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The rapid evolution of viruses generates proteins that are essential for infectivity and replication but with unknown functions, due to extreme sequence divergence1. Here, using a database of 67,715 newly predicted protein structures from 4,463 eukaryotic viral species, we found that 62% of viral proteins are structurally distinct and lack homologues in the AlphaFold database2,3. Among the remaining 38% of viral proteins, many have non-viral structural analogues that revealed surprising similarities between human pathogens and their eukaryotic hosts. Structural comparisons suggested putative functions for up to 25% of unannotated viral proteins, including those with roles in the evasion of innate immunity. In particular, RNA ligase T-like phosphodiesterases were found to resemble phage-encoded proteins that hydrolyse the host immune-activating cyclic dinucleotides 3',3'- and 2',3'-cyclic GMP-AMP (cGAMP). Experimental analysis showed that RNA ligase T homologues encoded by avian poxviruses similarly hydrolyse cGAMP, showing that RNA ligase T-mediated targeting of cGAMP is an evolutionarily conserved mechanism of immune evasion that is present in both bacteriophage and eukaryotic viruses. Together, the viral protein structural database and analyses presented here afford new opportunities to identify mechanisms of virus-host interactions that are common across the virome.

Type I interferon (IFN-I) and IFN-γ foster antitumor immunity by facilitating T cell responses. Paradoxically, IFNs may promote T cell exhaustion by activating immune checkpoints. The downstream regulators of these disparate responses are incompletely understood. Here, we describe how interferon regulatory factor 1 (IRF1) orchestrates these opposing effects of IFNs. IRF1 expression in tumor cells blocks Toll-like receptor- and IFN-I-dependent host antitumor immunity by preventing interferon-stimulated gene (ISG) and effector programs in immune cells. In contrast, expression of IRF1 in the host is required for antitumor immunity. Mechanistically, IRF1 binds distinctly or together with STAT1 at promoters of immunosuppressive but not immunostimulatory ISGs in tumor cells. Overexpression of programmed cell death ligand 1 (PD-L1) in Irf1-/- tumors only partially restores tumor growth, suggesting multifactorial effects of IRF1 on antitumor immunity. Thus, we identify that IRF1 expression in tumor cells opposes host IFN-I- and IRF1-dependent antitumor immunity to facilitate immune escape and tumor growth.

The endoplasmic reticulum aminopeptidase associated with antigen processing (ERAAP) plays a crucial role in shaping the peptide-major histocompatibility complex (MHC) class I repertoire and maintaining immune surveillance. While murine cytomegalovirus (MCMV) has multiple strategies for manipulating the antigen processing pathway to evade immune responses, the host has also developed ways to counter viral immune evasion. In this study, we find that MCMV modulates ERAAP and induces an interferon γ (IFN-γ)-producing CD8+ T cell effector response that targets uninfected ERAAP-deficient cells. We observe that ERAAP downregulation during infection leads to the presentation of the self-peptide FL9 on non-classical Qa-1b, thereby eliciting Qa-1b-restricted QFL T cells to proliferate in the liver and spleen of infected mice. QFL T cells upregulate effector markers upon MCMV infection and are sufficient to reduce viral load after transfer to immunodeficient mice. Our study highlights the consequences of ERAAP dysfunction during viral infection and provides potential targets for anti-viral therapies.

Upon pathogen detection, macrophages normally stay sessile in tissues while dendritic cells (DCs) migrate to secondary lymphoid tissues. The obligate intracellular protozoan Toxoplasma gondii exploits the trafficking of mononuclear phagocytes for dissemination via unclear mechanisms. We report that, upon T. gondii infection, macrophages initiate the expression of transcription factors normally attributed to DCs, upregulate CCR7 expression with a chemotactic response, and perform systemic migration when adoptively transferred into mice. We show that parasite effector GRA28, released by the MYR1 secretory pathway, cooperates with host chromatin remodelers in the host cell nucleus to drive the chemotactic migration of parasitized macrophages. During in vivo challenge studies, bone marrow-derived macrophages infected with wild-type T. gondii outcompeted those challenged with MYR1- or GRA28-deficient strains in migrating and reaching secondary organs. This work reveals how an intracellular parasite hijacks chemotaxis in phagocytes and highlights a remarkable migratory plasticity in differentiated cells of the mononuclear phagocyte system.

Brain metastasis (BrM) is the most common form of brain cancer, characterized by neurologic disability and an abysmal prognosis. Unfortunately, our understanding of the biology underlying human BrMs remains rudimentary. Here, we present an integrative analysis of >100,000 malignant and non-malignant cells from 15 human parenchymal BrMs, generated by single-cell transcriptomics, mass cytometry, and complemented with mouse model- and in silico approaches. We interrogated the composition of BrM niches, molecularly defined the blood-tumor interface, and revealed stromal immunosuppressive states enriched with infiltrated T cells and macrophages. Specific single-cell interrogation of metastatic tumor cells provides a framework of 8 functional cell programs that coexist or anticorrelate. Collectively, these programs delineate two functional BrM archetypes, one proliferative and the other inflammatory, that are evidently shaped through tumor-immune interactions. Our resource provides a foundation to understand the molecular basis of BrM in patients with tumor cell-intrinsic and host environmental traits.

INTRODUCTION: Human cytomegalovirus (HCMV) is a global health threat due to its ubiquity and lifelong persistence in infected people. During latency, host CD8+ T cell responses to HCMV continue to increase in a phenomenon known as memory inflation. We used murine CMV (MCMV) as a model for HCMV to characterize the memory inflation response to wild-type MCMV (KP) and a latency-defective mutant (ΔM33stop), which lacks M33, an MCMV chemokine receptor homolog. M33 is essential for normal reactivation from latency and this was leveraged to determine whether reactivation in vivo contributes to T cell memory inflation. METHODS: Mice were infected with wild-type or mutant MCMV and T cell responses were analyzed by flow cytometry at acute and latent time points. Ex vivo reactivation and cytotoxicity assays were carried out to further investigate immunity and virus replication. Quantitative reverse-transcriptase polymerase chain reaction (q-RTPCR) was used to examine gene expression during reactivation. MHC expression on infected cells was analyzed by flow cytometry. Finally, T cells were depleted from latently-infected B cell-deficient mice to examine the in vivo difference in reactivation between wild-type and ΔM33stop. RESULTS: We found that ΔM33stop triggers memory inflation specific for peptides derived from the immediate-early protein IE1 but not the early protein m164, in contrast to wild-type MCMV. During ex vivo reactivation, gene expression in DM33stop-infected lung tissues was delayed compared to wild-type virus. Normal gene expression was partially rescued by substitution of the HCMV US28 open reading frame in place of the M33 gene. In vivo depletion of T cells in immunoglobulin heavy chain-knockout mice resulted in reactivation of wild-type MCMV, but not ΔM33stop, confirming the role of M33 during reactivation from latency. Further, we found that M33 induces isotype-specific downregulation of MHC class I on the cell surface suggesting previously unappreciated roles in immune evasion. DISCUSSION: Our results indicate that M33 is more polyfunctional than previously appreciated. In addition to its role in reactivation, which had been previously described, we found that M33 alters viral gene expression, host T cell memory inflation, and MHC class I expression. US28 was able to partially complement most functions of M33, suggesting that its role in HCMV infection may be similarly pleotropic.

