host-switching in most strains. Our in vitro experimental framework for estimating cellular growth dynamics of Wolbachia strains in different host species, and cell types provides the first strategy for parameterizing endosymbiont and host cell biology at high resolution. This toolset will be crucial to our application of these bacteria as biological control agents in novel hosts and ecosystems. Author summary: Wolbachia pipientis is one of the most common bacterial endosymbionts due to its ability to manipulate host reproduction, and it has become a useful biological control tool for mosquito populations. Wolbachia is passed from mother to offspring, however the bacterium can also "jump" to new hosts via horizontal transmission. When a Wolbachia strain successfully infects a new host, it may encounter a resident strain, possibly resulting in a superinfection of both strains, or replacement of the resident strain by the new strain. Here, we use a Drosophila melanogaster cell culture system to study the dynamics of mixed Wolbachia infections consisting of the high-fidelity wMel and promiscuous wRi strains. The wMel strain consistently outcompetes the wRi strain, regardless of wMel's initial frequency in D. melanogaster cells. This competitive advantage is independent of host species. While both strains significantly impede host cell division, only the wMel strain is able to rapidly expand into uninfected cells. Our results suggest that the wRi strain is pathogenic in nature and a poor cellular symbiont, and it is retained in natural infections because cell lineages are not expendable or replaceable in development. These findings provide insights into mixed infection outcomes, which are crucial for the use of the bacteria in biological control. [ABSTRACT FROM AUTHOR]

Human activities such as agriculturalization and domestication have led to the emergence of many new pathogens via host-switching events between humans, domesticated and wild animals. Staphylococcus aureus is a multi-host opportunistic pathogen with a global healthcare and economic burden. Recently, it was discovered that laboratory and wild rodents can be colonised and infected with S. aureus, but the origins and zoonotic potential of rodent S. aureus is unknown. In order to trace their evolutionary history, we employed a dataset of 1249 S. aureus genome sequences including 393 of isolates from rodents and other small mammals (including newly determined sequences for 305 isolates from 7 countries). Among laboratory mouse populations, we identified multiple widespread rodent-specific S. aureus clones that likely originated in humans. Phylogeographic analysis of the most common murine lineage CC88 suggests that it emerged in the 1980s in laboratory mouse facilities most likely in North America, from where it spread to institutions around the world, via the distribution of mice for research. In contrast, wild rodents (mice, voles, squirrels) were colonized with a unique complement of S. aureus lineages that are widely disseminated across Europe. In order to investigate the molecular basis for S. aureus adaptation to rodent hosts, genome-wide association analysis was carried out revealing a unique complement of bacteriophages associated with a rodent host ecology. Of note, we identified novel prophages and pathogenicity islands in rodent-derived S. aureus that conferred the potential for coagulation of rodent plasma, a key phenotype of abscess formation and persistence. Our findings highlight the remarkable capacity of S. aureus to expand into new host populations, driven by the acquisition of genes promoting survival in new host-species. Author summary: New pathogens typically emerge after host-switching events, threatening public health and food security. Here we used an evolutionary genomic approach to reveal the origins of Staphylococcus aureus in laboratory and wild rodent populations. S. aureus in laboratory rodents appears to have originated in humans and following host jump events has spread around the world via the distribution of mice for research purposes. Distinct clones of S. aureus widely distributed around Europe are associated with wild rodent populations. We found unique types of prophages in rodent S. aureus that have played a central role in their emergence and expansion including some with genes encoding putative virulence factors. In particular, we identified novel prophages and pathogenicity islands that conferred the potential for coagulation of rodent plasma, a key phenotype of abscess formation and persistence. Taken together, our findings highlight the remarkable capacity of S. aureus to expand into new host populations, driven by the acquisition of genes acquired via mobile genetic elements. [ABSTRACT FROM AUTHOR]

