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While simian immunodeficiency virus (SIV) infection is non-pathogenic in naturally infected African nonhuman primate hosts, experimental or accidental infection in rhesus macaques often leads to AIDS. Baboons, widely distributed throughout Africa, do not naturally harbor SIV, and experimental infection of baboons with SIVmac results in transient low-level viral replication. Elucidation of mechanisms of natural immunity in baboons could uncover new targets of antiviral intervention. We tested the hypothesis that an SIVmac adapted to replicate in baboon primary cells will gain the capacity to establish chronic infections in vivo. Here, we generated SIVmac variants in baboon cells through serial passage in PBMC from different donors (SIVbn-PBMC s1), in PBMC from the same donors (SIVbn-PBMC s2), or in isolated CD4 cells from the same donors used for series 2 (SIVbn-CD4). While SIVbn-PBMC s1 and SIVbn-CD4 demonstrated increased replication capacity, SIVbn-PBMC s2 did not. Pharmacological blockade of CCR5 revealed SIVbn-PBMC s1 could more efficiently use available CCR5 than SIVmac, a trait we hypothesize arose to circumvent receptor occupation by chemokines. Sequencing analysis showed that all three viruses accumulated different types of mutations, and that more non-synonymous mutations became fixed in SIVbn-PBMC s1 than SIVbn-PBMC s2 and SIVbn-CD4, supporting the notion of stronger fitness pressure in PBMC from different genetic backgrounds. Testing the individual contribution of several newly fixed SIV mutations suggested that is the additive effect of these mutations in SIVbn-PBMC s1 that contributed to its enhanced fitness, as recombinant single mutant viruses showed no difference in replication capacity over the parental SIVmac239 strain. The replicative capacity of SIVbn-PBMC passage 4 (P4) s1 was tested in vivo by infecting baboons intravenously with SIVbn-PBMC P4 s1 or SIVmac251. While animals infected with SIVmac251 showed the known pattern of transient low-level viremia, animals infected with SIVbn-PBMC P4 s1 had undetectable viremia or viral DNA in lymphoid tissue. These studies suggest that adaptation of SIV to grow in baboon primary cells results in mutations that confer increased replicative capacity in the artificial environment of cell culture but make the virus unable to avoid the restrictive factors generated by a complex multicellular organism.

The Gram-negative pathogen Stenotrophomonas maltophilia causes a wide range of human infections. It causes particularly serious lung infections in individuals with cystic fibrosis, leading to high mortality rates. This pathogen is resistant to most known antibiotics and harbors a plethora of virulence factors, including lytic enzymes and serine proteases, that cause acute infection in host organisms. S. maltophilia also establishes chronic infections through biofilm formation. The biofilm environment protects the bacteria from external threats and harsh conditions and is therefore vital for the long-term pathogenesis of the microbe. While studies have identified several genes that mediate S. maltophilia’s initial colonization and biofilm formation, the cascade of events initiated by these factors is poorly understood. Consequently, understanding these and other virulence factors can yield exciting new targets for novel therapeutics.

IntroductionHuman brucellosis, a Brucella infection caused most common zoonosis in the world, remains a serious public health burden in China. Brucella chronic infection always causes immunosuppressive status and results in severe organ or tissue damages. The aim of this work was to study the role of the myeloid-derived suppressor cells (MDSCs) in human chronic brucellosis.MethodsFifty cases of chronic brucellosis and 40 healthy individual controls were enrolled in this study. We analyzed the frequency and subsets of MDSCs in PBMC between the chronic brucellosis and healthy control groups by flow cytometry. Furthermore, we also measured the inflammatory-related cytokines in serum samples and the MDSCs inhibition ability to the proliferation of T cells in vitro.ResultsWe found that the frequency of MDSCs in peripheral blood and the level of IL-6 and IL-10 Th2 cytokines and Arginase-1 were significantly increased in chronic brucellosis patients. In addition, we also found that the T cell function was suppressed in vitro by co-culturing with MDSCs from brucellosis patients.ConclusionOur study described an increase of immunosuppressive MDSCs in peripheral blood of chronic brucellosis patients. These results contribute to the understanding of Brucella persistent infection, which may provide an insight for effective treatment of chronic brucellosis patients in clinical practice.

Bacterial biofilms can be found in most environments on our planet, and the human body is no exception. Consisting of microbial cells encased in a matrix of extracellular polymers, biofilms enable bacteria to sequester themselves in favorable niches, while also increasing their ability to resist numerous stresses and survive under hostile circumstances. In recent decades, biofilms have increasingly been recognized as a major contributor to the pathogenesis of chronic infections. However, biofilms also occur in or on certain tissues in healthy individuals, and their constituent species are not restricted to canonical pathogens. In this review, we discuss the evidence for where, when, and what types of biofilms occur in the human body, as well as the diverse ways in which they can impact host health under homeostatic and dysbiotic states.

Staphylococcus epidermidis (S. epidermidis) is a clinically important conditioned pathogen that can cause a troublesome chronic implant-related infection once a biofilm is formed. The nitric oxide synthase (NOS) gene, which is responsible for endogenous nitric oxide synthesis, has already been found in the genome of S. epidermidis; however, the specific mechanisms associated with the effects of NOS on S. epidermidis pathogenicity are still unknown. The purpose of the current study was to investigate whether the NOS gene has an impact on biofilm formation in S. epidermidis. Bioinformatics analysis of the NOS gene was performed, and homologous recombination was subsequently employed to delete this gene. The effects of the NOS gene on biofilm formation of S. epidermidis and its underlying mechanisms were analyzed by bacterial growth assays, biofilm semiquantitative determination, Triton X-100-induced autolysis assays, and bacterial biofilm dispersal assays. Additionally, the transcription levels of fbe, aap, icaA, icaR and sigB, which are related to biofilm formation, were further investigated by qRT-PCR following NOS deletion. Phylogenetic analysis revealed that the NOS gene was conserved between bacterial species originating from different genera. The NOS deletion strain of S. epidermidis 1457 and its counterpart were successfully constructed. Disruption of the NOS gene resulted in significantly enhanced biofilm formation, slightly retarded bacterial growth, a markedly decreased autolysis rate, and drastically weakened bacterial biofilm dispersal. Our data showed that the fbe, aap and icaA genes were significantly upregulated, while the icaR and sigB genes were significantly downregulated, compared with the wild strain. Therefore, these data strongly suggested that the NOS gene can negatively regulate biofilm formation in S. epidermidis by affecting biofilm aggregation and dispersal.