Cellular respiration is essential for multiple bacterial pathogens and a validated antibiotic target. In addition to driving oxidative phosphorylation, bacterial respiration has a variety of ancillary functions that obscure its contribution to pathogenesis. We find here that the intracellular pathogen Listeria monocytogenes encodes two respiratory pathways which are partially functionally redundant and indispensable for pathogenesis. Loss of respiration decreased NAD+ regeneration, but this could be specifically reversed by heterologous expression of a water-forming NADH oxidase (NOX). NOX expression fully rescued intracellular growth defects and increased L. monocytogenes loads >1000-fold in a mouse infection model. Consistent with NAD+ regeneration maintaining L. monocytogenes viability and enabling immune evasion, a respiration-deficient strain exhibited elevated bacteriolysis within the host cytosol and NOX expression rescued this phenotype. These studies show that NAD+ regeneration represents a major role of L. monocytogenes respiration and highlight the nuanced relationship between bacterial metabolism, physiology, and pathogenesis.

Few patients with triple negative breast cancer (TNBC) benefit from immune checkpoint inhibitors with complete and durable remissions being quite rare. Oncogenes can regulate tumor immune infiltration, however whether oncogenes dictate diminished response to immunotherapy and whether these effects are reversible remains poorly understood. Here, we report that TNBCs with elevated MYC expression are resistant to immune checkpoint inhibitor therapy. Using mouse models and patient data, we show that MYC signaling is associated with low tumor cell PD-L1, low overall immune cell infiltration, and low tumor cell MHC-I expression. Restoring interferon signaling in the tumor increases MHC-I expression. By combining a TLR9 agonist and an agonistic antibody against OX40 with anti-PD-L1, mice experience tumor regression and are protected from new TNBC tumor outgrowth. Our findings demonstrate that MYC-dependent immune evasion is reversible and druggable, and when strategically targeted, may improve outcomes for patients treated with immune checkpoint inhibitors.

Glioblastoma multiforme (GBM) remains the top challenge to radiotherapy with only 25% one-year survival after diagnosis. Here, we reveal that co-enhancement of mitochondrial fatty acid oxidation (FAO) enzymes (CPT1A, CPT2 and ACAD9) and immune checkpoint CD47 is dominant in recurrent GBM patients with poor prognosis. A glycolysis-to-FAO metabolic rewiring is associated with CD47 anti-phagocytosis in radioresistant GBM cells and regrown GBM after radiation in syngeneic mice. Inhibition of FAO by CPT1 inhibitor etomoxir or CRISPR-generated CPT1A-/-, CPT2-/-, ACAD9-/- cells demonstrate that FAO-derived acetyl-CoA upregulates CD47 transcription via NF-κB/RelA acetylation. Blocking FAO impairs tumor growth and reduces CD47 anti-phagocytosis. Etomoxir combined with anti-CD47 antibody synergizes radiation control of regrown tumors with boosted macrophage phagocytosis. These results demonstrate that enhanced fat acid metabolism promotes aggressive growth of GBM with CD47-mediated immune evasion. The FAO-CD47 axis may be targeted to improve GBM control by eliminating the radioresistant phagocytosis-proofing tumor cells in GBM radioimmunotherapy.

To investigate the evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the immune population, we coincupi bated the authentic virus with a highly neutralizing plasma from a COVID-19 convalescent patient. The plasma fully neutralized the virus for seven passages, but, after 45 d, the deletion of F140 in the spike N-terminal domain (NTD) N3 loop led to partial breakthrough. At day 73, an E484K substitution in the receptor-binding domain (RBD) occurred, followed, at day 80, by an insertion in the NTD N5 loop containing a new glycan sequon, which generated a variant completely resistant to plasma neutralization. Computational modeling predicts that the deletion and insertion in loops N3 and N5 prevent binding of neutralizing antibodies. The recent emergence in the United Kingdom, South Africa, Brazil, and Japan of natural variants with similar changes suggests that SARS-CoV-2 has the potential to escape an effective immune response and that vaccines and antibodies able to control emerging variants should be developed.

An ideal therapeutic anti-SARS-CoV-2 antibody would resist viral escape1-3, have activity against diverse sarbecoviruses4-7, and be highly protective through viral neutralization8-11 and effector functions12,13. Understanding how these properties relate to each other and vary across epitopes would aid the development of therapeutic antibodies and guide vaccine design. Here we comprehensively characterize escape, breadth and potency across a panel of SARS-CoV-2 antibodies targeting the receptor-binding domain (RBD). Despite a trade-off between in vitro neutralization potency and breadth of sarbecovirus binding, we identify neutralizing antibodies with exceptional sarbecovirus breadth and a corresponding resistance to SARS-CoV-2 escape. One of these antibodies, S2H97, binds with high affinity across all sarbecovirus clades to a cryptic epitope and prophylactically protects hamsters from viral challenge. Antibodies that target the angiotensin-converting enzyme 2 (ACE2) receptor-binding motif (RBM) typically have poor breadth and are readily escaped by mutations despite high neutralization potency. Nevertheless, we also characterize a potent RBM antibody (S2E128) with breadth across sarbecoviruses related to SARS-CoV-2 and a high barrier to viral escape. These data highlight principles underlying variation in escape, breadth and potency among antibodies that target the RBD, and identify epitopes and features to prioritize for therapeutic development against the current and potential future pandemics.

The PD-L1 overexpression is an important event of immune escape and metastasis in triple-negative breast cancer (TNBC), but the molecular mechanism remains to be determined. Interferon gamma (IFNγ) represents a major driving force behind PD-L1 expression in tumor microenvironment, and histone deacetylase 2 (HDAC2) is required for IFN signaling. Here, we investigated the regulation of HDAC2 on the IFNγ-induced PD-L1 expression in TNBC cells. We found the HDAC2 and PD-L1 expression in TNBC was significantly higher than that in non-TNBC, and HDAC2 was positively correlated with PD-L1 expression. HDAC2 promoted PD-L1 induction by upregulating the phosphorylation of JAK1, JAK2, and STAT1, as well as the translocation of STAT1 to the nucleus and the recruitment of STAT1 to the PD-L1 promoter. Meanwhile, HDAC2 was recruited to the PD-L1 promoter by STAT1, and HDAC2 knockout compromised IFNγ-induced upregulation of H3K27, H3K9 acetylation, and the BRD4 recruitment in PD-L1 promoter. In addition, significant inhibition of proliferation, colony formation, migration, and cell cycle of TNBC cells were observed following knockout of HDAC2 in vitro. Furthermore, HDAC2 knockout reduced IFNγ-induced PD-L1 expression, lymphocyte infiltration, and retarded tumor growth and metastasis in the breast cancer mouse models. This study may provide evidence that HDAC2 promotes IFNγ-induced PD-L1 expression, suggesting a way for enhanced antitumor immunity when targeting the HDAC2 in TNBC.

The emerging field of regenerative cell therapy is still limited by the few cell types that can reliably be differentiated from pluripotent stem cells and by the immune hurdle of commercially scalable allogeneic cell therapeutics. Here, we show that gene-edited, immune-evasive cell grafts can survive and successfully treat diseases in immunocompetent, fully allogeneic recipients. Transplanted endothelial cells improved perfusion and increased the likelihood of limb preservation in mice with critical limb ischemia. Endothelial cell grafts transduced to express a transgene for alpha1-antitrypsin (A1AT) successfully restored physiologic A1AT serum levels in mice with genetic A1AT deficiency. This cell therapy prevented both structural and functional changes of emphysematous lung disease. A mixture of endothelial cells and cardiomyocytes was injected into infarcted mouse hearts, and both cell types orthotopically engrafted in the ischemic areas. Cell therapy led to an improvement in invasive hemodynamic heart failure parameters. Our study supports the development of hypoimmune, universal regenerative cell therapeutics for cost-effective treatments of major diseases.