c-FLIP functions as a dual regulator of apoptosis and inflammation, yet its implications in Zika virus (ZIKV) infection remain partially understood, especially in the context of ZIKV-induced congenital Zika syndrome (CZS) where both apoptosis and inflammation play pivotal roles. Our findings demonstrate that c-FLIP promotes ZIKV infection in placental cells and myeloid-derived macrophages, involving inflammation and caspase-8/3-mediated apoptosis. Moreover, our observations reveal that c-FLIP augments ZIKV infection in multiple tissues, including blood cell, spleen, uterus, testis, and the brain of mice. Notably, the partial deficiency of c-FLIP provides protection to embryos against ZIKV-induced CZS, accompanied by a reduction in caspase-3-mediated apoptosis. Additionally, we have found a distinctive parental effect of c-FLIP influencing ZIKV replication in fetal heads. In summary, our study reveals the critical role of c-FLIP as a positive regulator in caspase-8/3-mediated apoptosis during ZIKV infection, significantly contributing to the development of CZS. Author summary: Zika virus (ZIKV) infection in pregnant women can lead to the development of Congenital Zika Syndrome (CZS) in infants, resulting in complications such as microcephaly, intrauterine growth restriction (IUGR), and miscarriages. Although the mechanisms of apoptosis and inflammatory responses in ZIKV-induced CZS are not fully understood, our study investigated the role of c-FLIP, a critical regulator of apoptosis and inflammation during ZIKV infection and its associated CZS. In both human trophoblast cells and murine-derived macrophages, we observed that c-FLIP facilitated ZIKV infection by modulating caspase-8/3-mediated apoptosis. Mice deficient in c-FLIP exhibited reduced ZIKV replication and a decrease in inflammatory cytokine production. Importantly, c-FLIP deficiency demonstrated an inhibitory effect on CZS in ZIKV-infected pregnant mice. Additionally, c-FLIP displayed a parental influence on ZIKV replication in the fetal head by triggering caspase-3 activation. This research emphasizes the significance of c-FLIP in regulating caspase-8/3 and its profound impact on ZIKV-induced CZS, providing valuable insights into the role of apoptosis in ZIKV vertical transmission and fetal development. [ABSTRACT FROM AUTHOR]

One of the greatest challenges encountered by enteric pathogens is responding to rapid changes of nutrient availability in host. However, the mechanisms by which pathogens sense gastrointestinal signals and exploit available host nutrients for proliferation remain largely unknown. Here, we identified a two-component system in Vibrio parahaemolyticus, TtrRS, which senses environmental tetrathionate and subsequently activates the transcription of the ttrRS-ttrBCA-tsdBA gene cluster to promote V. parahaemolyticus colonization of adult mice. We demonstrated that TsdBA confers the ability of thiosulfate oxidation to produce tetrathionate which is sensed by TtrRS. TtrRS autoregulates and directly activates the transcription of the ttrBCA and tsdBA gene clusters. Activated TtrBCA promotes bacterial growth under micro-aerobic conditions by inducing the reduction of both tetrathionate and thiosulfate. TtrBCA and TsdBA activation by TtrRS is important for V. parahaemolyticus to colonize adult mice. Therefore, TtrRS and their target genes constitute a tetrathionate-responsive genetic circuit to exploit the host available sulfur compounds, which further contributes to the intestinal colonization of V. parahaemolyticus. Author summary: Access to host nutrients is regarded as a critical step for the pathogen infection. However, the human gastrointestinal tract is colonized by an enormous number of microbes, which limits pathogens' access to nutrient supplies. Sulfur is one of the most abundant elements on Earth and is also essential to all living organisms. The mechanisms by which bacteria, especially intestinal pathogens, regulate the bioavailability of utilizable sulfur in the host are poorly understood. Here, we characterized a tetrathionate-responsive genetic circuit, which was involved in the utilization of sulfur compounds including tetrathionate and thiosulfate in V. parahaemolyticus, and thus contributed to the bacterial intestinal colonization. Our study provides new insight into the ability of bacterial pathogens to sense gastrointestinal signals and exploit host nutrients for their colonization, which contributes to a better understanding of the pathogenesis of enteric pathogens. [ABSTRACT FROM AUTHOR]