DNA methylation is an epigenetic signature consisting of a methyl group at the 5’ cytosine of CpG dinucleotides. Modifications in DNA methylation pattern have been detected in cancer and infectious diseases and may be associated with gene expression changes. In cancer development DNA methylation aberrations are early events whereas in infectious diseases these epigenetic changes may be due to host/pathogen interaction. In particular, in leishmaniasis, a parasitic disease caused by the protozoan Leishmania, DNA methylation alterations have been detected in macrophages upon infection with Leishmania donovani and in skin lesions from patients with cutaneous leishmaniasis. Interestingly, different types of cancers, such as cutaneous malignant lesions, lymphoma and hepatocellular carcinoma, have been diagnosed in patients with a history of leishmaniasis. In fact, it is known that there exists an association between cancer and infectious diseases. Leishmania infection may increase susceptibility to develop cancer, but the mechanisms involved are not entirely clear. Considering these aspects, in this review we discuss the hypothesis that DNA methylation alterations induced by Leishmania may trigger tumorigenesis in long term infection since these epigenetic modifications may enhance and accumulate during chronic leishmaniasis.

Plasmodium multigene families are thought to play important roles in the pathogenesis of malaria. Plasmodium interspersed repeat (pir) genes comprise the largest multigene family in many Plasmodium species. However, their expression pattern and localisation remain to be elucidated. Understanding protein subcellular localisation is fundamental to reveal the functional importance and cell-cell interactions of the PIR proteins. Here, we use the rodent malaria parasite, Plasmodium chabaudi chabaudi, as a model to investigate the localisation pattern of this gene family. We found that most PIR proteins are co-expressed in clusters during acute and chronic infection; members of the S7 clade are predominantly expressed during the acute-phase, whereas members of the L1 clade dominate the chronic-phase of infection. Using peptide antisera specific for S7 or L1 PIRS, we show that these PIRs have different localisations within the infected red blood cells. S7 PIRs are exported into the infected red blood cell cytoplasm where they are co-localised with parasite-induced host cell modifications termed Maurer’s clefts, whereas L1 PIRs are localised on or close to the parasitophorous vacuolar membrane. This localisation pattern changes following mosquito transmission and during progression from acute- to chronic-phase of infection. The presence of PIRs in Maurer’s clefts, as seen for Plasmodium falciparum RIFIN and STEVOR proteins, might suggest trafficking of the PIRs on the surface of the infected erythrocytes. However, neither S7 nor L1 PIR proteins detected by the peptide antisera are localised on the surface of infected red blood cells, suggesting that they are unlikely to be targets of surface variant-specific antibodies or to be directly involved in adhesion of infected red blood cells to host cells, as described for Plasmodium falciparum VAR proteins. The differences in subcellular localisation of the two major clades of Plasmodium chabaudi PIRs across the blood cycle, and the apparent lack of expression on the red cell surface strongly suggest that the function(s) of this gene family may differ from those of other multigene families of Plasmodium, such as the var genes of Plasmodium falciparum.

Toxoplasma gondii bradyzoites establish chronic infections within their host cells. Recent studies have demonstrated that several parasite effector proteins are translocated to host cells during the bradyzoite stage of chronic infection. To understand the interaction between host cells and bradyzoites at the transcriptomic landscape level, we utilized single-cell RNA-sequencing (scRNA-Seq) to characterize the bradyzoite-induced host cell response. Distinct gene expression profiles were observed in infected host, cells with low parasite mapped reads, and mock (non-exposed) control cells. Gene set enrichment analysis showed that c-Myc and NF-κB signaling and energy metabolic pathways were upregulated by infection. Type I and II interferon response pathways were upregulated in cells with low parasite mapped reads compared to the non-exposed host control cells, and this upregulation effect was reversed in infected cells. Differences were observed in the host cells depending on the differentiation status of the parasites, as determined by BAG1 and SAG1 expression. NF-κB, inflammatory response pathways, and IFN-γ response pathways were downregulated in host cells containing T. gondiiBAG1+/SAG1−, whereas this downregulation effect was reversed in case of T. gondiiBAG1−/SAG1+. We also identified two distinct host cell subsets that contained T. gondiiBAG1+/SAG1−, one of which displayed distinct transcriptomes with upregulated c-Myc expression. Overall, these data clearly demonstrate that host cell transcriptional alteration by bradyzoite infection is different from that of tachyzoite infection, indicating fine-tuning of the host immune response.

Infectious osteomyelitis associated with periprosthetic joint infections is often recalcitrant to treatment and has a high rate of recurrence. In the case of Staphylococcus aureus, the most common pathogen in all forms of osteomyelitis, this may be attributed in part to residual intracellular infection of host cells, yet this is not generally considered in the treatment strategy. Osteocytes represent a unique cell type in this context due to their abundance, their formation of a syncytium throughout the bone that could facilitate bacterial spread and their relative inaccessibility to professional immune cells. As such, there is potential value in studying the host-pathogen interactions in the context of this cell type in a replicable and scalable in vitro model. Here, we examined the utility of the human osteosarcoma cell line SaOS2 differentiated to an osteocyte-like stage (SaOS2-OY) as an intracellular infection model for S. aureus. We demonstrate that S. aureus is capable of generating stable intracellular infections in SaOS2-OY cells but not in undifferentiated, osteoblast-like SaOS2 cells (SaOS2-OB). In SaOS2-OY cells, S. aureus transitioned towards a quasi-dormant small colony variant (SCV) growth phenotype over a 15-day post-infection period. The infected cells exhibited changes in the expression of key immunomodulatory mediators that are consistent with the infection response of primary osteocytes. Thus, SaOS2-OY is an appropriate cell line model that may be predictive of the interactions between S. aureus and human osteocytes, and this will be useful for studying mechanisms of persistence and for testing the efficacy of potential antimicrobial strategies.