We report severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike ΔH69/V70 in multiple independent lineages, often occurring after acquisition of receptor binding motif replacements such as N439K and Y453F, known to increase binding affinity to the ACE2 receptor and confer antibody escape. In vitro, we show that, although ΔH69/V70 itself is not an antibody evasion mechanism, it increases infectivity associated with enhanced incorporation of cleaved spike into virions. ΔH69/V70 is able to partially rescue infectivity of spike proteins that have acquired N439K and Y453F escape mutations by increased spike incorporation. In addition, replacement of the H69 and V70 residues in the Alpha variant B.1.1.7 spike (where ΔH69/V70 occurs naturally) impairs spike incorporation and entry efficiency of the B.1.1.7 spike pseudotyped virus. Alpha variant B.1.1.7 spike mediates faster kinetics of cell-cell fusion than wild-type Wuhan-1 D614G, dependent on ΔH69/V70. Therefore, as ΔH69/V70 compensates for immune escape mutations that impair infectivity, continued surveillance for deletions with functional effects is warranted.

Lack of effective immune infiltration represents a significant barrier to immunotherapy in solid tumors. Thus, solid tumor-enriched death receptor-5 (DR5) activating antibodies, which generates tumor debulking by extrinsic apoptotic cytotoxicity, remains a crucial alternate therapeutic strategy. Over past few decades, many DR5 antibodies moved to clinical trials after successfully controlling tumors in immunodeficient tumor xenografts. However, DR5 antibodies failed to significantly improve survival in phase-II trials, leading in efforts to generate second generation of DR5 agonists to supersize apoptotic cytotoxicity in tumors. Here we have discovered that clinical DR5 antibodies activate an unexpected immunosuppressive PD-L1 stabilization pathway, which potentially had contributed to their limited success in clinics. The DR5 agonist stimulated caspase-8 signaling not only activates ROCK1 but also undermines proteasome function, both of which contributes to increased PD-L1 stability on tumor cell surface. Targeting DR5-ROCK1-PD-L1 axis markedly increases immune effector T-cell function, promotes tumor regression, and improves overall survival in animal models. These insights have identified a potential clinically viable combinatorial strategy to revive solid cancer immunotherapy using death receptor agonism.

Orf8, one of the most puzzling genes in the SARS lineage of coronaviruses, marks a unique and striking difference in genome organization between SARS-CoV-2 and SARS-CoV-1. Here, using sequence comparisons, we unequivocally reveal the distant sequence similarities between SARS-CoV-2 Orf8 with its SARS-CoV-1 counterparts and the X4-like genes of coronaviruses, including its highly divergent "paralog" gene Orf7a, whose product is a potential immune antagonist of known structure. Supervised sequence space walks unravel identity levels that drop below 10% and yet exhibit subtle conservation patterns in this novel superfamily, characterized by an immunoglobulin-like beta sandwich topology. We document the high accuracy of the sequence space walk process in detail and characterize the subgroups of the superfamily in sequence space by systematic annotation of gene and taxon groups. While SARS-CoV-1 Orf7a and Orf8 genes are most similar to bat virus sequences, their SARS-CoV-2 counterparts are closer to pangolin virus homologs, reflecting the fine structure of conservation patterns within the SARS-CoV-2 genomes. The divergence between Orf7a and Orf8 is exceptionally idiosyncratic, since Orf7a is more constrained, whereas Orf8 is subject to rampant change, a peculiar feature that may be related to hitherto-unknown viral infection strategies. Despite their common origin, the Orf7a and Orf8 protein families exhibit different modes of evolutionary trajectories within the coronavirus lineage, which might be partly attributable to their complex interactions with the mammalian host cell, reflected by a multitude of functional associations of Orf8 in SARS-CoV-2 compared to a very small number of interactions discovered for Orf7a.IMPORTANCE Orf8 is one of the most puzzling genes in the SARS lineage of coronaviruses, including SARS-CoV-2. Using sophisticated sequence comparisons, we confirm its origins from Orf7a, another gene in the lineage that appears as more conserved, compared to Orf8. Orf7a is a potential immune antagonist of known structure, while a deletion of Orf8 was shown to decrease the severity of the infection in a cohort study. The subtle sequence similarities imply that Orf8 has the same immunoglobulin-like fold as Orf7a, confirmed by structure determination. We characterize the subgroups of this superfamily and demonstrate the highly idiosyncratic divergence patterns during the evolution of the virus.

The generation of reliable mAb of unique and desired specificities serves as a valuable technology to study protein expression and function. However, standard approaches to mAb generation usually involve large-scale protein purification and intensive screening. In this study, we describe an optimized high-throughput proof-of-principle method for the expanded generation, enrichment, and screening of mouse hybridomas secreting mAb specific for a protein of interest. Briefly, we demonstrate that small amounts of a biotinylated protein of interest can be used to generate tetramers for use as prime-boost immunogens, followed by selective enrichment of Ag-specific B cells by magnetic sorting using the same tetramers prior to hybridoma generation. This serves two purposes: 1) to effectively expand both low- and high-affinity B cells specific for the antigenic bait during immunization and 2) to minimize subsequent laborious hybridoma efforts by positive selection of Ag-specific, Ab-secreting cells prior to hybridoma fusion and validation screening. Finally, we employ a rapid and inexpensive screening technology, CELLISA, a high-throughput validation method that uses a chimeric Ag fused to the CD3ζ signaling domain expressed on enzyme-generating reporter cells; these reporters can detect specific mAb in hybridoma supernatants via plate-bound Ab-capture arrays, thereby easing screening. Using this strategy, we generated and characterized novel mouse mAb specific for a viral immunoevasin, the mouse CMV m12 protein, and suggest that these mAb may protect mice from CMV infection via passive immunity.

The Stimulator of Interferon Genes (STING) pathway initiates potent immune responses upon recognition of DNA. To initiate signaling, serine 365 (S365) in the C-terminal tail (CTT) of STING is phosphorylated, leading to induction of type I interferons (IFNs). Additionally, evolutionary conserved responses such as autophagy also occur downstream of STING, but their relative importance during in vivo infections remains unclear. Here we report that mice harboring a serine 365-to-alanine (S365A) mutation in STING are unexpectedly resistant to Herpes Simplex Virus (HSV)-1, despite lacking STING-induced type I IFN responses. By contrast, resistance to HSV-1 is abolished in mice lacking the STING CTT, suggesting that the STING CTT initiates protective responses against HSV-1, independently of type I IFNs. Interestingly, we find that STING-induced autophagy is a CTT- and TBK1-dependent but IRF3-independent process that is conserved in the STING S365A mice. Thus, interferon-independent functions of STING mediate STING-dependent antiviral responses in vivo.

Trogocytosis is part of an emerging, exciting theme of cell-cell interactions both within and between species, and it is relevant to host-pathogen interactions in many different contexts. Trogocytosis is a process in which one cell physically extracts and ingests "bites" of cellular material from another cell. It was first described in eukaryotic microbes, where it was uncovered as a mechanism by which amoebae kill cells. Trogocytosis is potentially a fundamental form of eukaryotic cell-cell interaction, since it also occurs in multicellular organisms, where it has functions in the immune system, in the central nervous system, and during development. There are numerous scenarios in which trogocytosis occurs and an ever-evolving list of functions associated with this process. Many aspects of trogocytosis are relevant to microbial pathogenesis. It was recently discovered that immune cells perform trogocytosis to kill Trichomonas vaginalis parasites. Additionally, through trogocytosis, Entamoeba histolytica acquires and displays human cell membrane proteins, enabling immune evasion. Intracellular bacteria seem to exploit host cell trogocytosis, since they can use it to spread from cell to cell. Thus, a picture is emerging in which trogocytosis plays critical roles in normal physiology, infection, and disease.

