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Powassan viruses (POWV) are emergent tick-borne flaviviruses that cause severe neurologic disease in humans. Subcutaneous inoculation of C57BL/6 mice with POWV (strain LI9) resulted in overt brain damage resembling spongiform encephalitis. Noting higher POWV lethality in older mice, we assessed neurovirulence as a function of age. We found that POWV LI9 inoculation was lethal in 80% of 50 wk old mice, 10-15 dpi, and that lethality was sequentially reduced in 40, 30, 20, 10 wk old mice to SignificanceOur findings establish a novel age-dependent lethal animal model to study encephalitic POWV diseasein vivo. These initial findings demonstrate that following peripheral inoculation, non-neuroadapted POWV LI9 is neuroinvasive and enters the brains of young and aged mice. However, POWV LI9 lethality is strictly age-dependent and correlated with increased viral load in the brains of aged mice. POWV rapidly directs neuronal loss and spongiform lesions, microglial activation and causes prolonged inflammation that fails to clear POWV from the brains of aged mice. Our results provide a lethal murine model of POWV neurovirulence that mirrors the prevalence of severe human POWV encephalitis in the elderly. This lethal murine POWV model provides mechanisms for defining POWV protective responses of the young, revealing determinants of age-dependent POWV lethality and evaluating potential POWV therapeutics.SUMMARYPowassan virus is an emerging tick-borne flavivirus linked to severe neurologic disease in aged individuals. Here we describe an age-dependent mouse model of POWV pathogenesis.SUBJECTSPowassan virus, flavivirus, neurovirulence, neuroinvasion, neurotropic, spongiform encephalopathy, microgliosis, neuroinflammation

Ticks are hematophagous ectoparasites capable of transmitting multiple human pathogens. Environmental changes have supported the expansion of ticks into new geographical areas that have become the epicenters of tick-borne diseases (TBDs).

Since its discovery in the United States in 2017, the Asian longhorned tick (Haemaphysalis longicornis) has been detected in most eastern states between Rhode Island and Georgia. Long Island, east of New York City, a recognized high-risk area for tick-borne diseases, is geographically close to New Jersey and New York sites where H. longicornis was originally found. However, extensive tick surveys conducted in 2018 did not identify H. longicornis on Long Island. In stark contrast, our 2022 tick survey suggests that H. longicornis has rapidly invaded and expanded in multiple surveying sites on Long Island (12 out of 17 sites). Overall, the relative abundance of H. longicornis was similar to that of lone star ticks, Amblyomma americanum, a previously recognized tick species abundantly present on Long Island. Interestingly, our survey suggests that H. longicornis has expanded within the Appalachian forest ecological zone of Long Island's north shore compared to the Pine Barrens located on the south shore of Long Island. The rapid invasion and expansion of H. longicornis into an insular environment are different from the historical invasion and expansion of two native tick species, Ixodes scapularis (blacklegged tick or deer tick) and A. americanum, in Long Island. The implications of H. longicornis transmitting or introducing tick-borne pathogens of public health importance remain unknown.

Outbreaks of emerging viral pathogens like severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are a major medical challenge. There is a pressing need for antivirals that can be rapidly deployed to curb infection and dissemination. We determined the efficacy of interferon lambda-1 (IFN-λ) as a broad-spectrum antiviral agent to inhibit SARS-CoV-2 infection and reduce pathology in a mouse model of disease. IFN-λ significantly limited SARS-CoV-2 production in primary human bronchial epithelial cells in culture. Pretreatment of human lung cells with IFN-λ completely blocked infectious virus production, and treatment with IFN-λ at the time of infection inhibited virus production more than 10-fold. To interrogate the protective effects of IFN-λ in response to SARS-CoV-2 infection, transgenic mice expressing the human angiotensin-converting enzyme 2 (ACE-2) were tested. One dose of IFN-λ administered intranasally was found to reduce animal morbidity and mortality. Our study with SARS-CoV-2 also revealed a sex differential in disease outcome. Male mice had higher mortality, reflecting the more severe symptoms and mortality found in male patients infected with SARS-CoV-2. The results indicate that IFN-λ potentially can treat early stages of SARS-CoV-2 infection and decrease pathology, and this murine model can be used to investigate the sex differential documented in COVID-19. IMPORTANCE The COVID-19 pandemic has claimed millions of lives worldwide. In this report, we used a preclinical mouse model to investigate the prophylactic and therapeutic value of intranasal IFN-λ for this acute respiratory disease. Specific vaccines have been responsible for curbing the transmission of SARS-CoV-2 in developed nations. However, vaccines require time to generate and keep pace with antigenic variants. There is a need for broad-spectrum prophylactic and therapeutic agents to combat new emerging viral pathogens. Our mouse model suggests IFN-λ has clinical utility, and it reflects the well-documented finding that male COVID-19 patients manifest more severe symptoms and mortality. Understanding this sex bias is critical for considering therapeutic approaches to COVID-19.

