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Previously we have described the V2-directed CAP256-VRC26 lineage that includes broadly neutralizing antibodies (bNAbs) that neutralize globally diverse strains of HIV. We also identified highly mutated “off-track” lineage members that share high sequence identity to broad members but lack breadth. Here, we defined the mutations that limit the breadth of these antibodies and the probability of their emergence. Mutants and chimeras between two pairs of closely related antibodies were generated: CAP256.04 and CAP256.25 (30% and 63% breadth, respectively) and CAP256.20 and CAP256.27 (2% and 59% breadth). Antibodies were tested against 14 heterologous HIV-1 viruses and select mutants to assess breadth and epitope specificity. A single R100rA mutation in the third heavy chain complementarity-determining region (CDRH3) introduced breadth into CAP256.04, but all three CAP256.25 heavy chain CDRs were required for potency. In contrast, in the CAP256.20/27 chimeras, replacing only the CDRH3 of CAP256.20 with that of CAP256.27 completely recapitulated breadth and potency, likely through the introduction of three charge-reducing mutations. In this individual, the mutations that limited the breadth of the off-track antibodies were predicted to occur with higher a probability than those in the naturally paired bNAbs, suggesting a low barrier to the evolution of the off-track phenotype. Mapping studies to determine the viral immunotypes (or epitope variants) that selected off-track antibodies indicated that unlike broader lineage members, CAP256.20 preferentially neutralized viruses containing 169Q. This suggests that this globally rare immunotype, which was common in donor CAP256, drove the off-track phenotype. These data show that affinity maturation to counter globally rare viral immunotypes can drive antibodies within a broad lineage along multiple pathways towards strain-specificity. Defining developmental pathways towards and away from breadth may facilitate the selection of immunogens that elicit bNAbs and minimize off-track antibodies. [ABSTRACT FROM AUTHOR]

Human zinc deficiency increases susceptibility to bacterial infection. Although zinc supplementation therapies can reduce the impact of disease, the molecular basis for protection remains unclear. Streptococcus pneumoniae is a major cause of bacterial pneumonia, which is prevalent in regions of zinc deficiency. We report that dietary zinc levels dictate the outcome of S. pneumoniae infection in a murine model. Dietary zinc restriction impacts murine tissue zinc levels with distribution post-infection altered, and S. pneumoniae virulence and infection enhanced. Although the activation and infiltration of murine phagocytic cells was not affected by zinc restriction, their efficacy of bacterial control was compromised. S. pneumoniae was shown to be highly sensitive to zinc intoxication, with this process impaired in zinc restricted mice and isolated phagocytic cells. Collectively, these data show how dietary zinc deficiency increases sensitivity to S. pneumoniae infection while revealing a role for zinc as a component of host antimicrobial defences. [ABSTRACT FROM AUTHOR]

Rotavirus is the leading agent causing acute gastroenteritis in young children, with the P[8] genotype accounting for more than 80% of infections in humans. The molecular bases for binding of the VP8* domain from P[8] VP4 spike protein to its cellular receptor, the secretory H type-1 antigen (Fuc-α1,2-Gal-β1,3-GlcNAc; H1), and to its precursor lacto-N-biose (Gal-β1,3-GlcNAc; LNB) have been determined. The resolution of P[8] VP8* crystal structures in complex with H1 antigen and LNB and site-directed mutagenesis experiments revealed that both glycans bind to the P[8] VP8* protein through a binding pocket shared with other members of the P[II] genogroup (i.e.: P[4], P[6] and P[19]). Our results show that the L-fucose moiety from H1 only displays indirect contacts with P[8] VP8*. However, the induced conformational changes in the LNB moiety increase the ligand affinity by two-fold, as measured by surface plasmon resonance (SPR), providing a molecular explanation for the different susceptibility to rotavirus infection between secretor and non-secretor individuals. The unexpected interaction of P[8] VP8* with LNB, a building block of type-1 human milk oligosaccharides, resulted in inhibition of rotavirus infection, highlighting the role and possible application of this disaccharide as an antiviral. While key amino acids in the H1/LNB binding pocket were highly conserved in members of the P[II] genogroup, differences were found in ligand affinities among distinct P[8] genetic lineages. The variation in affinities were explained by subtle structural differences induced by amino acid changes in the vicinity of the binding pocket, providing a fine-tuning mechanism for glycan binding in P[8] rotavirus. [ABSTRACT FROM AUTHOR]

Staphylococcal enterotoxins (SEs) produced by Staphylococcus aureus are known as causative agents of emetic food poisoning. We previously demonstrated that SEA binds with submucosal mast cells and evokes mast cell degranulation in a small emetic house musk shrew model. Notably, primates have been recognized as the standard model for emetic assays and analysis of SE emetic activity. However, the mechanism involved in SEA-induced vomiting in primates has not yet been elucidated. In the present study, we established common marmosets as an emetic animal model. Common marmosets were administered classical SEs, including SEA, SEB and SEC, and exhibited multiple vomiting responses. However, a non-emetic staphylococcal superantigen, toxic shock syndrome toxin-1, did not induce emesis in these monkeys. These results indicated that the common marmoset is a useful animal model for assessing the emesis-inducing activity of SEs. Furthermore, histological analysis uncovered that SEA bound with submucosal mast cells and induced mast cell degranulation. Additionally, ex vivo and in vivo pharmacological results showed that SEA-induced histamine release plays a critical role in the vomiting response in common marmosets. The present results suggested that 5-hydroxytryptamine also plays an important role in the transmission of emetic stimulation on the afferent vagus nerve or central nervous system. We conclude that SEA induces histamine release from submucosal mast cells in the gastrointestinal tract and that histamine contributes to the SEA-induced vomiting reflex via the serotonergic nerve and/or other vagus nerve. [ABSTRACT FROM AUTHOR]

Clostridium difficile spore germination is critical for the transmission of disease. C. difficile spores germinate in response to cholic acid derivatives, such as taurocholate (TA), and amino acids, such as glycine or alanine. Although the receptor with which bile acids are recognized (germinant receptor) is known, the amino acid co-germinant receptor has remained elusive. Here, we used EMS mutagenesis to generate mutants with altered requirements for the amino acid co-germinant, similar to the strategy we used previously to identify the bile acid germinant receptor, CspC. Surprisingly, we identified strains that do not require co-germinants, and the mutant spores germinated in response to TA alone. Upon sequencing these mutants, we identified different mutations in yabG. In C. difficile, yabG expression is required for the processing of key germination components to their mature forms (e.g., CspBA to CspB and CspA). A defined yabG mutant exacerbated the EMS mutant phenotype. Building upon this work, we found that small deletions in cspA resulted in spores that germinated in the presence of TA alone without the requirement of a co-germinant. cspA encodes a pseudoprotease that was previously shown to be important for incorporation of the CspC germinant receptor. Herein, our study builds upon the role of CspA during C. difficile spore germination by providing evidence that CspA is important for recognition of co-germinants during C. difficile spore germination. Our work suggests that two pseudoproteases (CspC and CspA) likely function as the C. difficile germinant receptors. [ABSTRACT FROM AUTHOR]