Rickettsia are obligate intracellular bacteria that evade antimicrobial autophagy in the host cell cytosol by unknown mechanisms. Other cytosolic pathogens block different steps of autophagy targeting, including the initial step of polyubiquitin-coat formation. One mechanism of evasion is to mobilize actin to the bacterial surface. Here, we show that actin mobilization is insufficient to block autophagy recognition of the pathogen Rickettsia parkeri. Instead, R. parkeri employs outer membrane protein B (OmpB) to block ubiquitylation of the bacterial surface proteins, including OmpA, and subsequent recognition by autophagy receptors. OmpB is also required for the formation of a capsule-like layer. Although OmpB is dispensable for bacterial growth in endothelial cells, it is essential for R. parkeri to block autophagy in macrophages and to colonize mice because of its ability to promote autophagy evasion in immune cells. Our results indicate that OmpB acts as a protective shield to obstruct autophagy recognition, thereby revealing a distinctive bacterial mechanism to evade antimicrobial autophagy.

Group A Streptococcus (GAS) is a human-specific pathogen that evades the host immune response through the elaboration of multiple virulence factors. Although many of these factors have been studied, numerous proteins encoded by the GAS genome are of unknown function. Herein, we characterize a biomimetic red blood cell (RBC)-captured protein of unknown function-annotated subsequently as S protein-in GAS pathophysiology. S protein maintains the hydrophobic properties of GAS, and its absence reduces survival in human blood. S protein facilitates GAS coating with lysed RBCs to promote molecular mimicry, which increases virulence in vitro and in vivo. Proteomic profiling reveals that the removal of S protein from GAS alters cellular and extracellular protein landscapes and is accompanied by a decrease in the abundance of several key GAS virulence determinants. In vivo, the absence of S protein results in a striking attenuation of virulence and promotes a robust immune response and immunological memory.

Treponema is a diverse bacterial genus, the species of which can be pathogenic, symbiotic, or free living. These treponemes can cause various diseases in humans and other animals, such as periodontal disease, bovine digital dermatitis and animal skin lesions. However, the most important and well-studied disease of treponemes that affects humans is 'syphilis'. This disease is caused by Treponema pallidum subspecie pallidum with 11-12 million new cases around the globe on an annual basis. In this study we analyze the transportome of ten Treponema species, with emphasis on the types of encoded transport proteins and their substrates. Of the ten species examined, two (T. primitia and T. azonutricium) reside as symbionts in the guts of termites; six (T. pallidum, T. paraluiscuniculi, T. pedis, T. denticola, T. putidum and T. brennaborense) are pathogens of either humans or animals, and T. caldarium and T. succinifaciens are avirulent species, the former being thermophilic. All ten species have a repertoire of transport proteins that assists them in residing in their respective ecological niches. For instance, oral pathogens use transport proteins that take up nutrients uniquely present in their ecosystem; they also encode multiple multidrug/macromolecule exporters that protect against antimicrobials and aid in biofilm formation. Proteins of termite gut symbionts convert cellulose into other sugars that can be metabolized by the host. As often observed for pathogens and symbionts, several of these treponemes have reduced genome sizes, and their small genomes correlate with their dependencies on the host. Overall, the transportomes of T. pallidum and other pathogens have a conglomerate of parasitic lifestyle-assisting proteins. For example, a T. pallidum repeat protein (TprK) mediates immune evasion; outer membrane proteins (OMPs) allow nutrient uptake and end product export, and several ABC transporters catalyze sugar uptake, considered pivotal to parasitic lifestyles. Taken together, the results of this study yield new information that may help open new avenues of treponeme research.

Cancer cells develop mechanisms to escape immunosurveillance, among which modulating the expression of immune suppressive messenger RNAs is most well-documented. However, how this is molecularly achieved remains largely unresolved. Here, we develop an in vivo mouse model of liver cancer to study oncogene cooperation in immunosurveillance. We show that MYC overexpression (MYCTg) synergizes with KRASG12D to induce an aggressive liver tumor leading to metastasis formation and reduced mouse survival compared with KRASG12D alone. Genome-wide ribosomal footprinting of MYCTg;KRASG12 tumors compared with KRASG12D revealed potential alterations in translation of mRNAs, including programmed-death-ligand 1 (PD-L1). Further analysis revealed that PD-L1 translation is repressed in KRASG12D tumors by functional, non-canonical upstream open reading frames in its 5' untranslated region, which is bypassed in MYCTg;KRASG12D tumors to evade immune attack. We show that this mechanism of PD-L1 translational upregulation was effectively targeted by a potent, clinical compound that inhibits eIF4E phosphorylation, eFT508, which reverses the aggressive and metastatic characteristics of MYCTg;KRASG12D tumors. Together, these studies reveal how immune-checkpoint proteins are manipulated by distinct oncogenes at the level of mRNA translation, which can be exploited for new immunotherapies.

The epithelial cell layer of the major organs of the mammalian gastrointestinal (GI) tract is extensively invaginated into thousands of gland and crypt structures. These are lined by distinct sets of epithelial cells and may comprise discrete niches. The host maximizes the distance between the epithelial cell layer and GI-inhabiting microbes to limit inflammation, and these strategies also likely keep bacteria out of the glands and crypts. We discuss here the specific host processes that have been shown to restrict bacterial presence in the glands and crypts, specifically the immune system, acid, mucin, oxygen, and reactive oxygen species. Not surprisingly, microbes have evolved sophisticated strategies to overcome these host factors and reside close to the epithelium in the glands and crypts. Bacterial properties important for gland and crypt colonization include bacterial immunomodulatory molecules, chemotaxis, and the use of certain metabolites. Overall, these as-yet limited studies suggest there are specific host and bacterial properties that control gland and crypt colonization, contributing to the overall microbial spatial organization of the GI tract. However, there remains much to be discovered in this area.

The innate immune system is the first line of defense against invasive fungal infections. As a consequence, many successful fungal pathogens have evolved elegant strategies to interact with host immune cells. For example, Candida albicans undergoes a morphogenetic switch coupled to cell wall remodeling upon phagocytosis by macrophages and then induces macrophage pyroptosis, an inflammatory cell death program. To elucidate the genetic circuitry through which C. albicans orchestrates this host response, we performed the first large-scale analysis of C. albicans interactions with mammalian immune cells. We identified 98 C. albicans genes that enable macrophage pyroptosis without influencing fungal cell morphology in the macrophage, including specific determinants of cell wall biogenesis and the Hog1 signaling cascade. Using these mutated genes, we discovered that defects in the activation of pyroptosis affect immune cell recruitment during infection. Examining host circuitry required for pyroptosis in response to C. albicans infection, we discovered that inflammasome priming and activation can be decoupled. Finally, we observed that apoptosis-associated speck-like protein containing a CARD (ASC) oligomerization can occur prior to phagolysosomal rupture by C. albicans hyphae, demonstrating that phagolysosomal rupture is not the inflammasome activating signal. Taking the data together, this work defines genes that enable fungal cell wall remodeling and activation of macrophage pyroptosis independently of effects on morphogenesis and identifies macrophage signaling components that are required for pyroptosis in response to C. albicans infection.IMPORTANCECandida albicans is a natural member of the human mucosal microbiota that can also cause superficial infections and life-threatening systemic infections, both of which are characterized by inflammation. Host defense relies mainly on the ingestion and destruction of C. albicans by innate immune cells, such as macrophages and neutrophils. Although some C. albicans cells are killed by macrophages, most undergo a morphological change and escape by inducing macrophage pyroptosis. Here, we investigated the C. albicans genes and host factors that promote macrophage pyroptosis in response to intracellular fungi. This work provides a foundation for understanding how host immune cells interact with C. albicans and may lead to effective strategies to modulate inflammation induced by fungal infections.