Powassan viruses (POWVs) are neurovirulent tick-borne flaviviruses emerging in the Northeastern U.S., with a 2% prevalence in Long Island (LI) deer ticks (Ixodes scapularis). POWVs are transmitted in as little as 15 minutes of a tick bite, and enter the CNS to cause encephalitis (10% fatal) and long-term neuronal damage. POWV-LI9 and POWV-LI41 present in LI Ixodes ticks were isolated by directly inoculating VeroE6 cells with tick homogenates and detecting POWV infected cells by immunoperoxidase staining. Inoculated POWV-LI9 and LI41 were exclusively present in infected cell foci, indicative of spread cell to cell, despite growth in liquid culture without an overlay. Cloning and sequencing establish POWV-LI9 as a phylogenetically distinct lineage II POWV strain circulating in LI deer ticks. Primary human brain microvascular endothelial cells (hBMECs) and pericytes form a neurovascular complex that restricts entry into the CNS. We found that POWV-LI9, -LI41 and Lineage I POWV-LB, productively infect hBMECs and pericytes and that POWVs were basolaterally transmitted from hBMECs to lower chamber pericytes without permeabilizing polarized hBMECs. Synchronous POWV-LI9 infection of hBMECs and pericytes induced proinflammatory chemokines, interferon-β (IFNβ) and IFN-stimulated genes, with delayed IFNβ secretion by infected pericytes. IFN inhibited POWV infection, but despite IFN secretion a subset of POWV infected hBMECs and pericytes remained persistently infected. These findings suggest a potential mechanism for POWVs (LI9/LI41 and LB) to infect hBMECs, spread basolaterally to pericytes and enter the CNS. hBMEC and pericyte responses to POWV infection suggest a role for immunopathology in POWV neurovirulence and potential therapeutic targets for preventing POWV spread to neuronal compartments.ImportanceWe isolated POWVs from LI deer ticks (I. scapularis) directly in VeroE6 cells and sequencing revealed POWV-LI9 as a distinct lineage II POWV strain. Remarkably, inoculating VeroE6 cells with POWV containing tick homogenates resulted in infected cell foci in liquid culture, consistent with cell to cell spread. POWV-LI9, -LI41, and Lineage I POWV-LB strains infected hBMECs and pericytes that comprise neurovascular complexes. POWVs were nonlytically transmitted basolaterally from infected hBMECs to lower chamber pericytes, suggesting a mechanism for POWV transmission across BBB. POWV-LI9 elicited inflammatory responses from infected hBMEC and pericytes that may contribute to immune cell recruitment and neuropathogenesis. This study reveals a potential mechanism for POWVs to enter the CNS by infecting hBMECs and spreading basolaterally to abluminal pericytes. Our findings reveal that POWV-LI9 persists in cells that form a neurovascular complex spanning the BBB, and suggest potential therapeutic targets for preventing POWV spread to neuronal compartments.

The causative agent of plague, Yersinia pestis, uses a type III secretion system to selectively destroy immune cells in humans, thus enabling Y. pestis to reproduce in the bloodstream and be transmitted to new hosts through fleabites. The host factors that are responsible for the selective destruction of immune cells by plague bacteria are unknown. Here we show that LcrV, the needle cap protein of the Y. pestis type III secretion system, binds to the N-formylpeptide receptor (FPR1) on human immune cells to promote the translocation of bacterial effectors. Plague infection in mice is characterized by high mortality; however, Fpr1-deficient mice have increased survival and antibody responses that are protective against plague. We identified FPR1R190W as a candidate resistance allele in humans that protects neutrophils from destruction by the Y. pestis type III secretion system. Thus, FPR1 is a plague receptor on immune cells in both humans and mice, and its absence or mutation provides protection against Y. pestis. Furthermore, plague selection of FPR1 alleles appears to have shaped human immune responses towards other infectious diseases and malignant neoplasms. The receptor FPR1 on human immune cells interacts with Yersinia pestis, mutations in this receptor provide resistance against plague in humans and Fpr1 deficiency enhances survival in mice.