While simian immunodeficiency virus (SIV) infection is non-pathogenic in naturally infected African nonhuman primate hosts, experimental or accidental infection in rhesus macaques often leads to AIDS. Baboons, widely distributed throughout Africa, do not naturally harbor SIV, and experimental infection of baboons with SIVmac results in transient low-level viral replication. Elucidation of mechanisms of natural immunity in baboons could uncover new targets of antiviral intervention. We tested the hypothesis that an SIVmac adapted to replicate in baboon primary cells will gain the capacity to establish chronic infections in vivo . Here, we generated SIVmac variants in baboon cells through serial passage in PBMC from different donors (SIVbn-PBMC s1), in PBMC from the same donors (SIVbn-PBMC s2), or in isolated CD4 cells from the same donors used for series 2 (SIVbn-CD4). While SIVbn-PBMC s1 and SIVbn-CD4 demonstrated increased replication capacity, SIVbn-PBMC s2 did not. Pharmacological blockade of CCR5 revealed SIVbn-PBMC s1 could more efficiently use available CCR5 than SIVmac, a trait we hypothesize arose to circumvent receptor occupation by chemokines. Sequencing analysis showed that all three viruses accumulated different types of mutations, and that more non-synonymous mutations became fixed in SIVbn-PBMC s1 than SIVbn-PBMC s2 and SIVbn-CD4, supporting the notion of stronger fitness pressure in PBMC from different genetic backgrounds. Testing the individual contribution of several newly fixed SIV mutations suggested that is the additive effect of these mutations in SIVbn-PBMC s1 that contributed to its enhanced fitness, as recombinant single mutant viruses showed no difference in replication capacity over the parental SIVmac239 strain. The replicative capacity of SIVbn-PBMC passage 4 (P4) s1 was tested in vivo by infecting baboons intravenously with SIVbn-PBMC P4 s1 or SIVmac251. While animals infected with SIVmac251 showed the known pattern of transient low-level viremia, animals infected with SIVbn-PBMC P4 s1 had undetectable viremia or viral DNA in lymphoid tissue. These studies suggest that adaptation of SIV to grow in baboon primary cells results in mutations that confer increased replicative capacity in the artificial environment of cell culture but make the virus unable to avoid the restrictive factors generated by a complex multicellular organism., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Obregon-Perko, Mannino, Ladner, Hodara, Ebrahimi, Parodi, Callery, Palacios and Giavedoni.)

The ability of Salmonella enterica subspecies enterica serovar Typhi (S. Typhi) to cause chronic gallbladder infections is dependent on biofilm growth on cholesterol gallstones. Non-typhoidal Salmonella (e.g. S. Typhimurium) also utilize the biofilm state to persist in the host and the environment. How the pathogen maintains recalcitrance to the host response, and oxidative stress in particular, during chronic infection is poorly understood. Previous experiments demonstrated that S. Typhi and S. Typhimurium biofilms are tolerant to hydrogen peroxide (H2O2), but that mutations in the biofilm extracellular polymeric substances (EPSs) O antigen capsule, colanic acid, or Vi antigen reduce tolerance. Here, biofilm-mediated tolerance to oxidative stress was investigated using a combination of EPS and catalase mutants, as catalases are important detoxifiers of H2O2. Using co-cultured biofilms of wild-type (WT) bacteria with EPS mutants, it was demonstrated that colanic acid in S. Typhimurium and Vi antigen in S. Typhi have a community function and protect all biofilm-resident bacteria rather than to only protect the individual cells producing the EPSs. However, the H2O2 tolerance deficiency of a O antigen capsule mutant was unable to be compensated for by co-culture with WT bacteria. For curli fimbriae, both WT and mutant strains are tolerant to H2O2 though unexpectedly, co-cultured WT/mutant biofilms challenged with H2O2 resulted in sensitization of both strains, suggesting a more nuanced oxidative resistance alteration in these co-cultures. Three catalase mutant (katE, katG and a putative catalase) biofilms were also examined, demonstrating significant reductions in biofilm H2O2 tolerance for the katE and katG mutants. Biofilm co-culture experiments demonstrated that catalases exhibit a community function. We further hypothesized that biofilms are tolerant to H2O2 because the physical barrier formed by EPSs slows penetration of H2O2 into the biofilm to a rate that can be mitigated by intra-biofilm catalases. Compared to WT, EPS-deficient biofilms have a heighted response even to low-dose (2.5 mM) H2O2 challenge, confirming that resident bacteria of EPS-deficient biofilms are under greater stress and have limited protection from H2O2. Thus, these data provide an explanation for how Salmonella achieves tolerance to H2O2 by a combination of an EPS-mediated barrier and enzymatic detoxification.

Toxoplasma gondii is a resilient parasite that infects a multitude of warm-blooded hosts and results in a lifelong chronic infection requiring continuous responses by the host. Chronic infection is characterized by a balanced immune response and neuropathology that are driven by changes in gene expression. Previous research pertaining to these processes has been conducted in various mouse models, and much knowledge of infection-induced gene expression changes has been acquired through the use of high throughput sequencing techniques in different mouse strains and post-mortem human studies. However, lack of infection time course data poses a prominent missing link in the understanding of chronic infection, and there is still much that is unknown regarding changes in genes specifically relating to neuropathology and resulting repair mechanisms as infection progresses throughout the different stages of chronicity. In this paper, we present a targeted approach to gene expression analysis during T. gondii infection through the use of NanoString nCounter gene expression assays. Wild type C57BL/6 and BALB/c background mice were infected, and transcriptional changes in the brain were evaluated at 14, 28, and 56 days post infection. Results demonstrate a dramatic shift in both previously demonstrated and novel gene expression relating to neuropathology and resolution in C57BL/6 mice. In addition, comparison between BALB/c and C57BL/6 mice demonstrate initial differences in gene expression that evolve over the course of infection and indicate decreased neuropathology and enhanced repair in BALB/c mice. In conclusion, these studies provide a targeted approach to gene expression analysis in the brain during infection and provide elaboration on previously identified transcriptional changes and also offer insights into further understanding the complexities of chronic T. gondii infection.