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease localized to China, Japan, and Korea that is characterized by severe hemorrhage and a high fatality rate. Currently, no specific vaccine or treatment has been approved for this disease. To develop a therapeutic agent for SFTS, we isolated antibodies from a phage-displayed antibody library that was constructed from a patient who recovered from SFTS virus (SFTSV) infection. One antibody, designated as Ab10, was reactive to the Gn envelope glycoprotein of SFTSV and protected host cells and A129 mice from infection in both in vitro and in vivo experiments. Notably, Ab10 protected 80% of mice, even when injected 5 days after inoculation with a lethal dose of SFTSV. Using cross-linker assisted mass spectrometry and alanine scanning, we located the non-linear epitope of Ab10 on the Gn glycoprotein domain II and an unstructured stem region, suggesting that Ab10 may inhibit a conformational alteration that is critical for cell membrane fusion between the virus and host cell. Ab10 reacted to recombinant Gn glycoprotein in Gangwon/Korea/2012, HB28, and SD4 strains. Additionally, based on its epitope, we predict that Ab10 binds the Gn glycoprotein in 247 of 272 SFTSV isolates previously reported. Together, these data suggest that Ab10 has potential to be developed into a therapeutic agent that could protect against more than 90% of reported SFTSV isolates. [ABSTRACT FROM AUTHOR]

The cellular prion protein (PrPC) is a cell surface glycoprotein attached to the membrane by a glycosylphosphatidylinositol (GPI)-anchor and plays a critical role in transmissible, neurodegenerative and fatal prion diseases. Alterations in membrane attachment influence PrPC-associated signaling, and the development of prion disease, yet our knowledge of the role of the GPI-anchor in localization, processing, and function of PrPC in vivo is limited We exchanged the PrPC GPI-anchor signal sequence of for that of Thy-1 (PrPCGPIThy-1) in cells and mice. We show that this modifies the GPI-anchor composition, which then lacks sialic acid, and that PrPCGPIThy-1 is preferentially localized in axons and is less prone to proteolytic shedding when compared to PrPC. Interestingly, after prion infection, mice expressing PrPCGPIThy-1 show a significant delay to terminal disease, a decrease of microglia/astrocyte activation, and altered MAPK signaling when compared to wild-type mice. Our results are the first to demonstrate in vivo, that the GPI-anchor signal sequence plays a fundamental role in the GPI-anchor composition, dictating the subcellular localization of a given protein and, in the case of PrPC, influencing the development of prion disease. [ABSTRACT FROM AUTHOR]

Mucosal immunoglobulins comprise mainly secretory IgA antibodies (SIgAs), which are the major contributor to pathogen-specific immune responses in mucosal tissues. These SIgAs are highly heterogeneous in terms of their quaternary structure. A recent report shows that the polymerization status of SIgA defines their functionality in the human upper respiratory mucosa. Higher order polymerization of SIgA (i.e., tetramers) leads to a marked increase in neutralizing activity against influenza viruses. However, the precise molecular mechanisms underlying the effects of SIgA polymerization remain elusive. Here, we developed a method for generating recombinant tetrameric monoclonal SIgAs. We then compared the anti-viral activities of these tetrameric SIgAs, which possessed variable regions identical to that of a broadly neutralizing anti-influenza antibody F045-092 against influenza A viruses, with that of monomeric IgG or IgA. The tetrameric SIgA showed anti-viral inhibitory activity superior to that of other forms only when the antibody exhibits low-affinity binding to the target. By contrast, SIgA tetramerization did not substantially modify anti-viral activity against targets with high-affinity binding. Taken together, the data suggest that tetramerization of SIgA improved target breadth, but not peak potency of antiviral functions of the broadly neutralizing anti-influenza antibody. This phenomenon presumably represents one of the mechanisms by which SIgAs present in human respiratory mucosa prevent infection by antigen-drifted influenza viruses. Understanding the mechanisms involved in cross neutralization of viruses by SIgAs might facilitate the development of vaccine strategies against viral infection of mucosal tissues. [ABSTRACT FROM AUTHOR]

Antibodies to the prion protein, PrP, represent a promising therapeutic approach against prion diseases but the neurotoxicity of certain anti-PrP antibodies has caused concern. Here we describe scPOM-bi, a bispecific antibody designed to function as a molecular prion tweezer. scPOM-bi combines the complementarity-determining regions of the neurotoxic antibody POM1 and the neuroprotective POM2, which bind the globular domain (GD) and flexible tail (FT) respectively. We found that scPOM-bi confers protection to prion-infected organotypic cerebellar slices even when prion pathology is already conspicuous. Moreover, scPOM-bi prevents the formation of soluble oligomers that correlate with neurotoxic PrP species. Simultaneous targeting of both GD and FT was more effective than concomitant treatment with the individual molecules or targeting the tail alone, possibly by preventing the GD from entering a toxic-prone state. We conclude that simultaneous binding of the GD and flexible tail of PrP results in strong protection from prion neurotoxicity and may represent a promising strategy for anti-prion immunotherapy. [ABSTRACT FROM AUTHOR]

Manganese (Mn) is an essential micronutrient that is not readily available to pathogens during infection due to an active host defense mechanism known as nutritional immunity. To overcome this nutrient restriction, bacteria utilize high-affinity transporters that allow them to compete with host metal-binding proteins. Despite the established role of Mn in bacterial pathogenesis, little is known about the relevance of Mn in the pathophysiology of E. faecalis. Here, we identified and characterized the major Mn acquisition systems of E. faecalis. We discovered that the ABC-type permease EfaCBA and two Nramp-type transporters, named MntH1 and MntH2, work collectively to promote cell growth under Mn-restricted conditions. The simultaneous inactivation of EfaCBA, MntH1 and MntH2 (ΔefaΔmntH1ΔmntH2 strain) led to drastic reductions (>95%) in cellular Mn content, severe growth defects in body fluids (serum and urine) ex vivo, significant loss of virulence in Galleria mellonella, and virtually complete loss of virulence in rabbit endocarditis and murine catheter-associated urinary tract infection (CAUTI) models. Despite the functional redundancy of EfaCBA, MntH1 and MntH2 under in vitro or ex vivo conditions and in the invertebrate model, dual inactivation of efaCBA and mntH2 (ΔefaΔmntH2 strain) was sufficient to prompt maximal sensitivity to calprotectin, a Mn- and Zn-chelating host antimicrobial protein, and for the loss of virulence in mammalian models. Interestingly, EfaCBA appears to play a prominent role during systemic infection, whereas MntH2 was more important during CAUTI. The different roles of EfaCBA and MntH2 in these sites could be attributed, at least in part, to the differential expression of efaA and mntH2 in cells isolated from hearts or from bladders. Collectively, this study demonstrates that Mn acquisition is essential for the pathogenesis of E. faecalis and validates Mn uptake systems as promising targets for the development of new antimicrobials. [ABSTRACT FROM AUTHOR]

The digestive tract is the first organ affected by the ingestion of foodborne bacteria. While commensal bacteria become resident, opportunistic or virulent bacteria are eliminated from the gut by the local innate immune system. Here we characterize a new mechanism of defense, independent of the immune system, in Drosophila melanogaster. We observed strong contractions of longitudinal visceral muscle fibers for the first 2 hours following bacterial ingestion. We showed that these visceral muscle contractions are induced by immune reactive oxygen species (ROS) that accumulate in the lumen and depend on the ROS-sensing TRPA1 receptor. We then demonstrate that both ROS and TRPA1 are required in a subset of anterior enteroendocrine cells for the release of the DH31 neuropeptide which activates its receptor in the neighboring visceral muscles. The resulting contractions of the visceral muscles favors quick expulsion of the bacteria, limiting their presence in the gut. Our results unveil a precocious mechanism of defense against ingested opportunistic bacteria, whether they are Gram-positive like Bacillus thuringiensis or Gram-negative like Erwinia carotovora carotovora. Finally, we found that the human homolog of DH31, CGRP, has a conserved function in Drosophila. [ABSTRACT FROM AUTHOR]

The article focuses on the role of noncanonical placental Fc receptors in modulating transplacental transfer of maternal Immunoglobulin G (IgG). Topics discussed include role of IgG in protecting fetus against infectious diseases, identification of Fc region amino acid after mutational analyses, and production of maternal IgG-based therapeutics and vaccines.