Significance Genetic analysis of Rickettsia has been difficult. We developed a transposon and selection scheme to facilitate the isolation of Rickettsia conorii mutants with insertional lesions. Here, we demonstrate that the R. conorii polysaccharide synthesis operon (pso) encompasses genetic determinants for biosynthesis of the O antigen, which also affect the composition of outer-membrane proteins, invasion of host cells, and pathogenesis. The O antigen provides essential barrier functions and plays a major role in host–pathogen interactions. Our findings suggest that infected hosts develop protective immunity against R. conorii via the production of antibodies targeting the O antigen. Conservation of pso among rickettsial species suggests that it may play a universal role in O-antigen synthesis, disease pathogenesis, and the development of immunity., Rickettsial diseases have long been diagnosed with serum antibodies cross-reactive against Proteus vulgaris (Weil–Felix reaction). Although Weil–Felix antibodies are associated with the development of immunity, their rickettsial target and contribution to disease pathogenesis are not established. Here, we developed a transposon for insertional mutagenesis of Rickettsia conorii, isolating variants defective for replication in cultured cells and in spotted fever pathogenesis. Mutations in the polysaccharide synthesis operon (pso) abolish lipopolysaccharide O-antigen synthesis and Weil–Felix serology and alter outer-membrane protein assembly. Unlike wild-type R. conorii, pso mutants cannot elicit bactericidal antibodies that bind O antigen. The pso operon is conserved among rickettsial pathogens, suggesting that bactericidal antibodies targeting O antigen may generate universal immunity that could be exploited to develop vaccines against rickettsial diseases.

The LysM domain is found in all kingdoms of life. While their function in mammals is not known, LysM domains of bacteria and their phage parasites are associated with enzymes that cleave or remodel peptidoglycan., Staphylococcus aureus causes reiterative and chronic persistent infections. This can be explained by the formidable ability of this pathogen to escape immune surveillance mechanisms. Cells of S. aureus display the abundant staphylococcal protein A (SpA). SpA binds to immunoglobulin (Ig) molecules and coats the bacterial surface to prevent phagocytic uptake. SpA also binds and cross-links variable heavy 3 (VH3) idiotype (IgM) B cell receptors, promoting B cell expansion and the secretion of nonspecific VH3-IgM via a mechanism requiring CD4+ T cell help. SpA binding to antibodies is mediated by the N-terminal Ig-binding domains (IgBDs). The so-called region X, uncharacterized LysM domain, and C-terminal LPXTG sorting signal for peptidoglycan attachment complete the linear structure of the protein. Here, we report that both the LysM domain and the LPXTG motif sorting signal are required for the B cell superantigen activity of SpA in a mouse model of infection. SpA molecules purified from staphylococcal cultures are sufficient to exert B cell superantigen activity and promote immunoglobulin secretion as long as they carry intact LysM and LPXTG motif domains with bound peptidoglycan fragments. The LysM domain binds the glycan chains of peptidoglycan fragments, whereas the LPXTG motif is covalently linked to wall peptides lacking glycan. These findings emphasize the complexity of SpA interactions with B cell receptors.

Exposure to Staphylococcus aureus does not lead to immunity as evidenced by the persistent colonization of one third of the human population. S. aureus immune escape is mediated by factors that preempt complement activation, destroy phagocytes, and modify B and T cell responses. One such factor, Staphylococcal protein A (SpA) encompasses five Immunoglobulin binding domains (IgBDs) that associate with the Fcγ domain to block phagocytosis. IgBDs also associate with the Fab domain of VH3-idiotypic IgM which activates B cells with the resulting secretion of antibodies that cannot bind determinants of S. aureus. SpA crosslinking of VH3-idiotypic IgG and IgE receptors of mast cells and basophils promotes histamine release and anaphylaxis. Previous work demonstrated the safety, immunogenicity, and protective efficacy of SpAKKAA, a variant partially defective for VH3-idiotypic Ig cross-linking, in murine models of S. aureus. Compared to mice (10%), humans produce significantly more VH3-idiotypic B cells (50%), prompting a search for safer SpA variants that may be suitably developed as clinical-grade vaccines for efficacy testing in humans. Here, we report the identification of such variants.