The innate immune system is the first line of defense against invasive fungal infections. As a consequence, many successful fungal pathogens have evolved elegant strategies to interact with host immune cells. For example, Candida albicans undergoes a morphogenetic switch coupled to cell wall remodeling upon phagocytosis by macrophages and then induces macrophage pyroptosis, an inflammatory cell death program. To elucidate the genetic circuitry through which C. albicans orchestrates this host response, we performed the first large-scale analysis of C. albicans interactions with mammalian immune cells. We identified 98 C. albicans genes that enable macrophage pyroptosis without influencing fungal cell morphology in the macrophage, including specific determinants of cell wall biogenesis and the Hog1 signaling cascade. Using these mutated genes, we discovered that defects in the activation of pyroptosis affect immune cell recruitment during infection. Examining host circuitry required for pyroptosis in response to C. albicans infection, we discovered that inflammasome priming and activation can be decoupled. Finally, we observed that apoptosis-associated speck-like protein containing a CARD (ASC) oligomerization can occur prior to phagolysosomal rupture by C. albicans hyphae, demonstrating that phagolysosomal rupture is not the inflammasome activating signal. Taking the data together, this work defines genes that enable fungal cell wall remodeling and activation of macrophage pyroptosis independently of effects on morphogenesis and identifies macrophage signaling components that are required for pyroptosis in response to C. albicans infection.IMPORTANCECandida albicans is a natural member of the human mucosal microbiota that can also cause superficial infections and life-threatening systemic infections, both of which are characterized by inflammation. Host defense relies mainly on the ingestion and destruction of C. albicans by innate immune cells, such as macrophages and neutrophils. Although some C. albicans cells are killed by macrophages, most undergo a morphological change and escape by inducing macrophage pyroptosis. Here, we investigated the C. albicans genes and host factors that promote macrophage pyroptosis in response to intracellular fungi. This work provides a foundation for understanding how host immune cells interact with C. albicans and may lead to effective strategies to modulate inflammation induced by fungal infections.

Viruses alter cellular physiology and function to establish cellular environment conducive for viral proliferation. Viral immune evasion is an essential aspect of viral persistence and proliferation. The multifaceted mitochondria play a central role in many cellular events such as metabolism, bioenergetics, cell death, and innate immune signaling. Recent findings accentuate that viruses regulate mitochondrial function and dynamics to facilitate viral proliferation. In this review, we will discuss how viruses exploit mitochondrial dynamics to modulate mitochondria-mediated antiviral innate immune response during infection. This review will provide new insight to understanding the virus-mediated alteration of mitochondrial dynamics and functions to perturb host antiviral immune signaling.

Tuberculosis (TB) is a large global health problem, in part because of the long period of coevolution of the pathogen, Mycobacterium tuberculosis, and its human host. A major factor that sustains the global epidemic of TB is the lack of a sufficiently effective vaccine. While basic mechanisms of immunity that protect against TB have been identified, attempts to improve immunity to TB by vaccination have been disappointing. This Review discusses the mechanisms used by M. tuberculosis to evade innate and adaptive immunity and that likely limit the efficacy of vaccines developed to date. Despite multiple mechanisms of immune evasion, recent trials have indicated that effective TB vaccines remain an attainable goal. This Review discusses how knowledge from other systems can inform improvements on current vaccine approaches.

Leptospirosis is a widespread zoonosis, potentially severe in humans, caused by spirochetal bacteria, Leptospira interrogans (L. interrogans). Host defense mechanisms involved in leptospirosis are poorly understood. Recognition of lipopolysaccharide (LPS) and lipoproteins by Toll-Like Receptors (TLR)4 and TLR2 is crucial for clearance of leptospires in mice, yet the role of Nucleotide Oligomerization Domain (NOD)-like receptors (NOD)1 and NOD2, recognizing peptidoglycan (PG) fragments has not previously been examined. Here, we show that pathogenic leptospires escape from NOD1 and NOD2 recognition both in vitro and in vivo, in mice. We found that leptospiral PG is resistant to digestion by certain hydrolases and that a conserved outer membrane lipoprotein of unknown function, LipL21, specific for pathogenic leptospires, is tightly bound to the PG. Leptospiral PG prepared from a mutant not expressing LipL21 (lipl21-) was more readily digested than the parental or complemented strains. Muropeptides released from the PG of the lipl21- mutant, or prepared using a procedure to eliminate the LipL21 protein from the PG of the parental strain, were recognized in vitro by the human NOD1 (hNOD1) and NOD2 (hNOD2) receptors, suggesting that LipL21 protects PG from degradation into muropeptides. LipL21 expressed in E. coli also resulted in impaired PG digestion and NOD signaling. We found that murine NOD1 (mNOD1) did not recognize PG of L. interrogans. This result was confirmed by mass spectrometry showing that leptospiral PG was primarily composed of MurTriDAP, the natural agonist of hNOD1, and contained only trace amounts of the tetra muropeptide, the mNOD1 agonist. Finally, in transgenic mice expressing human NOD1 and deficient for the murine NOD1, we showed enhanced clearance of a lipl21- mutant compared to the complemented strain, or to what was observed in NOD1KO mice, suggesting that LipL21 facilitates escape from immune surveillance in humans. These novel mechanisms allowing L. interrogans to escape recognition by the NOD receptors may be important in circumventing innate host responses.

Antiviral innate host defenses against acute viral infections include suppression of host protein synthesis to restrict viral protein production. Less is known about mechanisms by which viral pathogens subvert host antiviral innate responses for establishing their replication and dissemination. We investigated early innate defense against human immunodeficiency virus (HIV) infection and viral evasion by utilizing human CD4+ T cell cultures in vitro and a simian immunodeficiency virus (SIV) model of AIDS in vivo Our data showed that early host innate defense against the viral infection involves GCN2-ATF4 signaling-mediated suppression of global protein synthesis, which is exploited by the virus for supporting its own replication during early viral infection and dissemination in the gut mucosa. Suppression of protein synthesis and induction of protein kinase GCN2-ATF4 signaling were detected in the gut during acute SIV infection. These changes diminished during chronic viral infection. HIV replication induced by serum deprivation in CD4+ T cells was linked to the induction of ATF4 that was recruited to the HIV long terminal repeat (LTR) to promote viral transcription. Experimental inhibition of GCN2-ATF4 signaling either by a specific inhibitor or by amino acid supplementation suppressed the induction of HIV expression. Enhancing ATF4 expression through selenium administration resulted in reactivation of latent HIV in vitro as well as ex vivo in the primary CD4+ T cells isolated from patients receiving suppressive antiretroviral therapy (ART). In summary, HIV/SIV exploits the early host antiviral response through GCN2-ATF4 signaling by utilizing ATF4 for activating the viral LTR transcription to establish initial viral replication and is a potential target for HIV prevention and therapy.IMPORTANCE Understanding how HIV overcomes host antiviral innate defense response in order to establish infection and dissemination is critical for developing prevention and treatment strategies. Most investigations focused on the viral pathogenic mechanisms leading to immune dysfunction following robust viral infection and dissemination. Less is known about mechanisms that enable HIV to establish its presence despite rapid onset of host antiviral innate response. Our novel findings provide insights into the viral strategy that hijacks the host innate response of the suppression of protein biosynthesis to restrict the virus production. The virus leverages transcription factor ATF4 expression during the GCN2-ATF4 signaling response and utilizes it to activate viral transcription through the LTR to support viral transcription and production in both HIV and SIV infections. This unique viral strategy is exploiting the innate response and is distinct from the mechanisms of immune dysfunction after the critical mass of viral loads is generated.