The GI tract is preferentially targeted during acute/early HIV-1 infection. Consequent damage to the gut plays a central role in HIV pathogenesis. The basis for preferential targeting of gut tissues is not well defined. Recombinant proteins and synthetic peptides derived from HIV and SIV gp120 bind directly to integrin α4β7, a gut-homing receptor. Using both cell-surface expressed α4β7 and a soluble α4β7 heterodimer we demonstrate that its specific affinity for gp120 is similar to its affinity for MAdCAM (its natural ligand). The gp120 V2 domain preferentially engages extended forms of α4β7 in a cation -sensitive manner and is inhibited by soluble MAdCAM. Thus, V2 mimics MAdCAM in the way that it binds to α4β7, providing HIV a potential mechanism to discriminate between functionally distinct subsets of lymphocytes, including those with gut-homing potential. Furthermore, α4β7 antagonists developed for the treatment of inflammatory bowel diseases, block V2 binding to α4β7. A 15-amino acid V2 -derived peptide is sufficient to mediate binding to α4β7. It includes the canonical LDV/I α4β7 binding site, a cryptic epitope that lies 7–9 amino acids amino terminal to the LDV/I, and residues K169 and I181. These two residues were identified in a sieve analysis of the RV144 vaccine trial as sites of vaccine -mediated immune pressure. HIV and SIV V2 mAbs elicited by both vaccination and infection that recognize this peptide block V2-α4β7 interactions. These mAbs recognize conformations absent from the β- barrel presented in a stabilized HIV SOSIP gp120/41 trimer. The mimicry of MAdCAM-α4β7 interactions by V2 may influence early events in HIV infection, particularly the rapid seeding of gut tissues, and supports the view that HIV replication in gut tissue is a central feature of HIV pathogenesis. [ABSTRACT FROM AUTHOR]

The GI tract is preferentially targeted during acute/early HIV-1 infection. Consequent damage to the gut plays a central role in HIV pathogenesis. The basis for preferential targeting of gut tissues is not well defined. Recombinant proteins and synthetic peptides derived from HIV and SIV gp120 bind directly to integrin α4β7, a gut-homing receptor. Using both cell-surface expressed α4β7 and a soluble α4β7 heterodimer we demonstrate that its specific affinity for gp120 is similar to its affinity for MAdCAM (its natural ligand). The gp120 V2 domain preferentially engages extended forms of α4β7 in a cation -sensitive manner and is inhibited by soluble MAdCAM. Thus, V2 mimics MAdCAM in the way that it binds to α4β7, providing HIV a potential mechanism to discriminate between functionally distinct subsets of lymphocytes, including those with gut-homing potential. Furthermore, α4β7 antagonists developed for the treatment of inflammatory bowel diseases, block V2 binding to α4β7. A 15-amino acid V2 -derived peptide is sufficient to mediate binding to α4β7. It includes the canonical LDV/I α4β7 binding site, a cryptic epitope that lies 7–9 amino acids amino terminal to the LDV/I, and residues K169 and I181. These two residues were identified in a sieve analysis of the RV144 vaccine trial as sites of vaccine -mediated immune pressure. HIV and SIV V2 mAbs elicited by both vaccination and infection that recognize this peptide block V2-α4β7 interactions. These mAbs recognize conformations absent from the β- barrel presented in a stabilized HIV SOSIP gp120/41 trimer. The mimicry of MAdCAM-α4β7 interactions by V2 may influence early events in HIV infection, particularly the rapid seeding of gut tissues, and supports the view that HIV replication in gut tissue is a central feature of HIV pathogenesis. [ABSTRACT FROM AUTHOR]

Enteropathogenic Escherichia coli (EPEC) use a needle-like injection apparatus known as the type III secretion system (T3SS) to deliver protein effectors into host cells. Effector translocation is highly stratified in EPEC with the translocated intimin receptor (Tir) being the first effector delivered into the host. CesT is a multi-cargo chaperone that is required for the secretion of Tir and at least 9 other effectors. However, the structural and mechanistic basis for differential effector recognition by CesT remains unclear. Here, we delineated the minimal CesT-binding region on Tir to residues 35–77 and determined the 2.74 Å structure of CesT bound to an N-terminal fragment of Tir. Our structure revealed that the CesT-binding region in the N-terminus of Tir contains an additional conserved sequence, distinct from the known chaperone-binding β-motif, that we termed the CesT-extension motif because it extends the β-sheet core of CesT. This motif is also present in the C-terminus of Tir that we confirmed to be a unique second CesT-binding region. Point mutations that disrupt CesT-binding to the N- or C-terminus of Tir revealed that the newly identified carboxy-terminal CesT-binding region was required for efficient Tir translocation into HeLa cells and pedestal formation. Furthermore, the CesT-extension motif was identified in the N-terminal region of NleH1, NleH2, and EspZ, and mutations that disrupt this motif reduced translocation of these effectors, and in some cases, overall effector stability, thus validating the universality of this CesT-extension motif. The presence of two CesT-binding regions in Tir, along with the presence of the CesT-extension motif in other highly translocated effectors, may contribute to differential cargo recognition by CesT. [ABSTRACT FROM AUTHOR]

The article provides an overview of the evolution of fungi in a tightly regulated PHO pathway to retain intracellular phosphate homeostasis. It explores the phosphate status as being conveyed to the nucleus through a core regulatory cyclin-dependent kinase (CDK) complex to impact a response at transcriptional level in Saccharomyces cerevisiae. It highlights the phosphate starvation response mechanism in S. cerevisiae as conserved essentially in Neurospora crassa.