Some respiratory viruses can cause a viral interference through the activation of the interferon (IFN) pathway that reduces the replication of another virus. Epidemiological studies of coinfections between SARS-CoV-2 and other respiratory viruses have been hampered by non-pharmacological measures applied to mitigate the spread of SARS-CoV-2 during the COVID-19 pandemic. With the ease of these interventions, SARS-CoV-2 and influenza A viruses can now co-circulate. It is thus of prime importance to characterize their interactions. In this work, we investigated viral interference effects between an Omicron variant and a contemporary influenza A/H3N2 strain, in comparison with an ancestral SARS-CoV-2 strain and the 2009 pandemic influenza A/H1N1 virus. We infected nasal human airway epitheliums with SARS-CoV-2 and influenza, either simultaneously or 24 h apart. Viral load was measured by RT-qPCR and IFN-α/β/λ1/λ2 proteins were quantified by immunoassay. Expression of four interferon-stimulated genes (ISGs; OAS1/IFITM3/ISG15/MxA) was also measured by RT-droplet digital PCR. Additionally, susceptibility of each virus to IFN-α/β/λ2 recombinant proteins was determined. Our results showed that influenza A, and especially A/H3N2, interfered with both SARS-CoV-2 viruses, but that SARS-CoV-2 did not significantly interfere with A/H3N2 or A/H1N1. Consistently with these results, influenza, and particularly the A/H3N2 strain, caused a higher production of IFN proteins and expression of ISGs than SARS-CoV-2. SARS-CoV-2 induced a marginal IFN production and reduced the IFN response during coinfections with influenza. All viruses were susceptible to exogenous IFNs, with the ancestral SARS-CoV-2 and Omicron being less susceptible to type I and type III IFNs, respectively. Thus, influenza A causes a viral interference towards SARS-CoV-2 most likely through an IFN response. The opposite is not necessarily true, and a concurrent infection with both viruses leads to a lower IFN response. Taken together, these results help us to understand how SARS-CoV-2 interacts with another major respiratory pathogen. Author summary: During the COVID-19 pandemic, non-pharmacological measures were able to reduce the spread of SARS-CoV-2 and most respiratory viruses. Since the ease of these measures, SARS-CoV-2 variants and other viruses, such as influenza A, have started to co-circulate and can now infect a same host and interact with each other. These interactions can lead to attenuated or aggravated infections and can affect the timing of epidemics. Therefore, it is very important to elucidate how the new SARS-CoV-2 interacts with other viruses to better predict their implications in human health and their epidemic activity. Our work contributes to better understand these interactions using viruses that have likely co-circulated after lifting mitigation interventions, i.e., SARS-CoV-2 Omicron variant and a contemporary influenza A/H3N2 strain. We studied how each virus may affect the other virus' growth and how these interactions were associated with the innate immune response of the host. We found that a prior infection with influenza A can decrease the growth of SARS-CoV-2 while the latter reduces the innate immune response. Our results help to understand the interplay between SARS-CoV-2 and influenza A in the host and may improve mathematical models predicting epidemics. [ABSTRACT FROM AUTHOR]

Apoptosis, a major form of programmed cell death, is an essential component of host defense against invading intracellular pathogens. Viruses encode inhibitors of apoptosis to evade host responses during infection, and to support their own replication and survival. Therefore, hosts and their viruses are entangled in a constant evolutionary arms race to control apoptosis. Until now, apoptosis in the context of the antiviral immune system has been almost exclusively studied in vertebrates. This limited phyletic sampling makes it impossible to determine whether a similar mechanism existed in the last common ancestor of animals. Here, we established assays to probe apoptosis in the sea anemone Nematostella vectensis, a model species of Cnidaria, a phylum that diverged approximately 600 million years ago from the rest of animals. We show that polyinosinic:polycytidylic acid (poly I:C), a synthetic long double-stranded RNA mimicking viral RNA and a primary ligand for the vertebrate RLR melanoma differentiation-associated protein 5 (MDA5), is sufficient to induce apoptosis in N. vectensis. Furthermore, at the transcriptomic level, apoptosis related genes are significantly enriched upon poly(I:C) exposure in N. vectensis as well as bilaterian invertebrates. Our phylogenetic analysis of caspase