Rickettsia amblyommatis belongs to the spotted fever group of Rickettsia and infects Amblyomma americanum (Lone Star ticks) for transmission to offspring and mammals. Historically, the geographic range of A. americanum was restricted to the southeastern USA. However, recent tick surveys identified the progressive northward invasion of A. americanum, contributing to the increased number of patients with febrile illnesses of unknown etiology after a tick bite in the northeastern USA. While serological evidence strongly suggests that patients are infected with R. amblyommatis, the virulence potential of R. amblyommatis is not well established. Here, we performed a bioinformatic analysis of three genome sequences of R. amblyommatis and identified the presence of multiple putative virulence genes whose products are implicated for spotted fever pathogenesis. Similar to other pathogenic spotted fever rickettsiae, R. amblyommatis replicated intracellularly within the cytoplasm of tissue culture cells. Interestingly, R. amblyommatis displayed defective attachment to microvascular endothelial cells. The attachment defect and slow growth rate of R. amblyommatis required relatively high intravenous infectious doses to produce dose-dependent morbidity and mortality in C3H mice. In summary, our results corroborate clinical evidence that R. amblyommatis can cause mild disease manifestation in some patients., Rickettsia amblyommatis, an obligate intracellular pathogen transmitted by Lone Star ticks (Amblyomma americanum), infects endothelial cells and causes spotted fever pathogenesis in mice.

Exposure to Staphylococcus aureus does not lead to immunity as evidenced by the persistent colonization of one third of the human population. S. aureus immune escape is mediated by factors that preempt complement activation, destroy phagocytes, and modify B and T cell responses. One such factor, Staphylococcal protein A (SpA) encompasses five Immunoglobulin binding domains (IgBDs) that associate with the Fcγ domain to block phagocytosis. IgBDs also associate with Fab encoded by V(H)3 clan related genes. SpA binding to V(H)3-IgM that serves as a B cell receptor results in B cell expansion and secretion of antibodies with no specificity for S. aureus. SpA crosslinking of V(H)3-IgG and V(H)3-IgE bound to cognate receptors of mast cells and basophils promotes histamine release and anaphylaxis. Earlier work developed a prototype variant SpA(KKAA) with four amino acid substitutions in each IgBD. When tested in animal models, SpA(KKAA) elicited neutralizing antibodies and protection against infection. We show here that SpA(KKAA) retains crosslinking activity for V(H)3-IgG and V(H)3-IgE. We use a rational approach to design and test 67 new SpA variants for loss of V(H)3 binding and anaphylactic activities. We identify two detoxified candidates that elicit SpA-neutralizing antibodies and protect animals from S. aureus colonization and bloodstream infection. The new detoxified SpA candidates bear three instead of four amino acid substitutions thus increasing the development of SpA-specific antibodies. We propose that detoxified SpA variants unable to crosslink V(H)3-idiotypic immunoglobulin may be suitably developed as clinical-grade vaccines for safety and efficacy testing in humans.

Significance All currently licensed antibodies against bacteria target exotoxins. For most pathogens, neutralization of toxin(s) is not sufficient to prevent bacterial replication. Antibodies against surface determinants represent better candidates to enhance opsonophagocytic killing, but the mechanisms of action of such antibodies have not been systematically studied. Staphylococcal protein A is a conserved surface protein of Staphylococcus aureus and a crucial virulence determinant that manipulates B-cell responses and blocks deposition of opsonin. Monoclonal antibodies directed against SpA represent potential therapeutic agents as well as a formidable tool to identify and optimize effector functions of antibodies that can promote bacterial clearance., Antibodies may bind to bacterial pathogens or their toxins to control infections, and their effector activity is mediated through the recruitment of complement component C1q or the engagement with Fcγ receptors (FcγRs). For bacterial pathogens that rely on a single toxin to cause disease, immunity correlates with toxin neutralization. Most other bacterial pathogens, including Staphylococcus aureus, secrete numerous toxins and evolved multiple mechanisms to escape opsonization and complement killing. Several vaccine candidates targeting defined surface antigens of S. aureus have failed to meet clinical endpoints. It is unclear that such failures can be solely attributed to the poor selection of antibody targets. Thus far, studies to delineate antibody-mediated uptake and killing of Gram-positive pathogens remain extremely limited. Here, we exploit 3F6-hIgG1, a human monoclonal antibody that binds and neutralizes the abundant surface-exposed Staphylococcal protein A (SpA). We find that galactosylation of 3F6-hIgG1 that favors C1q recruitment is indispensable for opsonophagocytic killing of staphylococci and for protection against bloodstream infection in animals. However, the simple removal of fucosyl residues, which results in reduced C1q binding and increased engagement with FcγR, maintains the opsonophagocytic killing and protective attributes of the antibody. We confirm these results by engineering 3F6-hIgG1 variants with biased binding toward C1q or FcγRs. While the therapeutic benefit of monoclonal antibodies against infectious disease agents may be debatable, the functional characterization of such antibodies represents a powerful tool for the development of correlates of protection that may guide future vaccine trials.