Inducing broad spectrum neutralizing antibodies against challenging pathogens such as HIV-1 is a major vaccine design goal, but may be hindered by conformational instability within viral envelope glycoproteins (Env). Chemical cross-linking is widely used for vaccine antigen stabilization, but how this process affects structure, antigenicity and immunogenicity is poorly understood and its use remains entirely empirical. We have solved the first cryo-EM structure of a cross-linked vaccine antigen. The 4.2 Å structure of HIV-1 BG505 SOSIP soluble recombinant Env in complex with a CD4 binding site-specific broadly neutralizing antibody (bNAb) Fab fragment reveals how cross-linking affects key properties of the trimer. We observed density corresponding to highly specific glutaraldehyde (GLA) cross-links between gp120 monomers at the trimer apex and between gp120 and gp41 at the trimer interface that had strikingly little impact on overall trimer conformation, but critically enhanced trimer stability and improved Env antigenicity. Cross-links were also observed within gp120 at sites associated with the N241/N289 glycan hole that locally modified trimer antigenicity. In immunogenicity studies, the neutralizing antibody response to cross-linked trimers showed modest but significantly greater breadth against a global panel of difficult-to-neutralize Tier-2 heterologous viruses. Moreover, the specificity of autologous Tier-2 neutralization was modified away from the N241/N289 glycan hole, implying a novel specificity. Finally, we have investigated for the first time T helper cell responses to next-generation soluble trimers, and report on vaccine-relevant immunodominant responses to epitopes within BG505 that are modified by cross-linking. Elucidation of the structural correlates of a cross-linked viral glycoprotein will allow more rational use of this methodology for vaccine design, and reveals a strategy with promise for eliciting neutralizing antibodies needed for an effective HIV-1 vaccine. [ABSTRACT FROM AUTHOR]

The extensive glycosylation of HIV-1 envelope (Env) glycoprotein leaves few glycan-free holes large enough to admit broadly neutralizing antibodies (bnAb). Consequently, most bnAbs must inevitably make some glycan contacts and avoid clashes with others. To investigate how Env glycan maturation regulates HIV sensitivity to bnAbs, we modified HIV-1 pseudovirus (PV) using various glycoengineering (GE) tools. Promoting the maturation of α-2,6 sialic acid (SA) glycan termini increased PV sensitivity to two bnAbs that target the V2 apex and one to the interface between Env surface gp120 and transmembrane gp41 subunits, typically by up to 30-fold. These effects were reversible by incubating PV with neuraminidase. The same bnAbs were unusually potent against PBMC-produced HIV-1, suggesting similar α-2,6 hypersialylated glycan termini may occur naturally. Overexpressing β-galactosyltransferase during PV production replaced complex glycans with hybrid glycans, effectively 'thinning' trimer glycan coverage. This increased PV sensitivity to some bnAbs but ablated sensitivity to one bnAb that depends on complex glycans. Other bnAbs preferred small glycans or galactose termini. For some bnAbs, the effects of GE were strain-specific, suggesting that GE had context-dependent effects on glycan clashes. GE was also able to increase the percent maximum neutralization (i.e. saturation) by some bnAbs. Indeed, some bnAb-resistant strains became highly sensitive with GE—thus uncovering previously unknown bnAb breadth. As might be expected, the activities of bnAbs that recognize glycan-deficient or invariant oligomannose epitopes were largely unaffected by GE. Non-neutralizing antibodies were also unaffected by GE, suggesting that trimers remain compact. Unlike mature bnAbs, germline-reverted bnAbs avoided or were indifferent to glycans, suggesting that glycan contacts are acquired as bnAbs mature. Together, our results suggest that glycovariation can greatly impact neutralization and that knowledge of the optimal Env glycoforms recognized by bnAbs may assist rational vaccine design. [ABSTRACT FROM AUTHOR]

Despite extensive genetic diversity of HIV-1 in chronic infection, a single or few maternal virus variants become the founders of an infant’s infection. These transmitted/founder (T/F) variants are of particular interest, as a maternal or infant HIV vaccine should raise envelope (Env) specific IgG responses capable of blocking this group of viruses. However, the maternal or infant factors that contribute to selection of infant T/F viruses are not well understood. In this study, we amplified HIV-1 env genes by single genome amplification from 16 mother-infant transmitting pairs from the U.S. pre-antiretroviral era Women Infant Transmission Study (WITS). Infant T/F and representative maternal non-transmitted Env variants from plasma were identified and used to generate pseudoviruses for paired maternal plasma neutralization sensitivity analysis. Eighteen out of 21 (85%) infant T/F Env pseudoviruses were neutralization resistant to paired maternal plasma. Yet, all infant T/F viruses were neutralization sensitive to a panel of HIV-1 broadly neutralizing antibodies and variably sensitive to heterologous plasma neutralizing antibodies. Also, these infant T/F pseudoviruses were overall more neutralization resistant to paired maternal plasma in comparison to pseudoviruses from maternal non-transmitted variants (p = 0.012). Altogether, our findings suggest that autologous neutralization of circulating viruses by maternal plasma antibodies select for neutralization-resistant viruses that initiate peripartum transmission, raising the speculation that enhancement of this response at the end of pregnancy could further reduce infant HIV-1 infection risk. [ABSTRACT FROM AUTHOR]

HIV-1 envelope glycoprotein (Env) mediates virus attachment and entry into the host cells. Like other membrane-bound and secreted proteins, HIV-1 Env contains at its N terminus a signal peptide (SP) that directs the nascent Env to the endoplasmic reticulum (ER) where Env synthesis and post-translational modifications take place. SP is cleaved during Env biosynthesis but potentially influences the phenotypic traits of the Env protein. The Env SP sequences of HIV-1 isolates display high sequence variability, and the significance of such variability is unclear. We postulate that changes in the Env SP influence Env transport through the ER-Golgi secretory pathway and Env folding and/or glycosylation that impact on Env incorporation into virions, receptor binding and antibody recognition. We first evaluated the consequences of mutating the charged residues in the Env SP in the context of infectious molecular clone HIV-1 REJO.c/2864. Results show that three different mutations affecting histidine at position 12 affected Env incorporation into virions that correlated with reduction of virus infectivity and DC-SIGN-mediated virus capture and transmission. Mutations at positions 8, 12, and 15 also rendered the virus more resistant to neutralization by monoclonal antibodies against the Env V1V2 region. These mutations affected the oligosaccharide composition of N-glycans as shown by changes in Env reactivity with specific lectins and by mass spectrometry. Increased neutralization resistance and N-glycan composition changes were also observed when analogous mutations were introduced to another HIV-1 strain, JRFL. To the best of our knowledge, this is the first study showing that certain residues in the HIV-1 Env SP can affect virus neutralization sensitivity by modulating oligosaccharide moieties on the Env N-glycans. The HIV-1 Env SP sequences thus may be under selective pressure to balance virus infectiousness with virus resistance to the host antibody responses. (289 words) [ABSTRACT FROM AUTHOR]