Within the species of Salmonella enterica, there is significant diversity represented among the numerous subspecies and serovars. Collectively, these account for microbes with variable host ranges, from common plant and animal colonizers to extremely pathogenic and human-specific serovars. Despite these differences, many Salmonella species find commonality in the ability to form biofilms and the ability to cause acute, latent, or chronic disease. The exact outcome of infection depends on many factors such as the growth state of Salmonella, the environmental conditions encountered at the time of infection, as well as the infected host and immune response elicited. Here, we review the numerous biofilm lifestyles of Salmonella (on biotic and abiotic surfaces) and how the production of extracellular polymeric substances not only enhances long-term persistence outside the host but also is an essential function in chronic human infections. Furthermore, careful consideration is made for the events during initial infection that allow for gut transcytosis which, in conjunction with host immune functions, often determine the progression of disease. Both typhoidal and non-typhoidal salmonellae can cause chronic and/or secondary infections, thus the adaptive immune responses to both types of bacteria are discussed with particular attention to the differences between Salmonella Typhi, Salmonella Typhimurium, and invasive non-typhoidal Salmonella that can result in differential immune responses. Finally, while strides have been made in our understanding of immunity to Salmonella in the lymphoid organs, fewer definitive studies exist for intestinal and hepatobiliary immunity. By examining our current knowledge and what remains to be determined, we provide insight into new directions in the field of Salmonella immunity, particularly as it relates to chronic infection.

NK cells regulate CD4+ and CD8+ T cells in acute viral infection, vaccination, and the tumor microenvironment. NK cells also become exhausted in chronic activation settings. The mechanisms causing these ILC responses and their impact on adaptive immunity are unclear. CD8+ T cell exhaustion develops during chronic Toxoplasma gondii (T. gondii) infection resulting in parasite reactivation and death. How chronic T. gondii infection impacts the NK cell compartment is not known. We demonstrate that NK cells do not exhibit hallmarks of exhaustion. Their numbers are stable and they do not express high PD1 or LAG3. NK cell depletion with anti-NK1.1 is therapeutic and rescues chronic T. gondii infected mice from CD8+ T cell exhaustion dependent death, increases survival after lethal secondary challenge and alters cyst burdens in brain. Anti-NK1.1 treatment increased polyfunctional CD8+ T cell responses in spleen and brain and reduced CD8+ T cell apoptosis in spleen. Chronic T. gondii infection promotes the development of a modified NK cell compartment, which does not exhibit normal NK cell characteristics. NK cells are Ly49 and TRAIL negative and are enriched for expression of CD94/NKG2A and KLRG1. These NK cells are found in both spleen and brain. They do not produce IFNγ, are IL-10 negative, do not increase PDL1 expression, but do increase CD107a on their surface. Based on the NK cell receptor phenotype we observed NKp46 and CD94-NKG2A cognate ligands were measured. Activating NKp46 (NCR1-ligand) ligand increased and NKG2A ligand Qa-1b expression was reduced on CD8+ T cells. Blockade of NKp46 rescued the chronically infected mice from death and reduced the number of NKG2A+ cells. Immunization with a single dose non-persistent 100% protective T. gondii vaccination did not induce this cell population in the spleen, suggesting persistent infection is essential for their development. We hypothesize chronic T. gondii infection induces an NKp46 dependent modified NK cell population that reduces functional CD8+ T cells to promote persistent parasite infection in the brain. NK cell targeted therapies could enhance immunity in people with chronic infections, chronic inflammation and cancer.

Small colony variants (SCV) of Staphylococcus aureus have been reported as implicated in chronic infections. Here, we investigated the genomic and transcriptomic changes involved in the evolution from a wild-type to a SCV from in a patient with prosthetic joint infection relapse. The SCV presented a stable phenotype with no classical auxotrophy and the emergence of rifampicin resistance. Whole Genome Sequencing (WGS) analysis showed only the loss of a 42.5 kb phage and 3 deletions, among which one targeting the rpoB gene, known to be the target of rifampicin and to be associated to SCV formation in the context of a constitutively active stringent response. Transcriptomic analysis highlighted a specific signature in the SCV strain including a complex, multi-level strategy of survival and adaptation to chronicity within the host including a protection from the inflammatory response, an evasion of the immune response, a constitutively activated stringent response and a scavenging of iron sources.

Introduction: Human brucellosis, a Brucella infection caused most common zoonosis in the world, remains a serious public health burden in China. Brucella chronic infection always causes immunosuppressive status and results in severe organ or tissue damages. The aim of this work was to study the role of the myeloid-derived suppressor cells (MDSCs) in human chronic brucellosis., Methods: Fifty cases of chronic brucellosis and 40 healthy individual controls were enrolled in this study. We analyzed the frequency and subsets of MDSCs in PBMC between the chronic brucellosis and healthy control groups by flow cytometry. Furthermore, we also measured the inflammatory-related cytokines in serum samples and the MDSCs inhibition ability to the proliferation of T cells in vitro ., Results: We found that the frequency of MDSCs in peripheral blood and the level of IL-6 and IL-10 Th2 cytokines and Arginase-1 were significantly increased in chronic brucellosis patients. In addition, we also found that the T cell function was suppressed in vitro by co-culturing with MDSCs from brucellosis patients., Conclusion: Our study described an increase of immunosuppressive MDSCs in peripheral blood of chronic brucellosis patients. These results contribute to the understanding of Brucella persistent infection, which may provide an insight for effective treatment of chronic brucellosis patients in clinical practice., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Hou, Kong, Li, Xu, Chen, Zhang, Zhang, Li, Fu, Li and Wang.)