A hallmark of Staphylococcus aureus disease in humans is persistent infections without development of protective immune responses. Infected patients generate VH3 plasmablast expansions and increased VH3 idiotype Ig; however, the mechanisms for staphylococcal modification of immune responses are not known. We report here that S. aureus-infected mice generate VH3 antibody expansions via a mechanism requiring MHC-restricted antigen presentation to CD4(+) T cells and staphylococcal protein A (SpA), a cell wall-anchored surface molecule that binds Fcγ and VH3 variant heavy chains of Ig. VH3 expansion occurred with peptidoglycan-linked SpA from the bacterial envelope but not with recombinant SpA, and optimally required five tandem repeats of its Ig-binding domains. Signaling via receptor-interacting serine/threonine protein kinase 2 (RIPK2) was essential for implementing peptidoglycan-linked SpA superantigen activity. VH3 clan IgG from S. aureus-infected or SpA-treated animals was not pathogen-specific, suggesting that SpA cross-linking of VH3 idiotype B-cell receptors and activation via attached peptidoglycan are the determinants of staphylococcal escape from adaptive immune responses.

Vaccines and antibody therapeutics targeting staphylococcal surface molecules have failed to achieve clinical efficacy against MRSA infection. Here, Thomer et al. show that the R domain of prothrombin directs fibrinogen to the surface of S. aureus, which generates a protective coat for the pathogen, inhibiting phagocytosis by immune cells. The use of R-specific antibodies allows for immune cell recognition and protects mice against lethal bloodstream infections by broad spectrum MRSA isolates., Host immunity against bacteria typically involves antibodies that recognize the microbial surface and promote phagocytic killing. Methicillin-resistant Staphylococcus aureus (MRSA) is a frequent cause of lethal bloodstream infection; however, vaccines and antibody therapeutics targeting staphylococcal surface molecules have thus far failed to achieve clinical efficacy. S. aureus secretes coagulase (Coa), which activates host prothrombin and generates fibrin fibrils that protect the pathogen against phagocytosis by immune cells. Because of negative selection, the coding sequence for the prothrombin-binding D1-D2 domain is highly variable and does not elicit cross-protective immune responses. The R domain, tandem repeats of a 27-residue peptide that bind fibrinogen, is conserved at the C terminus of all Coa molecules, but its functional significance is not known. We show here that the R domain enables bloodstream infections by directing fibrinogen to the staphylococcal surface, generating a protective fibrin shield that inhibits phagocytosis. The fibrin shield can be marked with R-specific antibodies, which trigger phagocytic killing of staphylococci and protect mice against lethal bloodstream infections caused by a broad spectrum of MRSA isolates. These findings emphasize the critical role of coagulase in staphylococcal escape from opsonophagocytic killing and as a protective antigen for S. aureus vaccines.

Staphylococcus aureus, a bacterial commensal of the human nares and skin, is a frequent cause of soft tissue and bloodstream infections. A hallmark of staphylococcal infections is their frequent recurrence, even when treated with antibiotics and surgical intervention, which demonstrates the bacterium's ability to manipulate innate and adaptive immune responses. In this Review, we highlight how S. aureus virulence factors inhibit complement activation, block and destroy phagocytic cells and modify host B cell and T cell responses, and we discuss how these insights might be useful for the development of novel therapies against infections with antibiotic resistant strains such as methicillin-resistant S. aureus.

Staphylococcus aureus is a cause of sepsis and meningitis in very-low-birth-weight (VLBW) infants. Clinical trials with S. aureus specific antibodies failed to protect VLBW neonates, which may be due to the immune evasive attributes of staphylococcal protein A (SpA). Here we show that mouse monoclonal antibody SpAKKAA-mAb 3F6, which neutralizes the immunoglobulin Fcγ-binding and B cell receptor crosslinking attributes of SpA, protects neonatal mice against S. aureus sepsis and raises protective immunity against subsequent staphylococcal infection. We developed a humanized SpAKKAA-mAb that protects neonatal mice against S. aureus sepsis and may therefore be subjected to clinical testing in VLBW neonates.