The hepatitis C virus (HCV) envelope glycoproteins E1 and E2 form a non-covalently linked heterodimer on the viral surface that mediates viral entry. E1, E2 and the heterodimer complex E1E2 are candidate vaccine antigens, but are technically challenging to study because of difficulties in producing natively folded proteins by standard protein expression and purification methods. To better comprehend the antigenicity of these proteins, a library of alanine scanning mutants comprising the entirety of E1E2 (555 residues) was created for evaluating the role of each residue in the glycoproteins. The mutant library was probed, by a high-throughput flow cytometry-based assay, for binding with the co-receptor CD81, and a panel of 13 human and mouse monoclonal antibodies (mAbs) that target continuous and discontinuous epitopes of E1, E2, and the E1E2 complex. Together with the recently determined crystal structure of E2 core domain (E2c), we found that several residues in the E2 back layer region indirectly impact binding of CD81 and mAbs that target the conserved neutralizing face of E2. These findings highlight an unexpected role for the E2 back layer in interacting with the E2 front layer for its biological function. We also identified regions of E1 and E2 that likely located at or near the interface of the E1E2 complex, and determined that the E2 back layer also plays an important role in E1E2 complex formation. The conformation-dependent reactivity of CD81 and the antibody panel to the E1E2 mutant library provides a global view of the influence of each amino acid (aa) on E1E2 expression and folding. This information is valuable for guiding protein engineering efforts to enhance the antigenic properties and stability of E1E2 for vaccine antigen development and structural studies. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Norovirus is the leading cause of gastroenteritis worldwide. Despite recent developments in norovirus propagation in cell culture, these viruses are still challenging to grow routinely. Moreover, little is known on how norovirus infects the host cells, except that histo-blood group antigens (HBGAs) are important binding factors for infection and cell entry. Antibodies that bind at the HBGA pocket and block attachment to HBGAs are believed to neutralize the virus. However, additional neutralization epitopes elsewhere on the capsid likely exist and impeding the intrinsic structural dynamics of the capsid could be equally important. In the current study, we investigated a panel of Nanobodies in order to probe functional epitopes that could trigger capsid rearrangement and/ or interfere with HBGA binding interactions. The precise binding sites of six Nanobodies (Nano-4, Nano-14, Nano-26, Nano-27, Nano-32, and Nano-42) were identified using X-ray crystallography. We showed that these Nanobodies bound on the top, side, and bottom of the norovirus protruding domain. The impact of Nanobody binding on norovirus capsid morphology was analyzed using electron microscopy and dynamic light scattering. We discovered that distinct Nanobody epitopes were associated with varied changes in particle structural integrity and assembly. Interestingly, certain Nanobody-induced capsid morphological changes lead to the capsid protein degradation and viral RNA exposure. Moreover, Nanobodies employed multiple inhibition mechanisms to prevent norovirus attachment to HBGAs, which included steric obstruction (Nano-14), allosteric interference (Nano-32), and violation of normal capsid morphology (Nano-26 and Nano-85). Finally, we showed that two Nanobodies (Nano-26 and Nano-85) not only compromised capsid integrity and inhibited VLPs attachment to HBGAs, but also recognized a broad panel of norovirus genotypes with high affinities. Consequently, Nano-26 and Nano-85 have a great potential to function as novel therapeutic agents against human noroviruses. [ABSTRACT FROM AUTHOR]

Antibody-dependent enhancement of viral infection is a well-described phenomenon that is based on the cellular uptake of infectious virus-antibody complexes following their interaction with Fcγ receptors expressed on myeloid cells. Here we describe a novel mechanism of antibody-mediated enhancement of infection by a flavivirus (tick-borne encephalitis virus) in transformed and primary human cells, which is independent of the presence of Fcγ receptors. Using chemical cross-linking and immunoassays, we demonstrate that the monoclonal antibody (mab) A5, recognizing an epitope at the interface of the dimeric envelope protein E, causes dimer dissociation and leads to the exposure of the fusion loop (FL). Under normal conditions of infection, this process is triggered only after virus uptake by the acidic pH in endosomes, resulting in the initiation of membrane fusion through the interaction of the FL with the endosomal membrane. Analysis of virus binding and cellular infection, together with inhibition by the FL-specific mab 4G2, indicated that the FL, exposed after mab A5- induced dimer-dissociation, mediated attachment of the virus to the plasma membrane also at neutral pH, thereby increasing viral infectivity. Since antibody-induced enhancement of binding was not only observed with cells but also with liposomes, it is likely that increased infection was due to FL-lipid interactions and not to interactions with cellular plasma membrane proteins. The novel mechanism of antibody-induced infection enhancement adds a new facet to the complexity of antibody interactions with flaviviruses and may have implications for yet unresolved effects of polyclonal antibody responses on biological properties of these viruses. [ABSTRACT FROM AUTHOR]

Poxviruses have evolved unique proteins and mechanisms to counteract the nuclear factor κB (NF-κB) signaling pathway, which is an essential regulatory pathway of host innate immune responses. Here, we describe a NF-κB inhibitory virion protein of orf virus (ORFV), ORFV073, which functions very early in infected cells. Infection with ORFV073 gene deletion virus (OV-IA82Δ073) led to increased accumulation of NF-κB essential modulator (NEMO), marked phosphorylation of IκB kinase (IKK) subunits IKKα and IKKβ, IκBα and NF-κB subunit p65 (NF-κB-p65), and to early nuclear translocation of NF-κB-p65 in virus-infected cells (≤ 30 min post infection). Expression of ORFV073 alone was sufficient to inhibit TNFα induced activation of the NF-κB signaling in uninfected cells. Consistent with observed inhibition of IKK complex activation, ORFV073 interacted with the regulatory subunit of the IKK complex NEMO. Infection of sheep with OV-IA82Δ073 led to virus attenuation, indicating that ORFV073 is a virulence determinant in the natural host. Notably, ORFV073 represents the first poxviral virion-associated NF-κB inhibitor described, highlighting the significance of viral inhibition of NF-κB signaling very early in infection. [ABSTRACT FROM AUTHOR]

Myeloid-derived suppressor cells (MDSCs) are expanded in tumor microenvironments, including that of Epstein–Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC). The link between MDSC expansion and EBV infection in NPC is unclear. Here, we show that EBV latent membrane protein 1 (LMP1) promotes MDSC expansion in the tumor microenvironment by promoting extra-mitochondrial glycolysis in malignant cells, which is a scenario for immune escape initially suggested by the frequent, concomitant detection of abundant LMP1, glucose transporter 1 (GLUT1) and CD33+ MDSCs in tumor sections. The full process has been reconstituted in vitro. LMP1 promotes the expression of multiple glycolytic genes, including GLUT1. This metabolic reprogramming results in increased expression of the Nod-like receptor family protein 3 (NLRP3) inflammasome, COX-2 and P-p65 and, consequently, increased production of IL-1β, IL-6 and GM-CSF. Finally, these changes in the environment of malignant cells result in enhanced NPC-derived MDSC induction. One key step is the physical interaction of LMP1 with GLUT1 to stabilize the GLUT1 protein by blocking its K48-ubiquitination and p62-dependent autolysosomal degradation. This work indicates that LMP1-mediated glycolysis regulates IL-1β, IL-6 and GM-CSF production through the NLRP3 inflammasome, COX-2 and P-p65 signaling pathways to enhance tumor-associated MDSC expansion, which leads to tumor immunosuppression in NPC. [ABSTRACT FROM AUTHOR]

Dysbiosis, or the imbalance in the structural and/or functional properties of the microbiome, is at the origin of important infectious inflammatory diseases such as inflammatory bowel disease (IBD) and periodontal disease. Periodontitis is a polymicrobial inflammatory disease that affects a large proportion of the world's population and has been associated with a wide variety of systemic health conditions, such as diabetes, cardiovascular and respiratory diseases. Dysbiosis has been identified as a key element in the development of the disease. However, the precise mechanisms and environmental signals that lead to the initiation of dysbiosis in the human microbiome are largely unknown. In a series of previous in vivo studies using metatranscriptomic analysis of periodontitis and its progression we identified several functional signatures that were highly associated with the disease. Among them, potassium ion transport appeared to be key in the process of pathogenesis. To confirm its importance we performed a series of in vitro experiments, in which we demonstrated that potassium levels a increased the virulence of the oral community as a whole and at the same time altering the immune response of gingival epithelium, increasing the production of TNF-α and reducing the expression of IL-6 and the antimicrobial peptide human β-defensin 3 (hBD-3). These results indicate that levels of potassium in the periodontal pocket could be an important element in of dysbiosis in the oral microbiome. They are a starting point for the identification of key environmental signals that modify the behavior of the oral microbiome from a symbiotic community to a dysbiotic one. [ABSTRACT FROM AUTHOR]

The article offers information on Candida auris, a multidrug-resistant (MDR) yeast, as the emerging cause of nosocomial pathogen globally, particularly the multidrug-resistant fungal infections in hospitals around the world. Topics include the misidentification of C. auris in the routine microbiology laboratory, the virulence of C. auris due to genetic predisposition, and the emergence of C. auris as public health risk.