The Gram-negative pathogen Stenotrophomonas maltophilia causes a wide range of human infections. It causes particularly serious lung infections in individuals with cystic fibrosis, leading to high mortality rates. This pathogen is resistant to most known antibiotics and harbors a plethora of virulence factors, including lytic enzymes and serine proteases, that cause acute infection in host organisms. S. maltophilia also establishes chronic infections through biofilm formation. The biofilm environment protects the bacteria from external threats and harsh conditions and is therefore vital for the long-term pathogenesis of the microbe. While studies have identified several genes that mediate S. maltophilia's initial colonization and biofilm formation, the cascade of events initiated by these factors is poorly understood. Consequently, understanding these and other virulence factors can yield exciting new targets for novel therapeutics., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Bhaumik, Aungkur and Anderson.)

Bacterial biofilms can be found in most environments on our planet, and the human body is no exception. Consisting of microbial cells encased in a matrix of extracellular polymers, biofilms enable bacteria to sequester themselves in favorable niches, while also increasing their ability to resist numerous stresses and survive under hostile circumstances. In recent decades, biofilms have increasingly been recognized as a major contributor to the pathogenesis of chronic infections. However, biofilms also occur in or on certain tissues in healthy individuals, and their constituent species are not restricted to canonical pathogens. In this review, we discuss the evidence for where, when, and what types of biofilms occur in the human body, as well as the diverse ways in which they can impact host health under homeostatic and dysbiotic states., Competing Interests: Author EP and M-WT were employed by Genentech, Inc. This study received funding from Genentech, Inc. The funder had the following involvement with the study: employer of the authors., (Copyright © 2023 Perry and Tan.)

TFF1, a mucin-associated secreted peptide of gastric mucous cells, is known as a protective agent for stomach epithelium under different stimuli, but its role upon Helicobacter infection is still not clear. In this paper we characterized TFFs expression, with particular attention to TFF1, under Helicobacter infection in gastric cell lines. A mouse model was used to distinguish TFF1 mRNA expression between acute and chronic stages of Helicobacter infection. Our results show that TFF1 expression is induced in infected cells; in addition, the inflammatory response upon Helicobacter infection is inversely associated to pre-existing TFF1 protein levels. In infected mice, TFF1 is initially upregulated in gastric antrum in the acute phase of infection, along with IL-1β and IL-6. Then, expression of TFF1 is gradually silenced when the infection becomes chronic and IFN-γ, CXCL5, and CXCL15 reach higher levels. Our data suggest that TFF1 might help cells to counteract bacteria colonization and the development of a chronic inflammation.

Brucella is a facultative intracellular pathogen that causes zoonotic infection known as brucellosis which results in abortion and infertility in natural host. Humans, especially in low income countries, can acquire infection by direct contact with infected animal or by consumption of animal products and show high morbidity, severe economic losses and public health problems. However for survival, host cells develop complex immune mechanisms to defeat and battle against attacking pathogens and maintain a balance between host resistance and Brucella virulence. On the other hand as a successful intracellular pathogen, Brucella has evolved multiple strategies to evade immune response mechanisms to establish persistent infection and replication within host. In this review, we mainly summarize the Stealth strategies employed by Brucella to modulate innate and the adaptive immune systems, autophagy, apoptosis and possible role of small noncoding RNA in the establishment of chronic infection. The purpose of this review is to give an overview for recent understanding how this pathogen evades immune response mechanisms of host, which will facilitate to understanding the pathogenesis of brucellosis and the development of novel, more effective therapeutic approaches to treat brucellosis.

Staphylococcus epidermidis (S. epidermidis) is a clinically important conditioned pathogen that can cause a troublesome chronic implant-related infection once a biofilm is formed. The nitric oxide synthase ( NOS ) gene, which is responsible for endogenous nitric oxide synthesis, has already been found in the genome of S. epidermidis ; however, the specific mechanisms associated with the effects of NOS on S. epidermidis pathogenicity are still unknown. The purpose of the current study was to investigate whether the NOS gene has an impact on biofilm formation in S. epidermidis . Bioinformatics analysis of the NOS gene was performed, and homologous recombination was subsequently employed to delete this gene. The effects of the NOS gene on biofilm formation of S. epidermidis and its underlying mechanisms were analyzed by bacterial growth assays, biofilm semiquantitative determination, Triton X-100-induced autolysis assays, and bacterial biofilm dispersal assays. Additionally, the transcription levels of fbe , aap , icaA , icaR and sigB , which are related to biofilm formation, were further investigated by qRT-PCR following NOS deletion. Phylogenetic analysis revealed that the NOS gene was conserved between bacterial species originating from different genera. The NOS deletion strain of S. epidermidis 1457 and its counterpart were successfully constructed. Disruption of the NOS gene resulted in significantly enhanced biofilm formation, slightly retarded bacterial growth, a markedly decreased autolysis rate, and drastically weakened bacterial biofilm dispersal. Our data showed that the fbe , aap and icaA genes were significantly upregulated, while the icaR and sigB genes were significantly downregulated, compared with the wild strain. Therefore, these data strongly suggested that the NOS gene can negatively regulate biofilm formation in S. epidermidis by affecting biofilm aggregation and dispersal., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer FY declared a past co-authorship with the author LR to the handling editor., (Copyright © 2022 Wang, Rao, Huang, Ma, Yang, Yu, Sun and Ge.)

DNA methylation is an epigenetic signature consisting of a methyl group at the 5' cytosine of CpG dinucleotides. Modifications in DNA methylation pattern have been detected in cancer and infectious diseases and may be associated with gene expression changes. In cancer development DNA methylation aberrations are early events whereas in infectious diseases these epigenetic changes may be due to host/pathogen interaction. In particular, in leishmaniasis, a parasitic disease caused by the protozoan Leishmania , DNA methylation alterations have been detected in macrophages upon infection with Leishmania donovani and in skin lesions from patients with cutaneous leishmaniasis. Interestingly, different types of cancers, such as cutaneous malignant lesions, lymphoma and hepatocellular carcinoma, have been diagnosed in patients with a history of leishmaniasis. In fact, it is known that there exists an association between cancer and infectious diseases. Leishmania infection may increase susceptibility to develop cancer, but the mechanisms involved are not entirely clear. Considering these aspects, in this review we discuss the hypothesis that DNA methylation alterations induced by Leishmania may trigger tumorigenesis in long term infection since these epigenetic modifications may enhance and accumulate during chronic leishmaniasis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Vega-Benedetti, Loi and Zavattari.)