Staphylococcus aureus , an invasive pathogen of humans and animals, requires a specialized ESS pathway to secrete proteins (EsxA, EsxB, EsxC, and EsxD) during infection. Expression of ess genes is required for S. aureus establishment of persistent abscess lesions following bloodstream infection; however, the mechanisms whereby effectors of the ESS pathway implement their virulence strategies were heretofore not known. Here, we show that EssE forms a complex with other members of the ESS secretion pathway and its substrates, promoting the secretion of EsxA, EsxB, EsxC, EsxD, and EssD. During bloodstream infection of mice, the S. aureus essE mutant displays defects in host cytokine responses, specifically in the production of interleukin-12 (IL-12) (p40/p70) and the suppression of RANTES (CCL5), activators of T H 1 T cell responses and immune cell chemotaxis, respectively. Thus, essE -mediated secretion of protein effectors via the ESS pathway may enable S. aureus to manipulate host immune responses by modifying the production of cytokines. IMPORTANCE Staphylococcus aureus and other firmicutes evolved a specialized ESS ( E sxA/ESAT-6-like s ecretion s ystem) pathway for the secretion of small subsets of proteins lacking canonical signal peptides. The molecular mechanisms for ESS-dependent secretion and their functional purpose are still unknown. We demonstrate here that S. aureus EssE functions as a membrane assembly platform for elements of the secretion machinery and their substrates. Furthermore, S. aureus EssE-mediated secretion contributes to the production or the suppression of specific cytokines during host infection, thereby modifying immune responses toward this pathogen.

The risk for Staphylococcus aureus bloodstream infection (BSI) is increased in immunocompromised individuals, including patients with hematologic malignancy and/or chemotherapy. Due to the emergence of antibiotic-resistant strains, designated methicillin-resistant S. aureus (MRSA), staphylococcal BSI in cancer patients is associated with high mortality; however, neither a protective vaccine nor pathogen-specific immunotherapy is currently available. Here, we modeled staphylococcal BSI in leukopenic CD-1 mice that had been treated with cyclophosphamide, a drug for leukemia and lymphoma patients. Cyclophosphamide-treated mice were highly sensitive to S. aureus BSI and developed infectious lesions lacking immune cell infiltrates. Virulence factors of S. aureus that are key for disease establishment in immunocompetent hosts—α-hemolysin (Hla), iron-regulated surface determinants (IsdA and IsdB), coagulase (Coa), and von Willebrand factor binding protein (vWbp)—are dispensable for the pathogenesis of BSI in leukopenic mice. In contrast, sortase A mutants, which cannot assemble surface proteins, display delayed time to death and increased survival in this model. A vaccine with four surface antigens (ClfA, FnBPB, SdrD, and SpA KKAA ), which was identified by genetic vaccinology using sortase A mutants, raised antigen-specific immune responses that protected leukopenic mice against staphylococcal BSI.

Pauli et al. find that the active B cell response in patients infected with Staphylococcus aureus is predominately focused on a single antigen, SpA, which acts as a B cell superantigen. SpA-reactive antibodies dominate the response despite the presence of circulating B cells specific for other bacterial surface antigens., Staphylococcus aureus bacterial infection commonly results in chronic or recurrent disease, suggesting that humoral memory responses are hampered. Understanding how S. aureus subverts the immune response is critical for the rescue of host natural humoral immunity and vaccine development. S. aureus expresses the virulence factor Protein A (SpA) on all clinical isolates, and SpA has been shown in mice to expand and ablate variable heavy 3 (VH3) idiotype B cells. The effects of SpA during natural infection, however, have not been addressed. Acutely activated B cells, or plasmablasts (PBs), were analyzed to dissect the ongoing immune response to infection through the production of monoclonal antibodies (mAbs). The B cells that were activated by infection had a highly limited response. When screened against multiple S. aureus antigens, only high-affinity binding to SpA was observed. Consistently, PBs underwent affinity maturation, but their B cell receptors demonstrated significant bias toward the VH3 idiotype. These data suggest that the superantigenic activity of SpA leads to immunodominance, limiting host responses to other S. aureus virulence factors that would be necessary for protection and memory formation.

Envelope biogenesis in bacteria involves synthesis of intermediates that are tethered to the lipid carrier undecaprenol-phosphate. LytR-CpsA-Psr (LCP) enzymes have been proposed to catalyze the transfer of undecaprenol-linked intermediates onto the C6-hydroxyl of MurNAc in peptidoglycan, thereby promoting attachment of wall teichoic acid (WTA) in bacilli and staphylococci and capsular polysaccharides (CPS) in streptococci. S. aureus encodes three lcp enzymes, and a variant lacking all three genes (Δlcp) releases WTA from the bacterial envelope and displays a growth defect. Here, we report that the type 5 capsular polysaccharide (CP5) of Staphylococcus aureus Newman is covalently attached to the glycan strands of peptidoglycan. Cell wall attachment of CP5 is abrogated in the Δlcp variant, a defect that is best complemented via expression of lcpC in trans. CP5 synthesis and peptidoglycan attachment are not impaired in the tagO mutant, suggesting that CP5 synthesis does not involve the GlcNAc-ManNAc linkage unit of WTA and may instead utilize another Wzy-type ligase to assemble undecaprenyl-phosphate intermediates. Thus, LCP enzymes of S. aureus are promiscuous enzymes that attach secondary cell wall polymers with discrete linkage units to peptidoglycan.