Broadly-neutralizing monoclonal antibodies (bNAbs) may guide vaccine development for highly variable viruses including hepatitis C virus (HCV), since they target conserved viral epitopes that could serve as vaccine antigens. However, HCV resistance to bNAbs could reduce the efficacy of a vaccine. HC33.4 and AR4A are two of the most potent anti-HCV human bNAbs characterized to date, binding to highly conserved epitopes near the amino- and carboxy-terminus of HCV envelope (E2) protein, respectively. Given their distinct epitopes, it was surprising that these bNAbs showed similar neutralization profiles across a panel of natural HCV isolates, suggesting that some viral polymorphisms may confer resistance to both bNAbs. To investigate this resistance, we developed a large, diverse panel of natural HCV envelope variants and a novel computational method to identify bNAb resistance polymorphisms in envelope proteins (E1 and E2). By measuring neutralization of a panel of HCV pseudoparticles by 10 μg/mL of each bNAb, we identified E1E2 variants with resistance to one or both bNAbs, despite 100% conservation of the AR4A binding epitope across the panel. We discovered polymorphisms outside of either binding epitope that modulate resistance to both bNAbs by altering E2 binding to the HCV co-receptor, scavenger receptor B1 (SR-B1). This study is focused on a mode of neutralization escape not addressed by conventional analysis of epitope conservation, highlighting the contribution of extra-epitopic polymorphisms to bNAb resistance and presenting a novel mechanism by which HCV might persist even in the face of an antibody response targeting multiple conserved epitopes. [ABSTRACT FROM AUTHOR]

Mucins are heavily glycosylated proteins that give mucus its gel-like properties. Moreover, the glycans decorating the mucin protein core can alter the protective properties of the mucus barrier. To investigate whether these alterations could be parasite-induced we utilized the Trichuris muris (T. muris) infection model, using different infection doses and strains of mice that are resistant (high dose infection in BALB/c and C57BL6 mice) or susceptible (high dose infection in AKR and low dose infection in BALB/c mice) to chronic infection by T. muris. During chronicity, within the immediate vicinity of the T. muris helminth the goblet cell thecae contained mainly sialylated mucins. In contrast, the goblet cells within the epithelial crypts in the resistant models contained mainly sulphated mucins. Maintained mucin sulphation was promoted by TH2-immune responses, in particular IL-13, and contributed to the protective properties to the mucus layer, making it less vulnerable to degradation by T. muris excretory secretory products. Mucin sulphation was markedly reduced in the caecal goblet cells in the sulphate anion transporter-1 (Sat-1) deficient mice. We found that Sat-1 deficient mice were susceptible to chronic infection despite a strong TH2-immune response. Lower sulphation levels lead to decreased efficiency of establishment of T. muris infection, independent of egg hatching. This study highlights the complex process by which immune-regulated alterations in mucin glycosylation occur following T. muris infection, which contributes to clearance of parasitic infection. [ABSTRACT FROM AUTHOR]

Cyclic GMP-AMP (cGAMP) synthase (cGAS, a.k.a. MB21D1), a cytosolic DNA sensor, catalyzes formation of the second messenger 2’3’-cGAMP that activates the stimulator of interferon genes (STING) signaling. How the cGAS activity is modulated remains largely unknown. Here, we demonstrate that sentrin/SUMO-specific protease 7 (SENP7) interacted with and potentiated cGAS activation. The small ubiquitin-like modifier (SUMO) was conjugated onto the lysine residues 335, 372 and 382 of cGAS, which suppressed its DNA-binding, oligomerization and nucleotidyl-transferase activities. SENP7 reversed this inhibition via catalyzing the cGAS de-SUMOylation. Consistently, silencing of SENP7 markedly impaired the IRF3-responsive gene expression induced by cGAS-STING axis. SENP7-knockdown mice were more susceptible to herpes simplex virus 1 (HSV-1) infection. SENP7 was significantly up-regulated in patients with SLE. Our study highlights the temporal modulation of the cGAS activity via dynamic SUMOylation, uncovering a novel mechanism for fine-tuning the STING signaling in innate immunity. [ABSTRACT FROM AUTHOR]

Ebola (EBOV) and Marburg (MARV) viruses are members of the Filoviridae family which cause outbreaks of hemorrhagic fever. The filovirus VP40 matrix protein is essential for virus assembly and budding, and its PPxY L-domain motif interacts with WW-domains of specific host proteins, such as Nedd4 and ITCH, to facilitate the late stage of virus-cell separation. To identify additional WW-domain-bearing host proteins that interact with VP40, we used an EBOV PPxY-containing peptide to screen an array of 115 mammalian WW-domain-bearing proteins. Using this unbiased approach, we identified BCL2 Associated Athanogene 3 (BAG3), a member of the BAG family of molecular chaperone proteins, as a specific VP40 PPxY interactor. Here, we demonstrate that the WW-domain of BAG3 interacts with the PPxY motif of both EBOV and MARV VP40 and, unexpectedly, inhibits budding of both eVP40 and mVP40 virus-like particles (VLPs), as well as infectious VSV-EBOV recombinants. BAG3 is a stress induced protein that regulates cellular protein homeostasis and cell survival through chaperone-mediated autophagy (CMA). Interestingly, our results show that BAG3 alters the intracellular localization of VP40 by sequestering VP40 away from the plasma membrane. As BAG3 is the first WW-domain interactor identified that negatively regulates budding of VP40 VLPs and infectious virus, we propose that the chaperone-mediated autophagy function of BAG3 represents a specific host defense strategy to counteract the function of VP40 in promoting efficient egress and spread of virus particles. [ABSTRACT FROM AUTHOR]

Burkholderia dolosa is a member of the Burkholderia cepacia complex (BCC), which is a group of bacteria that cause chronic lung infection in patients with cystic fibrosis (CF) and can be associated with outbreaks carrying high morbidity and mortality. While investigating the genomic diversity of B. dolosa strains collected from an outbreak among CF patients, we previously identified fixL as a gene showing signs of strong positive selection. This gene has homology to fixL of the rhizobial FixL/FixJ two-component system. The goals of this study were to determine the functions of FixLJ and their role in virulence in B. dolosa. We generated a fixLJ deletion mutant and complemented controls in B. dolosa strain AU0158. Using a fixK-lacZ reporter we found that FixLJ was activated in low oxygen in multiple BCC species. In a murine pneumonia model, the B. dolosa fixLJ deletion mutant was cleared faster from the lungs and spleen than wild-type B. dolosa strain AU0158 at 7 days post infection. Interestingly, the fixLJ deletion mutant made more biofilm, albeit with altered structure, but was less motile than strain AU0158. Using RNA-seq with in vitro grown bacteria, we found ~11% of the genome was differentially expressed in the fixLJ deletion mutant relative to strain AU0158. Multiple flagella-associated genes were down-regulated in the fixLJ deletion mutant, so we also evaluated virulence of a fliC deletion mutant, which lacks a flagellum. We saw no difference in the ability of the fliC deletion mutant to persist in the murine model relative to strain AU0158, suggesting factors other than flagella caused the phenotype of decreased persistence. We found the fixLJ deletion mutant to be less invasive in human lung epithelial and macrophage-like cells. In conclusion, B. dolosa fixLJ is a global regulator that controls biofilm formation, motility, intracellular invasion/persistence, and virulence. [ABSTRACT FROM AUTHOR]