Brucella spp. are highly adapted intracellular pathogens of mammals that cause chronic infections while surving and replicating in host monocytes and macrophages. Although monocytes are normally susceptible to infection, pretreatment with pro-inflammatory cytokine interferon- (IFN-) activates cellular defense mechanisms that increase intracellular killing of Brucella and prevents bacterial replication. We examined the contribution of the IFN- inducible GTPase, LRG-47, to B. abortus 2308 infection in in vitro and in vivo murine models. Infecting nonactivated macrophages from LRG-47-/- mice revealed that loss of this host protein negatively effected the intracellular survival and replication of IgG opsonized B. abortus. In contrast, survival and replication of non-opsonized B. abortus was the same in both C57/B6 and LRG-47-/- peritoneal macrophages. Following IFN-γ activation of LRG-47-/- monocytes, IgG opsonized B. abortus survived better than non-opsonized bacteria. Similar experiments using macrophages from BALB/c and C57/B6 mice found no difference in intracellular survival or replication between non-opsonized and IgG opsonized B. abortus in either resting or IFN-γ activated monocytes. The differential fate of opsonized and non-opsonized B. abortus was only observed in macrophages collected from LRG-47-/- mice. Given the specific nature of the relationship between this host protein and the mechanism of Brucella internalization, LRG-47-/- mice were infected with B. abortus to assess whether the loss of the lrg47 protein would affect the ability of the bacteria to colonize or persist within the host. B. abortus were able to establish and maintain similar numbers of bacteria in both C57/B6 mice and LRG-47-/- through 3 weeks post intraperitoneal infection. By nine weeks p.i. fewer B. abortus were recovered from LRG-47-/- mice than controls, suggesting that the host protein has a positive role in maintaining long term persistence of the bacteria within the host.

Plasmodium multigene families are thought to play important roles in the pathogenesis of malaria. Plasmodium interspersed repeat (pir) genes comprise the largest multigene family in many Plasmodium species. However, their expression pattern and localisation remain to be elucidated. Understanding protein subcellular localisation is fundamental to reveal the functional importance and cell-cell interactions of the PIR proteins. Here, we use the rodent malaria parasite , Plasmodium chabaudi chabaudi , as a model to investigate the localisation pattern of this gene family. We found that most PIR proteins are co-expressed in clusters during acute and chronic infection; members of the S7 clade are predominantly expressed during the acute-phase, whereas members of the L1 clade dominate the chronic-phase of infection. Using peptide antisera specific for S7 or L1 PIRS, we show that these PIRs have different localisations within the infected red blood cells. S7 PIRs are exported into the infected red blood cell cytoplasm where they are co-localised with parasite-induced host cell modifications termed Maurer's clefts, whereas L1 PIRs are localised on or close to the parasitophorous vacuolar membrane. This localisation pattern changes following mosquito transmission and during progression from acute- to chronic-phase of infection. The presence of PIRs in Maurer's clefts, as seen for Plasmodium falciparum RIFIN and STEVOR proteins, might suggest trafficking of the PIRs on the surface of the infected erythrocytes. However, neither S7 nor L1 PIR proteins detected by the peptide antisera are localised on the surface of infected red blood cells, suggesting that they are unlikely to be targets of surface variant-specific antibodies or to be directly involved in adhesion of infected red blood cells to host cells, as described for Plasmodium falciparum VAR proteins. The differences in subcellular localisation of the two major clades of Plasmodium chabaudi PIRs across the blood cycle, and the apparent lack of expression on the red cell surface strongly suggest that the function(s) of this gene family may differ from those of other multigene families of Plasmodium , such as the var genes of Plasmodium falciparum ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Giorgalli, Cunningham, Broncel, Sait, Harrison, Hosking, Vandomme, Amis, Antonello, Sullivan, Uwadiae, Torella, Higgins and Langhorne.)

Toxoplasma gondii bradyzoites establish chronic infections within their host cells. Recent studies have demonstrated that several parasite effector proteins are translocated to host cells during the bradyzoite stage of chronic infection. To understand the interaction between host cells and bradyzoites at the transcriptomic landscape level, we utilized single-cell RNA-sequencing (scRNA-Seq) to characterize the bradyzoite-induced host cell response. Distinct gene expression profiles were observed in infected host, cells with low parasite mapped reads, and mock (non-exposed) control cells. Gene set enrichment analysis showed that c-Myc and NF-κB signaling and energy metabolic pathways were upregulated by infection. Type I and II interferon response pathways were upregulated in cells with low parasite mapped reads compared to the non-exposed host control cells, and this upregulation effect was reversed in infected cells. Differences were observed in the host cells depending on the differentiation status of the parasites, as determined by BAG1 and SAG1 expression. NF-κB, inflammatory response pathways, and IFN-γ response pathways were downregulated in host cells containing T. gondii BAG1+/SAG1- , whereas this downregulation effect was reversed in case of T. gondii BAG1-/SAG1+ . We also identified two distinct host cell subsets that contained T. gondii BAG1+/SAG1- , one of which displayed distinct transcriptomes with upregulated c-Myc expression. Overall, these data clearly demonstrate that host cell transcriptional alteration by bradyzoite infection is different from that of tachyzoite infection, indicating fine-tuning of the host immune response., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Sugi, Tomita, Kidaka, Kawai, Hayashida, Weiss and Yamagishi.)