Staphylococcal protein A (SpA) binds Fcγ and VH3 clan Fab domains of human and animal immunoglobulin (Ig) with each of its five Ig binding domains (IgBDs), thereby supporting Staphylococcus aureus escape from opsonophagocytic killing and suppressing adaptive B cell responses. The variant SpAKKAA cannot bind Ig yet retains antigenic properties that elicit SpA-neutralizing antibodies and disease protection in mice, whereas S. aureus infection or SpA-immunization cannot elicit neutralizing antibodies. As a test for this model, we analyzed here mAb 358A76, which was isolated from SpA-immunized mice. Unlike SpAKKAA-derived mAbs, mAb 358A76 binds only the first IgBD (E) but not any of the other four IgBDs (D-A-B-C) and its binding can neutralize only the E domain of SpA, which does not provide disease protection in mice. These results are in agreement with a model whereby wild-type SpA-immunization generates a limited antibody response without neutralizing and/or disease protective attributes.

The current epidemic of infections caused by antibiotic-resistant Gram-positive bacteria requires the discovery of new drug targets and the development of new therapeutics. Lipoteichoic acid (LTA), a cell wall polymer of Gram-positive bacteria, consists of 1,3-polyglycerol-phosphate linked to glycolipid. LTA synthase (LtaS) polymerizes polyglycerol-phosphate from phosphatidylglycerol, a reaction that is essential for the growth of Gram-positive bacteria. We screened small molecule libraries for compounds inhibiting growth of Staphylococcus aureus but not of Gram-negative bacteria. Compound 1771 [2-oxo-2-(5-phenyl-1,3,4-oxadiazol-2-ylamino)ethyl 2-naphtho[2,1- b ]furan-1-ylacetate] blocked phosphatidylglycerol binding to LtaS and inhibited LTA synthesis in S. aureus and in Escherichia coli expressing ltaS . Compound 1771 inhibited the growth of antibiotic-resistant Gram-positive bacteria and prolonged the survival of mice with lethal S. aureus challenge, validating LtaS as a target for the development of antibiotics.

Humans and animals are colonized by members of the genus Staphylococcus, however only some of these species evolved to cause invasive disease. The genetic basis for conversion of commensal staphylococci into pathogens is not known. We hypothesized that Staphylococcus aureus genes for coagulation and agglutination in vertebrate blood (coa, vwb and clfA) may support pathogenic conversion. Expression of coa and vwb in Staphylococcus epidermidis or Staphylococcus simulans supported a coagulase-positive phenotype but not the ability to cause disease in a mouse model of bloodstream infection. However, the simultaneous expression of coa, vwb and clfA in coagulase-negative staphylococci enabled bacterial agglutination in plasma and enhanced survival of S. simulans in human whole blood. Agglutination of S. simulans in the bloodstream of infected mice upon expression of coa, vwb and clfA provided also a mean for dissemination and replication in distal organs. Thus, the acquisition of genes for bacterial agglutination with fibrin appear sufficient for the conversion of commensal staphylococci into invasive pathogens.

Staphylococcus aureus – a commensal of the human skin, nares and gastrointestinal tract – is also a leading cause of bacterial skin and soft tissue infection (SSTIs), bacteremia, sepsis, peritonitis, pneumonia and endocarditis. Antibiotic-resistant strains, designated MRSA (methicillin-resistant S. aureus), are common and represent a therapeutic challenge. Current research and development efforts seek to address the challenge of MRSA infections through vaccines and immune therapeutics. Mice have been used as experimental models for S. aureus SSTI, bacteremia, sepsis, peritonitis and endocarditis. This work led to the identification of key virulence factors, candidate vaccine antigens or immune-therapeutics that still require human clinical testing to establish efficacy. Past failures of human clinical trials raised skepticism whether the mouse is an appropriate model for S. aureus disease in humans. S. aureus causes chronic-persistent infections that, even with antibiotic or surgical intervention, reoccur in humans and in mice. Determinants of S. aureus evasion from human innate and adaptive immune responses have been identified, however only some of these are relevant in mice. Future research must integrate these insights and refine the experimental mouse models for specific S. aureus diseases to accurately predict the failure or success for candidate vaccines and immune-therapeutics.