family genes in N. vectensis reveals conservation of all four caspase genes involved in apoptosis in mammals and revealed a cnidarian-specific caspase gene which was strongly upregulated. Altogether, our findings suggest that apoptosis in response to a viral challenge is a functionally conserved mechanism that can be traced back to the last common ancestor of Bilateria and Cnidaria. Author summary: Apoptosis is a programmed cell death mechanism used by vertebrates to efficiently block viral infection. The presence of long double-stranded RNA (dsRNA) in the cytosol is a key feature of DNA and RNA virus replication and is absent from uninfected mammalian host cells. Therefore, the ability to sense and respond to viral dsRNAs is crucial for organismal survival. Indeed, numerous studies in mammals have shown that dsRNA has the capacity to trigger a robust apoptosis as antiviral response. However, such studies were limited to vertebrates, and it remained largely unclear how such systems have evolved. Here, we used the sea anemone Nematostella vectensis, member of phylum Cnidaria (jellyfish, corals, hydroids and sea anemones), to address this topic. We demonstrated that indeed dsRNA is sufficient to induce apoptosis in Nematostella and uncovered a conserved network of genes involved in this process. Further, by comparing the results of gene expression analyses in sea anemones and other diverse animal groups such as oysters and lancelets, we show that apoptosis is prevalent in many animal groups and was already part of the response to dsRNA in the last common ancestor of Cnidaria and most other animals that lived 600 million years ago. [ABSTRACT FROM AUTHOR]

Vibriosis is one of the most serious diseases that commonly occurs in aquatic animals, thus, shaping a steady inherited resistance trait in organisms has received the highest priority in aquaculture. Whereas, the mechanisms underlying the development of such a resistance trait are mostly elusive. In this study, we constructed vibriosis-resistant and susceptible families of the Pacific white shrimp Litopenaeus vannamei after four generations of artificial selection. Microbiome sequencing indicated that shrimp can successfully develop a colonization resistance trait against Vibrio infections. This trait was characterized by a microbial community structure with specific enrichment of a single probiotic species (namely Shewanella algae), and notably, its formation was inheritable and might be memorized by host epigenetic remodeling. Regardless of the infection status, a group of genes was specifically activated in the resistant family through disruption of complete methylation. Specifically, hypo-methylation and hyper-expression of genes related to lactate dehydrogenase (LDH) and iron homeostasis might provide rich sources of specific carbon (lactate) and ions for the colonization of S. algae, which directly results in the reduction of Vibrio load in shrimp. Lactate feeding increased the survival of shrimp, while knockdown of LDH gene decreased the survival when shrimp was infected by Vibrio pathogens. In addition, treatment of shrimp with the methyltransferase inhibitor 5-azacytidine resulted in upregulations of LDH and some protein processing genes, significant enrichment of S. algae, and simultaneous reduction of Vibrio in shrimp. Our results suggest that the colonization resistance can be memorized as epigenetic information by the host, which has played a pivotal role in vibriosis resistance. The findings of this study will aid in disease control and the selection of superior lines of shrimp with high disease resistance. Author summary: Vibriosis is a bacterial disease that commonly occurs in animals, and causes high mortality and severe economic loss in aquaculture every year. Therefore, shaping a steady inherited resistance trait in organisms has received the highest priority in artificial selection. Whereas, the mechanisms underlying the development of such a resistance trait are mostly elusive. In this study, we found that penaeid shrimp can adopt a defense mechanism of colonization resistance against vibriosis. Wherein, a specific enrichment of a single probiotic species (Shewanella algae) in the intestine plays pivotal roles in reducing the Vibrio load and controlling vibriosis. Further evidence revealed that this process might be memorized and regulated by the host's epigenetic system. Activation of a subset of the host genes might provide proper niches for the enrichment of probiotics and simultaneous reduction of pathogens in the intestine through disruption of complete methylation. Together, our findings not only offer new insights into the key mechanisms in shaping disease resistance in shrimp, but also provide some clues for vibriosis control in aquaculture and for the selection of superior lines of shrimp with high disease resistance. [ABSTRACT FROM AUTHOR]