Infectious osteomyelitis associated with periprosthetic joint infections is often recalcitrant to treatment and has a high rate of recurrence. In the case of Staphylococcus aureus , the most common pathogen in all forms of osteomyelitis, this may be attributed in part to residual intracellular infection of host cells, yet this is not generally considered in the treatment strategy. Osteocytes represent a unique cell type in this context due to their abundance, their formation of a syncytium throughout the bone that could facilitate bacterial spread and their relative inaccessibility to professional immune cells. As such, there is potential value in studying the host-pathogen interactions in the context of this cell type in a replicable and scalable in vitro model. Here, we examined the utility of the human osteosarcoma cell line SaOS2 differentiated to an osteocyte-like stage (SaOS2-OY) as an intracellular infection model for S. aureus . We demonstrate that S. aureus is capable of generating stable intracellular infections in SaOS2-OY cells but not in undifferentiated, osteoblast-like SaOS2 cells (SaOS2-OB). In SaOS2-OY cells, S. aureus transitioned towards a quasi-dormant small colony variant (SCV) growth phenotype over a 15-day post-infection period. The infected cells exhibited changes in the expression of key immunomodulatory mediators that are consistent with the infection response of primary osteocytes. Thus, SaOS2-OY is an appropriate cell line model that may be predictive of the interactions between S. aureus and human osteocytes, and this will be useful for studying mechanisms of persistence and for testing the efficacy of potential antimicrobial strategies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Gunn, Zelmer, Kidd, Solomon, Roscioli, Yang and Atkins.)

The ability of Salmonella enterica subspecies enterica serovar Typhi ( S. Typhi) to cause chronic gallbladder infections is dependent on biofilm growth on cholesterol gallstones. Non-typhoidal Salmonella (e.g. S. Typhimurium) also utilize the biofilm state to persist in the host and the environment. How the pathogen maintains recalcitrance to the host response, and oxidative stress in particular, during chronic infection is poorly understood. Previous experiments demonstrated that S . Typhi and S . Typhimurium biofilms are tolerant to hydrogen peroxide (H 2 O 2 ), but that mutations in the biofilm extracellular polymeric substances (EPSs) O antigen capsule, colanic acid, or Vi antigen reduce tolerance. Here, biofilm-mediated tolerance to oxidative stress was investigated using a combination of EPS and catalase mutants, as catalases are important detoxifiers of H 2 O 2 . Using co-cultured biofilms of wild-type (WT) bacteria with EPS mutants, it was demonstrated that colanic acid in S . Typhimurium and Vi antigen in S . Typhi have a community function and protect all biofilm-resident bacteria rather than to only protect the individual cells producing the EPSs. However, the H 2 O 2 tolerance deficiency of a O antigen capsule mutant was unable to be compensated for by co-culture with WT bacteria. For curli fimbriae, both WT and mutant strains are tolerant to H 2 O 2 though unexpectedly, co-cultured WT/mutant biofilms challenged with H 2 O 2 resulted in sensitization of both strains, suggesting a more nuanced oxidative resistance alteration in these co-cultures. Three catalase mutant ( katE , katG and a putative catalase) biofilms were also examined, demonstrating significant reductions in biofilm H 2 O 2 tolerance for the katE and katG mutants. Biofilm co-culture experiments demonstrated that catalases exhibit a community function. We further hypothesized that biofilms are tolerant to H 2 O 2 because the physical barrier formed by EPSs slows penetration of H 2 O 2 into the biofilm to a rate that can be mitigated by intra-biofilm catalases. Compared to WT, EPS-deficient biofilms have a heighted response even to low-dose (2.5 mM) H 2 O 2 challenge, confirming that resident bacteria of EPS-deficient biofilms are under greater stress and have limited protection from H 2 O 2 . Thus, these data provide an explanation for how Salmonella achieves tolerance to H 2 O 2 by a combination of an EPS-mediated barrier and enzymatic detoxification., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Hahn, González and Gunn.)

Toxoplasma gondii is a resilient parasite that infects a multitude of warm-blooded hosts and results in a lifelong chronic infection requiring continuous responses by the host. Chronic infection is characterized by a balanced immune response and neuropathology that are driven by changes in gene expression. Previous research pertaining to these processes has been conducted in various mouse models, and much knowledge of infection-induced gene expression changes has been acquired through the use of high throughput sequencing techniques in different mouse strains and post-mortem human studies. However, lack of infection time course data poses a prominent missing link in the understanding of chronic infection, and there is still much that is unknown regarding changes in genes specifically relating to neuropathology and resulting repair mechanisms as infection progresses throughout the different stages of chronicity. In this paper, we present a targeted approach to gene expression analysis during T. gondii infection through the use of NanoString nCounter gene expression assays. Wild type C57BL/6 and BALB/c background mice were infected, and transcriptional changes in the brain were evaluated at 14, 28, and 56 days post infection. Results demonstrate a dramatic shift in both previously demonstrated and novel gene expression relating to neuropathology and resolution in C57BL/6 mice. In addition, comparison between BALB/c and C57BL/6 mice demonstrate initial differences in gene expression that evolve over the course of infection and indicate decreased neuropathology and enhanced repair in BALB/c mice. In conclusion, these studies provide a targeted approach to gene expression analysis in the brain during infection and provide elaboration on previously identified transcriptional changes and also offer insights into further understanding the complexities of chronic T. gondii infection., Competing Interests: CD is an employee of NanoString. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Bergersen, Barnes, Worth, David and Wilson.)

Within the species of Salmonella enterica , there is significant diversity represented among the numerous subspecies and serovars. Collectively, these account for microbes with variable host ranges, from common plant and animal colonizers to extremely pathogenic and human-specific serovars. Despite these differences, many Salmonella species find commonality in the ability to form biofilms and the ability to cause acute, latent, or chronic disease. The exact outcome of infection depends on many factors such as the growth state of Salmonella , the environmental conditions encountered at the time of infection, as well as the infected host and immune response elicited. Here, we review the numerous biofilm lifestyles of Salmonella (on biotic and abiotic surfaces) and how the production of extracellular polymeric substances not only enhances long-term persistence outside the host but also is an essential function in chronic human infections. Furthermore, careful consideration is made for the events during initial infection that allow for gut transcytosis which, in conjunction with host immune functions, often determine the progression of disease. Both typhoidal and non-typhoidal salmonellae can cause chronic and/or secondary infections, thus the adaptive immune responses to both types of bacteria are discussed with particular attention to the differences between Salmonella Typhi, Salmonella Typhimurium, and invasive non-typhoidal Salmonella that can result in differential immune responses. Finally, while strides have been made in our understanding of immunity to Salmonella in the lymphoid organs, fewer definitive studies exist for intestinal and hepatobiliary immunity. By examining our current knowledge and what remains to be determined, we provide insight into new directions in the field of Salmonella immunity, particularly as it relates to chronic infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Harrell, Hahn, D’Souza, Vasicek, Sandala, Gunn and McLachlan.)