Paired immune receptors display near-identical extracellular ligand-binding regions but have intracellular sequences with opposing signaling functions. While inhibitory receptors dampen cellular activation by recognizing self-associated molecules, the functions of activating counterparts are less clear. Here, we studied the inhibitory receptor Siglec-11 that shows uniquely human expression in brain microglia and engages endogenous polysialic acid to suppress inflammation. We demonstrated that the human-specific pathogen Escherichia coli K1 uses its polysialic acid capsule as a molecular mimic to engage Siglec-11 and escape killing. In contrast, engagement of the activating counterpart Siglec-16 increases elimination of bacteria. Since mice do not have paired Siglec receptors, we generated a model by replacing the inhibitory domain of mouse Siglec-E with the activating module of Siglec-16. Siglec-E16 enhanced proinflammatory cytokine expression and bacterial killing in macrophages and boosted protection against intravenous bacterial challenge. These data elucidate uniquely human interactions of a pathogen with Siglecs and support the long-standing hypothesis that activating counterparts of paired immune receptors evolved as a response to pathogen molecular mimicry of host ligands for inhibitory receptors.

Chlamydia trachomatis is an obligate intracellular pathogen that resides in a membrane-bound compartment, the inclusion. The bacteria secrete a unique class of proteins, Incs, which insert into the inclusion membrane and modulate the host-bacterium interface. We previously reported that IncE binds specifically to the Sorting Nexin 5 Phox domain (SNX5-PX) and disrupts retromer trafficking. Here, we present the crystal structure of the SNX5-PX:IncE complex, showing IncE bound to a unique and highly conserved hydrophobic groove on SNX5. Mutagenesis of the SNX5-PX:IncE binding surface disrupts a previously unsuspected interaction between SNX5 and the cation-independent mannose-6-phosphate receptor (CI-MPR). Addition of IncE peptide inhibits the interaction of CI-MPR with SNX5. Finally, C. trachomatis infection interferes with the SNX5:CI-MPR interaction, suggesting that IncE and CI-MPR are dependent on the same binding surface on SNX5. Our results provide new insights into retromer assembly and underscore the power of using pathogens to discover disease-related cell biology.

Despite the success of combination antiretroviral therapy (cART) for suppressing HIV and improving patients' quality of life, HIV persists in cART-treated patients and remains an incurable disease. Financial burdens and health consequences of lifelong cART treatment call for novel HIV therapies that result in a permanent cure. Cellular immunity is central in controlling HIV replication. However, HIV adopts numerous strategies to evade immune surveillance. Engineered immunity via genetic manipulation could offer a functional cure by generating cells that have enhanced antiviral activity and are resistant to HIV infection. Recently, encouraging reports from several human clinical trials using an anti-CD19 chimeric antigen receptor (CAR) modified T-cell therapy for treating B-cell malignancies have provided valuable insights and generated remarkable enthusiasm in engineered T-cell therapy. In this review, we discuss the development of HIV-specific chimeric antigen receptors and the use of stem cell based therapies to generate lifelong anti-HIV immunity.

Mycobacterium tuberculosis (Mtb) establishes a persistent infection, despite inducing antigen-specific T-cell responses. Although T cells arrive at the site of infection, they do not provide sterilizing immunity. The molecular basis of how Mtb impairs T-cell function is not clear. Mtb has been reported to block major histocompatibility complex class II (MHC-II) antigen presentation; however, no bacterial effector or host-cell target mediating this effect has been identified. We recently found that Mtb EsxH, which is secreted by the Esx-3 type VII secretion system, directly inhibits the endosomal sorting complex required for transport (ESCRT) machinery. Here, we showed that ESCRT is required for optimal antigen processing; correspondingly, overexpression and loss-of-function studies demonstrated that EsxH inhibited the ability of macrophages and dendritic cells to activate Mtb antigen-specific CD4+ T cells. Compared with the wild-type strain, the esxH-deficient strain induced fivefold more antigen-specific CD4+ T-cell proliferation in the mediastinal lymph nodes of mice. We also found that EsxH undermined the ability of effector CD4+ T cells to recognize infected macrophages and clear Mtb. These results provide a molecular explanation for how Mtb impairs the adaptive immune response.

To successfully replicate, viruses protect their genomic material from degradation by the host cell. RNA viruses must contend with numerous destabilizing host cell processes including mRNA decay pathways and viral RNA (vRNA) degradation resulting from the antiviral response. Members of the Picornaviridae family of small RNA viruses have evolved numerous diverse strategies to evade RNA decay, including incorporation of stabilizing elements into vRNA and re-purposing host stability factors. Viral proteins are deployed to disrupt and inhibit components of the decay machinery and to redirect decay machinery to the advantage of the virus. This review summarizes documented interactions of picornaviruses with cellular RNA decay pathways and processes.

Squamous cell carcinoma (SCC) is the second commonest type of skin cancer. Moreover, about 90% of head and neck cancers are SCCs. SCCs develop at a significantly higher rate under chronic immunosuppressive conditions, implicating a role of immune surveillance in controlling SCCs. It remains largely unknown how SCCs evade immune recognition. Here, we established a mouse model by injecting tumor cells derived from primary SCCs harboring KrasG12D mutation and Smad4 deletion into wild-type (wt) or CD8-/- recipients. We found comparable tumor growth between wt and CD8-/- recipients, indicating a complete escape of CD8+ T cell-mediated anti-tumor responses by these SCCs. Mechanistically, CD8+ T cells apparently were not defective in infiltrating tumors given their relatively increased percentage among tumor infiltrating lymphocytes (TILs). CD8+ TILs exhibited phenotypes of chronic activation and exhaustion, including overexpression of activation markers, co-expression of programmed cell death 1 (PD-1) and lymphocyte activation gene-3 (LAG-3), as well as TCRβ downregulation. Among CD4+ TILs, T regulatory cells (Tregs) were preferentially expanded. Contradictory to prior findings in melanoma, Treg expansion was independent of CD8+ T cells in our SCC model. Unexpectedly, CD8+ T cells were required for promoting NK cell infiltration within SCCs. Furthermore, we uncovered AKT-dependent lymphocyte-induced PD-L1 upregulation on SCCs, which was contributed greatly by combinatorial effects of CD8+ T and NK cells. Lastly, dual blockade of PD-1 and LAG-3 inhibited the tumor growth of SCCs. Thus, our findings identify novel immune evasion mechanisms of SCCs and suggest that immunosuppressive mechanisms operate in a cancer-type specific and context-dependent manner.

Intracellular bacteria are often clinically relevant pathogens that infect virtually every cell type found in host organisms. However, myeloid cells, especially macrophages, constitute the primary cells targeted by most species of intracellular bacteria. Paradoxically, macrophages possess an extensive antimicrobial arsenal and are efficient at killing microbes. In addition to their ability to detect and signal the presence of pathogens, macrophages sequester and digest microorganisms using the phagolysosomal and autophagy pathways or, ultimately, eliminate themselves through the induction of programmed cell death. Consequently, intracellular bacteria influence numerous host processes and deploy sophisticated strategies to replicate within these host cells. Although most intracellular bacteria have a unique intracellular life cycle, these pathogens are broadly categorized into intravacuolar and cytosolic bacteria. Following phagocytosis, intravacuolar bacteria reside in the host endomembrane system and, to some extent, are protected from the host cytosolic innate immune defenses. However, the intravacuolar lifestyle requires the generation and maintenance of unique specialized bacteria-containing vacuoles and involves a complex network of host-pathogen interactions. Conversely, cytosolic bacteria escape the phagolysosomal pathway and thrive in the nutrient-rich cytosol despite the presence of host cell-autonomous defenses. The understanding of host-pathogen interactions involved in the pathogenesis of intracellular bacteria will continue to provide mechanistic insights into basic cellular processes and may lead to the discovery of novel therapeutics targeting infectious and inflammatory diseases.