Malaria parasites (Plasmodium spp.) encounter markedly different (nutritional) environments during their complex life cycles in the mosquito and human hosts. Adaptation to these different host niches is associated with a dramatic rewiring of metabolism, from a highly glycolytic metabolism in the asexual blood stages to increased dependence on tricarboxylic acid (TCA) metabolism in mosquito stages. Here we have used stable isotope labelling, targeted metabolomics and reverse genetics to map stage-specific changes in Plasmodium berghei carbon metabolism and determine the functional significance of these changes on parasite survival in the blood and mosquito stages. We show that glutamine serves as the predominant input into TCA metabolism in both asexual and sexual blood stages and is important for complete male gametogenesis. Glutamine catabolism, as well as key reactions in intermediary metabolism and CoA synthesis are also essential for ookinete to oocyst transition in the mosquito. These data extend our knowledge of Plasmodium metabolism and point towards possible targets for transmission-blocking intervention strategies. Furthermore, they highlight significant metabolic differences between Plasmodium species which are not easily anticipated based on genomics or transcriptomics studies and underline the importance of integration of metabolomics data with other platforms in order to better inform drug discovery and design. [ABSTRACT FROM AUTHOR]

Bacterial type III protein secretion systems inject effector proteins into eukaryotic host cells in order to promote survival and colonization of Gram-negative pathogens and symbionts. Secretion across the bacterial cell envelope and injection into host cells is facilitated by a so-called injectisome. Its small hydrophobic export apparatus components SpaP and SpaR were shown to nucleate assembly of the needle complex and to form the central “cup” substructure of a Salmonella Typhimurium secretion system. However, the in vivo placement of these components in the needle complex and their function during the secretion process remained poorly defined. Here we present evidence that a SpaP pentamer forms a 15 Å wide pore and provide a detailed map of SpaP interactions with the export apparatus components SpaQ, SpaR, and SpaS. We further refine the current view of export apparatus assembly, consolidate transmembrane topology models for SpaP and SpaR, and present intimate interactions of the periplasmic domains of SpaP and SpaR with the inner rod protein PrgJ, indicating how export apparatus and needle filament are connected to create a continuous conduit for substrate translocation. [ABSTRACT FROM AUTHOR]

Metal acquisition is crucial for all cells and for the virulence of many bacterial pathogens. In particular, nickel is a virulence determinant for the human gastric pathogen Helicobacter pylori as it is the cofactor of two enzymes essential for in vivo colonization, urease and a [NiFe] hydrogenase. To import nickel despite its scarcity in the human body, H. pylori requires efficient uptake mechanisms that are only partially defined. Indeed, alternative ways of nickel entry were predicted to exist in addition to the well-described NixA permease. Using a genetic screen, we identified an ABC transporter, that we designated NiuBDE, as a novel H. pylori nickel transport system. Unmarked mutants carrying deletions of nixA, niuD and/or niuB, were constructed and used to measure (i) tolerance to toxic nickel exposure, (ii) intracellular nickel content by ICP-OES, (iii) transport of radioactive nickel and (iv) expression of a reporter gene controlled by nickel concentration. We demonstrated that NiuBDE and NixA function separately and are the sole nickel transporters in H. pylori. NiuBDE, but not NixA, also transports cobalt and bismuth, a metal currently used in H. pylori eradication therapy. Both NiuBDE and NixA participate in nickel-dependent urease activation at pH 5 and survival under acidic conditions mimicking those encountered in the stomach. However, only NiuBDE is able to carry out this activity at neutral pH and is essential for colonization of the mouse stomach. Phylogenomic analyses indicated that both nixA and niuBDE genes have been acquired via horizontal gene transfer by the last common ancestor of the gastric Helicobacter species. Our work highlights the importance of this evolutionary event for the emergence of Helicobacter gastric species that are adapted to the hostile environment of the stomach where the capacity of Helicobacter to import nickel and thereby activate urease needs to be optimized. [ABSTRACT FROM AUTHOR]

IFI16 (gamma-interferon-inducible protein 16), a predominantly nuclear protein involved in transcriptional regulation, also functions as an innate immune response DNA sensor and induces the IL-1β and antiviral type-1 interferon-β (IFN-β) cytokines. We have shown that IFI16, in association with BRCA1, functions as a sequence independent nuclear sensor of episomal dsDNA genomes of KSHV, EBV and HSV-1. Recognition of these herpesvirus genomes resulted in IFI16 acetylation, BRCA1-IFI16-ASC-procaspase-1 inflammasome formation, cytoplasmic translocation, and IL-1β generation. Acetylated IFI16 also interacted with cytoplasmic STING and induced IFN-β. However, the identity of IFI16 associated nuclear proteins involved in STING activation and the mechanism is not known. Mass spectrometry of proteins precipitated by anti-IFI16 antibodies from uninfected endothelial cell nuclear lysate revealed that histone H2B interacts with IFI16. Single and double proximity ligation microscopy, immunoprecipitation, EdU-genome labeled virus infection, and chromatin immunoprecipitation studies demonstrated that H2B is associated with IFI16 and BRCA1 in the nucleus in physiological conditions. De novo KSHV and HSV-1 infection as well as latent KSHV and EBV infection induces the cytoplasmic distribution of H2B-IFI16, H2B-BRCA1 and IFI16-ASC complexes. Vaccinia virus (dsDNA) cytoplasmic replication didn’t induce the redistribution of nuclear H2B-IFI16 or H2B into the cytoplasm. H2B is critical in KSHV and HSV-1 genome recognition by IFI16 during de novo infection. Viral genome sensing by IFI16-H2B-BRCA1 leads to BRCA1 dependent recruitment of p300, and acetylation of H2B and IFI16. BRCA1 knockdown or inhibition of p300 abrogated the acetylation of H2B-IFI16 or H2B. Ran-GTP protein mediated the translocation of acetylated H2B and IFI16 to the cytoplasm along with BRCA1 that is independent of IFI16-ASC inflammasome. ASC knockdown didn’t affect the acetylation of H2B, its cytoplasmic transportation, and the association of STING with IFI16 and H2B during KSHV infection. Absence of H2B didn’t affect IFI16-ASC association and cytoplasmic distribution and thus demonstrating that IFI16-H2B complex is independent of IFI16-ASC-procaspase-1-inflammasome complex formed during infection. The H2B-IFI16-BRCA1 complex interacted with cGAS and STING in the cytoplasm leading to TBK1 and IRF3 phosphorylation, nuclear translocation of pIRF3 and IFN-β production. Silencing of H2B, cGAS and STING inhibited IFN-β induction but not IL-1β secretion, and cGAMP activity is significantly reduced by H2B and IFI16 knockdown during infection. Silencing of ASC inhibited IL-1β secretion but not IFN-β secretion during de novo KSHV and HSV-1 infection. These studies identify H2B as an innate nuclear sensor mediating a novel extra chromosomal function, and reveal that two IFI16 complexes mediate KSHV and HSV-1 genome recognition responses, with recognition by the IFI16-BRCA1-H2B complex resulting in IFN-β responses and recognition by IFI16-BRCA1 resulting in inflammasome responses. [ABSTRACT FROM AUTHOR]