NK cells regulate CD4+ and CD8+ T cells in acute viral infection, vaccination, and the tumor microenvironment. NK cells also become exhausted in chronic activation settings. The mechanisms causing these ILC responses and their impact on adaptive immunity are unclear. CD8+ T cell exhaustion develops during chronic Toxoplasma gondii ( T. gondii ) infection resulting in parasite reactivation and death. How chronic T. gondii infection impacts the NK cell compartment is not known. We demonstrate that NK cells do not exhibit hallmarks of exhaustion. Their numbers are stable and they do not express high PD1 or LAG3. NK cell depletion with anti-NK1.1 is therapeutic and rescues chronic T. gondii infected mice from CD8+ T cell exhaustion dependent death, increases survival after lethal secondary challenge and alters cyst burdens in brain. Anti-NK1.1 treatment increased polyfunctional CD8+ T cell responses in spleen and brain and reduced CD8+ T cell apoptosis in spleen. Chronic T. gondii infection promotes the development of a modified NK cell compartment, which does not exhibit normal NK cell characteristics. NK cells are Ly49 and TRAIL negative and are enriched for expression of CD94/NKG2A and KLRG1. These NK cells are found in both spleen and brain. They do not produce IFNγ, are IL-10 negative, do not increase PDL1 expression, but do increase CD107a on their surface. Based on the NK cell receptor phenotype we observed NKp46 and CD94-NKG2A cognate ligands were measured. Activating NKp46 (NCR1-ligand) ligand increased and NKG2A ligand Qa-1b expression was reduced on CD8+ T cells. Blockade of NKp46 rescued the chronically infected mice from death and reduced the number of NKG2A+ cells. Immunization with a single dose non-persistent 100% protective T. gondii vaccination did not induce this cell population in the spleen, suggesting persistent infection is essential for their development. We hypothesize chronic T. gondii infection induces an NKp46 dependent modified NK cell population that reduces functional CD8+ T cells to promote persistent parasite infection in the brain. NK cell targeted therapies could enhance immunity in people with chronic infections, chronic inflammation and cancer., (Copyright © 2020 Ivanova, Krempels, Denton, Fettel, Saltz, Rach, Fatima, Mundhenke, Materi, Dunay and Gigley.)

Toxoplasma gondii is a prevalent parasite of mammals and birds including up to 30% of humans world-wide. Primary infection of immunocompetent hosts leads to a robust cell-mediated immune response, which controls but does not clear the infection, thus enabling long-term parasite persistence in brain and muscle tissues. Chronic toxoplasmosis in mice is associated with resistance to heterologous pathogens and this has been related to increased numbers of inflammatory monocytes. Here we have analyzed whether chronic T. gondii infection impacts the subset distribution and the phenotype of peripheral human monocytes in vivo and their responses to parasite infection in vitro . CD14 + monocytes from T. gondii -seropositive blood donors expressed significantly less FcγRIII (CD16) than those from seronegative controls, but they did not show a shift in the distribution of classical, intermediate and non-classical monocyte subpopulations. Percentages of CD62L + and CD64 + monocytes were however decreased and increased, respectively, in chronically infected individuals as compared to naïve controls. Infection of monocyte-enriched PBMCs from both seropositive and seronegative individuals with T. gondii led to an increase of CD14 + CD16 - classical monocytes and a decrease of CD14 + CD16 + double positive monocytes. Remarkably, after in vitro parasite infection, expression of the chemokine receptor CCR2 was severely impaired in monocytes from both, individuals with chronic toxoplasmosis and seronegative controls. In contrast, only monocytes from chronically infected humans but not those from controls dose-dependently up-regulated HLA-DR, DP, DQ expression following in vitro infection. Furthermore, monocyte-enriched PBMCs from seropositive individuals up-regulated IL-12 mRNA more vigorously after in vitro infection than cells from naïve controls. Collectively, our results establish that infection of humans with T. gondii exerts long-term effects on the phenotype and responsiveness of blood monocytes. This may have important implications for innate immune responses to T. gondii and unrelated pathogens.

Toxoplasma gondii is a successful protozoan parasite that cycles between definitive felid hosts and a broad range of intermediate hosts, including rodents and humans. Within intermediate hosts, this obligate intracellular parasite invades the small intestine, inducing an inflammatory response. Toxoplasma infects infiltrating immune cells, using them to spread systemically and reach tissues amenable to chronic infection. An intact immune system is necessary to control life-long chronic infection. Chronic infection is characterized by formation of parasite cysts, which are necessary for survival through the gastrointestinal tract of the next host. Thus, Toxoplasma must evade sterilizing immunity, but still rely on the host's immune response for survival and transmission. To do this, Toxoplasma exploits a central cost-benefit tradeoff in immunity: the need to escalate inflammation for pathogen clearance vs. the need to limit inflammation-induced bystander damage. What are the consequences of sustained inflammation on host biology? Many studies have focused on aspects of the immune response that directly target Toxoplasma growth and survival, commonly referred to as "resistance mechanisms." However, it is becoming clear that a parallel arm of the immune response has evolved to mitigate damage caused by the parasite directly (for example, egress-induced cell death) or bystander damage due to the inflammatory response (for example, reactive nitrogen species, degranulation). These so-called "disease tolerance" mechanisms promote tissue function and host survival without directly targeting the pathogen. Here we review changes to host metabolism, tissue structure, and immune function that point to disease tolerance mechanisms during Toxoplasma infection. We explore the impact tolerance programs have on the health of the host and parasite biology.