A strong genetic predictor of outcome following untreated HIV-1 infection is the carriage of specific alleles of human leukocyte antigens (HLAs) that present viral epitopes to T cells. Residual variation in outcome measures may be attributed, in part, to viral adaptation to HLA-restricted T cell responses. Variants of the endoplasmic reticulum aminopeptidases (ERAPs) influence the repertoire of T cell epitopes presented by HLA alleles as they trim pathogen-derived peptide precursors to optimal lengths for antigen presentation, along with other functions unrelated to antigen presentation. We investigated whether ERAP variants influence HLA-associated HIV-1 adaptation with demonstrable effects on overall HIV-1 disease outcome. Utilizing host and viral data of 249 West Australian individuals with HIV-1 subtype B infection, we identified a novel association between two linked ERAP2 single nucleotide polymorphisms (SNPs; rs2248374 and rs2549782) with plasma HIV RNA concentration (viral load) (P adjusted = 0.0024 for both SNPs). Greater HLA-associated HIV-1 adaptation in the HIV-1 Gag gene correlated significantly with higher viral load, lower CD4+ T cell count and proportion; P = 0.0103, P = 0.0061, P = 0.0061, respectively). When considered together, there was a significant interaction between the two ERAP2 SNPs and HLA-associated HIV-1 adaptation on viral load (P = 0.0111). In a comprehensive multivariate model, addition of ERAP2 haplotypes and HLA associated adaptation as an interaction term to known HLA and CCR5 determinants and demographic factors, increased the explanatory variance of population viral load from 17.67% to 45.1% in this dataset. These effects were not replicated in publicly available datasets with comparably sized cohorts, suggesting that any true global epistasis may be dependent on specific HLA-ERAP allelic combinations. Our data raises the possibility that ERAP2 variants may shape peptide repertoires presented to HLA class I-restricted T cells to modulate the degree of viral adaptation within individuals, in turn contributing to disease variability at the population level. Analyses of other populations and experimental studies, ideally with locally derived ERAP genotyping and HLA-specific viral adaptations are needed to elucidate this further. Author summary: HIV infection outcome is variable between individuals and understanding the factors that impact this variation is important for efforts towards a HIV cure or vaccine. Here, we found that the level of HIV in the blood is affected by whether an individual carries a specific form of ERAP2, a molecule that influences processing and presentation of the virus to the immune system, as well as the degree to which HIV has mutated to adapt to immune responses. We also show that the interaction of ERAP2 and other known genetic factors explains greater variation in infection outcome than these factors alone. These findings expand our knowledge of the potential importance of viral processing and presentation in the immune response to HIV. [ABSTRACT FROM AUTHOR]

Prions can exist as different strains that consist of conformational variants of the misfolded, pathogenic prion protein isoform PrPSc. Defined by stably transmissible biological and biochemical properties, strains have been identified in a spectrum of prion diseases, including chronic wasting disease (CWD) of wild and farmed cervids. CWD is highly contagious and spreads via direct and indirect transmission involving extraneural sites of infection, peripheral replication and neuroinvasion of prions. Here, we investigated the impact of infection route on CWD prion conformational selection and propagation. We used gene-targeted mouse models expressing deer PrP for intracerebral or intraperitoneal inoculation with fractionated or unfractionated brain homogenates from white-tailed deer, harboring CWD strains Wisc-1 or 116AG. Upon intracerebral inoculation, Wisc-1 and 116AG-inoculated mice differed in conformational stability of PrPSc. In brains of mice infected intraperitoneally with either inoculum, PrPSc propagated with identical conformational stability and fewer PrPSc deposits in most brain regions than intracerebrally inoculated animals. For either inoculum, PrPSc conformational stability in brain and spinal cord was similar upon intracerebral infection but significantly higher in spinal cords of intraperitoneally infected animals. Inoculation with fractionated brain homogenates