Chronic viruses and cancers thwart immune responses in humans by inducing T cell dysfunction. Using a murine chronic virus that models human infections, we investigated the function of the adhesion molecule, P-selectin glycoprotein ligand-1 (PSGL-1), that is upregulated on responding T cells. PSGL-1-deficient mice cleared the virus due to increased intrinsic survival of multifunctional effector T cells that had downregulated PD-1 as well as other inhibitory receptors. Notably, this response resulted in CD4(+)-T-cell-dependent immunopathology. Mechanistically, PSGL-1 ligation on exhausted CD8(+) T cells inhibited T cell receptor (TCR) and interleukin-2 (IL-2) signaling and upregulated PD-1, leading to diminished survival with TCR stimulation. In models of melanoma cancer in which T cell dysfunction occurs, PSGL-1 deficiency led to PD-1 downregulation, improved T cell responses, and tumor control. Thus, PSGL-1 plays a fundamental role in balancing viral control and immunopathology and also functions to regulate T cell responses in the tumor microenvironment.

HIV targets the gut mucosa early in infection, causing immune and epithelial barrier dysfunction and disease progression. However, gut mucosal sensing and innate immune signaling through mucosal pattern recognition receptors (PRRs) during HIV infection and disease progression are not well defined. Using the simian immunodeficiency virus (SIV)-infected rhesus macaque model of AIDS, we found a robust increase in PRRs and inflammatory cytokine gene expression during the acute SIV infection in both peripheral blood and gut mucosa, coinciding with viral replication. PRR expression remained elevated in peripheral blood following the transition to chronic SIV infection. In contrast, massive dampening of PRR expression was detected in the gut mucosa, despite the presence of detectable viral loads. Exceptionally, expression of Toll-like receptor 4 (TLR4) and TLR8 was downmodulated and diverged from expression patterns for most other TLRs in the gut. Decreased mucosal PRR expression was associated with increased abundance of several pathogenic bacterial taxa, including Pasteurellaceae members, Aggregatibacter and Actinobacillus, and Mycoplasmataceae family. Early antiretroviral therapy led to viral suppression but only partial maintenance of gut PRRs and cytokine gene expression. In summary, SIV infection dampens mucosal innate immunity through PRR dysregulation and may promote immune activation, gut microbiota changes, and ineffective viral clearance.

UnlabelledPersistent pathogens, such as herpes simplex virus 1 (HSV-1), have evolved a variety of immune evasion strategies to avoid being detected and destroyed by the host's immune system. A dynamic cross talk appears to occur between the HSV-1 latency-associated transcript (LAT), the only viral gene that is abundantly transcribed during latency, and the CD8(+)T cells that reside in HSV-1 latently infected human and rabbit trigeminal ganglia (TG). The reactivation phenotype of TG that are latently infected with wild-type HSV-1 or with LAT-rescued mutant (i.e., LAT(+)TG) is significantly higher than TG latently infected with LAT-null mutant (i.e., LAT(-)TG). Whether LAT promotes virus reactivation by selectively shaping a unique repertoire of HSV-specific CD8(+)T cells in LAT(+)TG is unknown. In the present study, we assessed the frequency, function, and exhaustion status of TG-resident CD8(+)T cells specific to 40 epitopes derived from HSV-1 gB, gD, VP11/12, and VP13/14 proteins, in human leukocyte antigen (HLA-A*0201) transgenic rabbits infected ocularly with LAT(+)versus LAT(-)virus. Compared to CD8(+)T cells from LAT(-)TG, CD8(+)T cells from LAT(+)TG (i) recognized a broader selection of nonoverlapping HSV-1 epitopes, (ii) expressed higher levels of PD-1, TIM-3, and CTLA-4 markers of exhaustion, and (iii) produced less tumor necrosis factor alpha, gamma interferon, and granzyme B. These results suggest a novel immune evasion mechanism by which the HSV-1 LAT may contribute to the shaping of a broader repertoire of exhausted HSV-specific CD8(+)T cells in latently infected TG, thus allowing for increased viral reactivation.ImportanceA significantly larger repertoire of dysfunctional (exhausted) HSV-specific CD8(+)T cells were found in the TG of HLA transgenic rabbits latently infected with wild-type HSV-1 or with LAT-rescued mutant (i.e., LAT(+)TG) than in a more restricted repertoire of functional HSV-specific CD8(+)T cells in the TG of HLA transgenic rabbits latently infected with LAT-null mutant (i.e., LAT(-)TG). These findings suggest that the HSV-1 LAT locus interferes with the host cellular immune response by shaping a broader repertoire of exhausted HSV-specific CD8(+)T cells within the latency/reactivation TG site.

Many bacterial pathogens can cause acute infections that are cleared with the onset of adaptive immunity, but a subset of these pathogens can establish persistent, and sometimes lifelong, infections. While bacteria that cause chronic infections are phylogenetically diverse, they share common features in their interactions with the host that enable a protracted period of colonization. This article will compare the persistence strategies of two chronic pathogens from the Proteobacteria, Brucella abortus and Salmonella enterica serovar Typhi, to consider how these two pathogens, which are very different at the genomic level, can utilize common strategies to evade immune clearance to cause chronic intracellular infections of the mononuclear phagocyte system.

The antimicrobial peptide LL-37 is generated upon proteolytic cleavage of cathelicidin and limits invading pathogens by directly targeting microbial membranes as well as stimulating innate immune cell function. However, some microbes evade LL-37-mediated defense. Notably, group A Streptococcus (GAS) strains belonging to the hypervirulent M1T1 serogroup are more resistant to human LL-37 than other GAS serogroups. We show that the GAS surface-associated M1 protein sequesters and neutralizes LL-37 antimicrobial activity through its N-terminal domain. M1 protein also binds the cathelicidin precursor hCAP-18, preventing its proteolytic maturation into antimicrobial forms. Exogenous M1 protein rescues M1-deficient GAS from killing by neutrophils and within neutrophil extracellular traps and neutralizes LL-37 chemotactic properties. M1 also binds murine cathelicidin, and its virulence contribution in a murine model of necrotizing skin infection is largely driven by its ability to neutralize this host defense peptide. Thus, cathelicidin resistance is essential for the pathogenesis of hyperinvasive M1T1 GAS.

The rise of the AIDS epidemic made the requirement for T cells in our continuous protection from pathogens critically apparent. The striking frequency with which AIDS patients exhibited profound neurological pathologies brought attention to many chronic infections that are latent within the immune-privileged CNS. One of the most common lethal opportunistic infections of these patients was with the protozoan parasite, Toxoplasma gondii. Reactivation of Toxoplasma cysts within the brain causes massive tissue destruction evidenced as multiple ring-enhancing lesions on MRI and is called toxoplasmic encephalitis (TE). TE is not limited to AIDS patients, but rather is a risk for all severely immunocompromised patients, including recipients of chemotherapy or transplant recipients. The lessons learned from these patient populations are supported by T cell depletion studies in mice. Such experiments have demonstrated that CD4+ and CD8+ T cells are required for protection against TE. Although it is clear that these T cell subsets work synergistically to fight infection, much evidence has been generated that suggests CD8+ T cells play a dominant role in protection during chronic toxoplasmosis. In other models of CNS inflammation, such as intracerebral infection with LCMV and experimental autoimmune encephalomyelitis (EAE), infiltration of T cells into the brain is harmful and even fatal. In the brain of the immunocompetent host, the well-regulated T cell response to T. gondii is therefore an ideal model to understand a controlled inflammatory response to CNS infection. This review will examine our current understanding of CD8+ T cells in the CNS during T. gondii infection in regards to the (1) mechanisms governing entry into the brain, (2) cues that dictate behavior within the brain, and (3) the functional and phenotypic properties exhibited by these cells.