The pneumococcus is one of the most prodigious producers of hydrogen peroxide amongst bacterial pathogens. Hydrogen peroxide production by the pneumococcus has been implicated in antibiotic synergism, competition between other bacterial colonizers of the nasopharynx, and damage to epithelial cells. However, the role during invasive disease has been less clear with mutants defective in hydrogen peroxide production demonstrating both attenuation and heightened invasive disease capacity depending upon strain and serotype background. This work resolves these conflicting observations by demonstrating that the main hydrogen peroxide producing enzyme of the pneumococcus, SpxB, is required for capsule formation in a strain dependent manner. Capsule production by strains harboring capsules with acetylated sugars was dependent upon the presence of spxB while capsule production in serotypes lacking such linkages were not. The spxB mutant had significantly lower steady-state cellular levels of acetyl-CoA, suggesting that loss of capsule arises from dysregulation of this intermediary metabolite. This conclusion is corroborated by deletion of pdhC, which also resulted in lower steady-state acetyl-CoA levels and phenocopied the capsule expression profile of the spxB mutant. Capsule and acetyl-CoA levels were restored in the spxB and lctO (lactate oxidase) double mutant, supporting the connection between central metabolism and capsule formation. Taken together, these data show that the defect in pathogenesis in the spxB mutant is due to a metabolic imbalance that attenuates capsule formation and not to reduced hydrogen peroxide formation. [ABSTRACT FROM AUTHOR]

We used pulse-labeling with the methionine analogue homopropargylglycine (HPG) to investigate spatiotemporal aspects of protein synthesis during herpes simplex virus (HSV) infection. In vivo incorporation of HPG enables subsequent selective coupling of fluorochrome-capture reagents to newly synthesised proteins. We demonstrate that HPG labeling had no effect on cell viability, on accumulation of test early or late viral proteins, or on overall virus yields. HPG pulse-labeling followed by SDS-PAGE analysis confirmed incorporation into newly synthesised proteins, while parallel processing by in situ cycloaddition revealed new insight into spatiotemporal aspects of protein localisation during infection. A striking feature was the rapid accumulation of newly synthesised proteins not only in a general nuclear pattern but additionally in newly forming sub-compartments represented by small discrete foci. These newly synthesised protein domains (NPDs) were similar in size and morphology to PML domains but were more numerous, and whereas PML domains were progressively disrupted, NPDs were progressively induced and persisted. Immediate-early proteins ICP4 and ICP0 were excluded from NPDs, but using an ICP0 mutant defective in PML disruption, we show a clear spatial relationship between NPDs and PML domains with NPDs frequently forming immediately adjacent and co-joining persisting PML domains. Further analysis of location of the chaperone Hsc70 demonstrated that while NPDs formed early in infection without overt Hsc70 recruitment, later in infection Hsc70 showed pronounced recruitment frequently in a coat-like fashion around NPDs. Moreover, while ICP4 and ICP0 were excluded from NPDs, ICP22 showed selective recruitment. Our data indicate that NPDs represent early recruitment of host and viral de novo translated protein to distinct structural entities which are precursors to the previously described VICE domains involved in protein quality control in the nucleus, and reveal new features from which we propose spatially linked platforms of newly synthesised protein processing after nuclear import. [ABSTRACT FROM AUTHOR]

The Staphylococcus aureus manganese transporter protein MntC is under investigation as a component of a prophylactic S.aureus vaccine. Passive immunization with monoclonal antibodies mAB 305-78-7 and mAB 305-101-8 produced using MntC was shown to significantly reduce S. aureus burden in an infant rat model of infection. Earlier interference mapping suggested that a total of 23 monoclonal antibodies generated against MntC could be subdivided into three interference groups, representing three independent immunogenic regions. In the current work binding epitopes for selected representatives of each of these interference groups (mAB 305-72-5 – group 1, mAB 305-78-7 – group 2, and mAB 305-101-8 – group 3) were mapped using Hydrogen-Deuterium Exchange Mass Spectrometry (DXMS). All of the identified epitopes are discontinuous, with binding surface formed by structural elements that are separated within the primary sequence of the protein but adjacent in the context of the three-dimensional structure. The approach was validated by co-crystallizing the Fab fragment of one of the antibodies (mAB 305-78-7) with MntC and solving the three-dimensional structure of the complex. X-ray results themselves and localization of the mAB 305-78-7 epitope were further validated using antibody binding experiments with MntC variants containing substitutions of key amino acid residues. These results provided insight into the antigenic properties of MntC and how these properties may play a role in protecting the hostagainst S. aureus infection by preventing the capture and transport of Mn2+, a key element that the pathogen uses to evade host immunity. [ABSTRACT FROM AUTHOR]

In the context of HIV vaccine design and development, HIV-1 spike mimetics displaying a range of stabilities were evaluated to determine whether more stable, well-ordered trimers would more efficiently elicit neutralizing antibodies. To begin, in vitro analysis of trimers derived from the cysteine-stabilized SOSIP platform or the uncleaved, covalently linked NFL platform were evaluated. These native-like trimers, derived from HIV subtypes A, B, and C, displayed a range of thermostabilities, and were “stress-tested” at varying temperatures as a prelude to in vivo immunogenicity. Analysis was performed both in the absence and in the presence of two different adjuvants. Since partial trimer degradation was detected at 37°C before or after formulation with adjuvant, we sought to remedy such an undesirable outcome. Cross-linking (fixing) of the well-ordered trimers with glutaraldehyde increased overall thermostability, maintenance of well-ordered trimer integrity without or with adjuvant, and increased resistance to solid phase-associated trimer unfolding. Immunization of unfixed and fixed well-ordered trimers into animals revealed that the elicited tier 2 autologous neutralizing activity correlated with overall trimer thermostability, or melting temperature (Tm). Glutaraldehyde fixation also led to higher tier 2 autologous neutralization titers. These results link retention of trimer quaternary packing with elicitation of tier 2 autologous neutralizing activity, providing important insights for HIV-1 vaccine design. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis (Mtb) must cope with exogenous oxidative stress imposed by the host. Unlike other antioxidant enzymes, Mtb’s thioredoxin reductase TrxB2 has been predicted to be essential not only to fight host defenses but also for in vitro growth. However, the specific physiological role of TrxB2 and its importance for Mtb pathogenesis remain undefined. Here we show that genetic inactivation of thioredoxin reductase perturbed several growth-essential processes, including sulfur and DNA metabolism and rapidly killed and lysed Mtb. Death was due to cidal thiol-specific oxidizing stress and prevented by a disulfide reductant. In contrast, thioredoxin reductase deficiency did not significantly increase susceptibility to oxidative and nitrosative stress. In vivo targeting TrxB2 eradicated Mtb during both acute and chronic phases of mouse infection. Deliberately leaky knockdown mutants identified the specificity of TrxB2 inhibitors and showed that partial inactivation of TrxB2 increased Mtb’s susceptibility to rifampicin. These studies reveal TrxB2 as essential thiol-reducing enzyme in Mtb in vitro and during infection, establish the value of targeting TrxB2, and provide tools to accelerate the development of TrxB2 inhibitors. [ABSTRACT FROM AUTHOR]