resulted in lower variance of survival times upon intraperitoneal compared to intracerebral infection. In summary, we demonstrate that extraneural infection mitigates the impact of PrPSc quaternary structure on infection and reduces conformational variability of PrPSc propagated in the brain. These findings provide new insights into the evolution of stable CWD strains in natural, extraneural transmissions. Author summary: Chronic wasting disease (CWD) is a prion disease spreading among wild and farmed cervids. Several strains of CWD prions have been identified that propagate stably upon intracerebral passage in experimental models. Understanding the emergence and adaptation of prion strains is critical for risk assessment of CWD cross-species transmission. We capitalize on our new gene-targeted mouse models of CWD that recapitulate key features of CWD pathogenesis in cervids. We infected these mice with whole of fractionated brain homogenates of deer containing previously characterized, distinct CWD strains by inoculation either into the brain or intraperitoneally. We found that the biochemical properties and pathological hallmarks of prions generated in central nervous system tissues of those mice differed, depending on route of inoculation. Furthermore, mouse survival times upon inoculation with fractionated brain homogenates were less variable upon intraperitoneal compared to intracerebral inoculation. Our findings shed new light on the impact of extraneural infection routes that mirror natural transmission and unravel peripheral infection as a selective barrier that restricts the conformational diversity of prions in the brain. [ABSTRACT FROM AUTHOR]

Filamentous cell growth is a vital property of fungal pathogens. The mechanisms of filamentation in the emerging multidrug-resistant fungal pathogen Candida auris are poorly understood. Here, we show that exposure of C. auris to glycerol triggers a rod-like filamentation-competent (RL-FC) phenotype, which forms elongated filamentous cells after a prolonged culture period. Whole-genome sequencing analysis reveals that all RL-FC isolates harbor a mutation in the C2H2 zinc finger transcription factor-encoding gene GFC1 (Gfc1 variants). Deletion of GFC1 leads to an RL-FC phenotype similar to that observed in Gfc1 variants. We further demonstrate that GFC1 mutation causes enhanced fatty acid β-oxidation metabolism and thereby promotes RL-FC/filamentous growth. This regulation is achieved through a Multiple Carbon source Utilizer (Mcu1)-dependent mechanism. Interestingly, both the evolved RL-FC isolates and the gfc1Δ mutant exhibit an enhanced ability to colonize the skin. Our results reveal that glycerol-mediated GFC1 mutations are beneficial during C. auris skin colonization and infection. Author summary: Candida auris is an emerging multidrug-resistant fungal pathogen that has rapidly spread worldwide in the past decade. This fungus can manifest filamentous morphology under certain conditions, such as passage through a mammalian host. Here, we demonstrate that exposure of C. auris to glycerol triggers a rod-like filamentation-competent (RL-FC) phenotype, resulting in elongated filamentous cells post a prolonged cultivation. Whole-genome sequencing analysis indicates that mutation of a C2H2 zinc finger transcription factor-encoding gene GFC1 is responsible for the generation of RL-FC/filamentous phenotype and enhances ability of skin colonization. We further substantiate that Multiple Carbon source Utilizer (Mcu1)-mediated fatty acid β-oxidation metabolism contributes to Gfc1-suppressed filamentous growth. Our findings link a single gene mutation to metabolic adaptation and acquisition of filamentous competence in C. auris, shedding light on how adaptive traits emerge in pathogens to enhance colonization and infection of the host. [ABSTRACT FROM AUTHOR]

AAV-mediated gene therapy typically requires a high dose of viral transduction, risking acute immune responses and patient safety, part of which is due to limited understanding of the host-viral interactions, especially post-transduction viral genome processing. Here, through a genome-wide CRISPR screen, we identified SMCHD1 (Structural Maintenance of Chromosomes Hinge Domain 1), an epigenetic modifier, as a critical broad-spectrum restricting host factor for post-entry AAV transgene expression. SMCHD1 knock-down by RNAi and CRISPRi or knock-out by CRISPR all resulted in significantly enhanced transgene expression across multiple viral serotypes, as well as for both single-strand and self-complementary AAV genome types. Mechanistically, upon viral transduction, SMCHD1 effectively repressed AAV transcription