Listeria monocytogenes is a facultative intracellular pathogen that escapes from phagosomes and grows in the cytosol of infected host cells. Most of the determinants that govern its intracellular life cycle are controlled by the transcription factor PrfA, including the pore-forming cytolysin listeriolysin O (LLO), two phospholipases C (PlcA and PlcB), and ActA. We constructed a strain that lacked PrfA but expressed LLO from a PrfA-independent promoter, thereby allowing the bacteria to gain access to the host cytosol. This strain did not grow efficiently in wild-type macrophages but grew normally in macrophages that lacked ATG5, a component of the autophagy LC3 conjugation system. This strain colocalized more with the autophagy marker LC3 (42% ± 7%) at 2 h postinfection, which constituted a 5-fold increase over the colocalization exhibited by the wild-type strain (8% ± 6%). While mutants lacking the PrfA-dependent virulence factor PlcA, PlcB, or ActA grew normally, a double mutant lacking both PlcA and ActA failed to grow in wild-type macrophages and colocalized more with LC3 (38% ± 5%). Coexpression of LLO and PlcA in a PrfA-negative strain was sufficient to restore intracellular growth and decrease the colocalization of the bacteria with LC3. In a cell-free assay, purified PlcA protein blocked LC3 lipidation, a key step in early autophagosome biogenesis, presumably by preventing the formation of phosphatidylinositol 3-phosphate (PI3P). The results of this study showed that avoidance of autophagy by L. monocytogenes primarily involves PlcA and ActA and that either one of these factors must be present for L. monocytogenes growth in macrophages.

Understanding how viruses subvert host immunity and persist is essential for developing strategies to eliminate infection. T cell exhaustion during chronic viral infection is well described, but effects on antibody-mediated effector activity are unclear. Herein, we show that increased amounts of immune complexes generated in mice persistently infected with lymphocytic choriomeningitis virus (LCMV) suppressed multiple Fcγ-receptor (FcγR) functions. The high amounts of immune complexes suppressed antibody-mediated cell depletion, therapeutic antibody-killing of LCMV infected cells and human CD20-expressing tumors, as well as reduced immune complex-mediated cross-presentation to T cells. Suppression of FcγR activity was not due to inhibitory FcγRs or high concentrations of free antibody, and proper FcγR functions were restored when persistently infected mice specifically lacked immune complexes. Thus, we identify a mechanism of immunosuppression during viral persistence with implications for understanding effective antibody activity aimed at pathogen control.

Pathogens have evolved clever strategies to evade and in some cases exploit the attacks of an activated immune system. Salmonella enterica is one such pathogen, exploiting multiple aspects of host defense to promote its replication in the host. Here we review recent findings on the mechanisms by which Salmonella establishes systemic and chronic infection, including strategies involving manipulation of innate immune signaling and inflammatory forms of cell death, as well as immune evasion by establishing residency in M2 macrophages. We also examine recent evidence showing that the oxidative environment and the high levels of antimicrobial proteins produced in response to localized Salmonella gastrointestinal infection enable the pathogen to successfully outcompete the resident gut microbiota.

Helicobacter pylori infects the human gastric mucosa causing a chronic infection that is the primary risk factor for gastric cancer development. Recent studies demonstrate that H. pylori promotes tolerogenic dendritic cell (DC) development indicating that this bacterium evades the host immune response. However, the signaling pathways involved in modulating DC activation during infection remain unclear. Here, we report that H. pylori infection activated the signal transducer and activator of transcription 3 (STAT3) pathway in murine bone marrow-derived DCs (BMDCs) and splenic DCs isolated ex vivo. Isogenic cagA-, cagE-, vacA- and urease-mutants exhibited levels of phosphoSTAT3 that were comparable to in the wild-type (WT) parent strain. H. pylori-infected BMDCs produced increased immunosuppressive IL-10, which activated STAT3 in an autocrine/paracrine fashion. Neutralization of IL-10 prevented H. pylori-mediated STAT3 activation in both BMDCs and splenic DCs. In addition, anti-IL-10 treatment of infected H. pylori-BMDCs was associated with increased CD86 and MHC II expression and enhanced proinflammatory IL-1β cytokine secretion. Finally, increased CD86 and MHC II expression was detected in H. pylori-infected STAT3 knockout DCs when compared to WT controls. Together, these results demonstrate that H. pylori infection induces IL-10 secretion in DCs, which activates STAT3, thereby modulating DC maturation and reducing IL-1β secretion. These findings identify a host molecular mechanism by which H. pylori can manipulate the innate immune response to potentially favor chronic infection and promote carcinogenesis.

Conventional pertussis animal models deliver hundreds of thousands of Bordetella pertussis bacteria deep into the lungs, rapidly inducing severe pneumonic pathology and a robust immune response. However, human infections usually begin with colonization and growth in the upper respiratory tract. We inoculated only the nasopharynx of mice to explore the course of infection in a more natural exposure model. Nasopharyngeal colonization resulted in robust growth in the upper respiratory tract but elicited little immune response, enabling prolonged and persistent infection. Immunization with human acellular pertussis vaccine, which prevents severe lung infections in the conventional pneumonic infection model, had little effect on nasopharyngeal colonization. Our infection model revealed that B. pertussis can efficiently colonize the mouse nasopharynx, grow and spread within and between respiratory organs, evade robust host immunity, and persist for months. This experimental approach can measure aspects of the infection processes not observed in the conventional pneumonic infection model. [ABSTRACT FROM AUTHOR]

BackgroundLeptospirosis, a re-emerging disease of global importance caused by pathogenic Leptospira spp., is considered the world's most widespread zoonotic disease. Rats serve as asymptomatic carriers of pathogenic Leptospira and are critical for disease spread. In such reservoir hosts, leptospires colonize the kidney, are shed in the urine, persist in fresh water and gain access to a new mammalian host through breaches in the skin.Methodology/principal findingsPrevious studies have provided evidence for post-translational modification (PTM) of leptospiral proteins. In the current study, we used proteomic analyses to determine the presence of PTMs on the highly abundant leptospiral protein, LipL32, from rat urine-isolated L. interrogans serovar Copenhageni compared to in vitro-grown organisms. We observed either acetylation or tri-methylation of lysine residues within multiple LipL32 peptides, including peptides corresponding to regions of LipL32 previously identified as epitopes. Intriguingly, the PTMs were unique to the LipL32 peptides originating from in vivo relative to in vitro grown leptospires. The identity of each modified lysine residue was confirmed by fragmentation pattern analysis of the peptide mass spectra. A synthetic peptide containing an identified tri-methylated lysine, which corresponds to a previously identified LipL32 epitope, demonstrated significantly reduced immunoreactivity with serum collected from leptospirosis patients compared to the peptide version lacking the tri-methylation. Further, a subset of the identified PTMs are in close proximity to the established calcium-binding and putative collagen-binding sites that have been identified within LipL32.Conclusions/significanceThe exclusive detection of PTMs on lysine residues within LipL32 from in vivo-isolated L. interrogans implies that infection-generated modification of leptospiral proteins may have a biologically relevant function during the course of infection. Although definitive determination of the role of these PTMs must await further investigations, the reduced immune recognition of a modified LipL32 epitope suggests the intriguing possibility that LipL32 modification represents a novel mechanism of immune evasion within Leptospira.