by the formation of an LRIF1-HP1-containing protein complex and directly binding with the AAV genome to maintain a heterochromatin-like state. SMCHD1-KO or LRIF1-KD could disrupt such a complex and thus result in AAV transcriptional activation. Together, our results highlight the host factor-induced chromatin remodeling as a critical inhibitory mechanism for AAV transduction and may shed light on further improvement in AAV-based gene therapy. Author summary: Adeno-associated virus (AAV) is one of the most commonly used carrier for gene therapy. However, current approved therapies typically use extremely high doses in order to achieve sufficient beneficial efficiency. This is partially due to a lack of understanding about host reponsese that restrict AAV-mediated gene delivery and expression. In this study, we carried out genome-wide CRISPR screens to identify potential host restricting factors for AAV transduction in human cells and identified SMCHD1 as one of the key epigenetic modifers that formed a complex with LRIF1, bound with HP1-associated AAV genome to form a heterochromatin-like states, and suppressed its transcription. Disruption of the SMCHD1-LRIF1 complex would increase the genome accessibility of AAVs and viral transduction, and significantly increase AAV-mediated transgene expression. Therefore, our results indicated that the host factor-induced chromatin remodeling might be a critical inhibitory mechanism for AAV transduction and may help to further improve AAV-based gene therapy. [ABSTRACT FROM AUTHOR]

The involvement of ©δ TCR-bearing lymphocytes in immunological memory has gained increasing interest due to their functional duality between adaptive and innate immunity. ©δ T effector memory (TEM) and central memory (TCM) subsets have been identified, but their respective roles in memory responses are poorly understood. In the present study, we used subsequent mouse cytomegalovirus (MCMV) infections of αβ T cell deficient mice in order to analyze the memory potential of ©δ T cells. As for CMV-specific αβ T cells, MCMV induced the accumulation of cytolytic, KLRG1+CX3CR1+ ©δ TEM that principally localized in infected organ vasculature. Typifying T cell memory, ©δ T cell expansion in organs and blood was higher after secondary viral challenge than after primary infection. Viral control upon MCMV reinfection was prevented when masking ©δ T-cell receptor, and was associated with a preferential amplification of private and unfocused TCR δ chain repertoire composed of a combination of clonotypes expanded post-primary infection and, more unexpectedly, of novel expanded clonotypes. Finally, long-term-primed ©δ TCM cells, but not ©δ TEM cells, protected T cell-deficient hosts against MCMV-induced death upon adoptive transfer, probably through their ability to survive and to generate TEM in the recipient host. This better survival potential of TCM cells was confirmed by a detailed scRNASeq analysis of the two ©δ T cell memory subsets which also revealed their similarity to classically adaptive αβ CD8 T cells. Overall, our study uncovered memory properties of long-lived TCM ©δ T cells that confer protection in a chronic infection, highlighting the interest of this T cell subset in vaccination approaches. Author summary: Cytomegalovirus (CMV) is a widespread, latent virus that can cause severe organ disease in immune-compromised patients. Anti-CMV memory immune responses are essential to control viral reactivation and/or reinfection events that commonly take place in solid organ transplantation. The role of ©δ T-cell receptor bearing lymphocytes could be crucial in this context where immunosuppressive/ablative treatments cause suboptimal and/or delayed αβ T cell responses. Here we asked whether ©δ T cells could compensate for the absence of αβ T cells in the long-term control of mouse CMV infection. Three months post-primary viral challenge in αβ-T cell deficient mice, ©δ T cells displayed similar features as cytolytic, CMV-specific αβ CD8 T cells. We showed that previous priming with CMV endowed ©δ T cells with an enhanced antiviral potential and that long-term maintenance of ©δ T cell-mediated antiviral protection was dependent on ©δ central memory T cells (TCM). As observed in human, the ©δ T cell response to a secondary CMV challenge generated a private TCR δ repertoire. Finally, our gene expression/accessibility single cell analysis revealed that ©δ TCM shared similar features as their αβ T cell counterpart. Our results sustain the adaptive-like properties of these unconventional T cells and reveal the interest of targeting ©δ TCM subset in novel antiviral vaccination approaches. [ABSTRACT FROM AUTHOR]