Staphylococcus aureus secretes products that convert host fibrinogen to fibrin and promote its agglutination with fibrin fibrils, thereby shielding bacteria from immune defenses. The agglutination reaction involves ClfA (clumping factor A), a surface protein with serine-aspartate (SD) repeats that captures fibrin fibrils and fibrinogen. Pathogenic staphylococci express several different SD proteins that are modified by two glycosyltransferases, SdgA and SdgB. Here, we characterized three genes of S. aureus, aggA, aggB (sdgA), and aggC (sdgB), and show that aggA and aggC contribute to staphylococcal agglutination with fibrin fibrils in human plasma. We demonstrate that aggB (sdgA) and aggC (sdgB) are involved in GlcNAc modification of the ClfA SD repeats. However, only sdgB is essential for GlcNAc modification, and an sdgB mutant is defective in the pathogenesis of sepsis in mice. Thus, GlcNAc modification of proteins promotes S. aureus replication in the bloodstream of mammalian hosts.

Heritable defects in human B cell/antibody development are not associated with increased susceptibility to Staphylococcus aureus infection. Protein A (SpA), a surface molecule of S. aureus , binds the Fcγ domain of immunoglobulin (Ig) and cross-links the Fab domain of V H 3-type B cell receptors (IgM). Here we generated S. aureus spa variants harboring amino acid substitutions at four key residues in each of the five Ig-binding domains of SpA. Wild-type S. aureus required SpA binding to Ig to resist phagocytosis and SpA-mediated B cell receptor cross-linking to block antibody development in mice. The spa KKAA mutant, which cannot bind Ig or IgM, was phagocytosed and elicited B cell responses to key virulence antigens that protected animals against lethal S. aureus challenge. The immune evasive attributes of S. aureus SpA were abolished in µMT mice lacking mature B cells and antibodies. Thus, while wild-type S. aureus escapes host immune surveillance, the spa KKAA variant elicits adaptive responses that protect against recurrent infection. IMPORTANCE Staphylococcus aureus causes recurrent skin and bloodstream infections without eliciting immunity. Heritable defects in neutrophil and T cell function, but not B cell or antibody development, are associated with increased incidence of S. aureus infection, and efforts to develop antibody-based S. aureus vaccines have thus far been unsuccessful. We show here that the Fcγ and V H 3-type Fab binding activities of staphylococcal protein A (SpA) are essential for S. aureus escape from host immune surveillance in mice. The virulence attributes of SpA in mice required mature B cells and immunoglobulin. These results suggest that antibodies and B cells play a key role in the pathogenesis of staphylococcal infections and provide insights into the development of a vaccine against S. aureus .

Staphylococcus aureus is a frequent cause of skin infection and sepsis in humans. Preclinical vaccine studies with S. aureus have used a mouse model with intraperitoneal challenge and survival determination as a measure for efficacy. To appreciate the selection of protective antigens in this model, we sought to characterize the pathological attributes of S. aureus infection in the peritoneal cavity. Testing C57BL/6J and BALB/c mice, >10 9 CFU of S. aureus Newman were needed to produce a lethal outcome in 90% of animals infected via intraperitoneal injection. Both necropsy and histopathology revealed the presence of intraperitoneal abscesses in the vicinity of inoculation sites. Abscesses were comprised of fibrin as well as collagen deposits and immune cells with staphylococci replicating at the center of these lesions. Animals that succumbed to challenge harbored staphylococci in abscess lesions and in blood. The establishment of lethal infections, but not the development of intraperitoneal abscesses, was dependent on S. aureus expression of alpha-hemolysin (Hla). Active immunization with nontoxigenic Hla H35L or passive immunization with neutralizing monoclonal antibodies protected mice against early lethal events associated with intraperitoneal S. aureus infection but did not affect the establishment of abscess lesions. These results characterize a mouse model for the study of intraperitoneal abscess formation by S. aureus , a disease that occurs frequently in humans undergoing continuous ambulatory peritoneal dialysis for end-stage renal disease.

Staphylococcus aureus is a leading cause of human soft tissue infections and bacterial sepsis. The emergence of antibiotic-resistant strains (methicillin-resistant S. aureus [MRSA]) has prompted research into staphylococcal vaccines and preventive measures. The envelope of S. aureus is decorated with staphylococcal protein A (SpA), which captures the Fcγ portion of immunoglobulins to prevent opsonophagocytosis and associates with the Fab portion of V H 3-type B cell receptors to trigger B cell superantigen activity. Nontoxigenic protein A (SpA KKAA ), when used as an immunogen in mice, stimulates humoral immune responses that neutralize the Fcγ and the V H 3 + Fab binding activities of SpA and provide protection from staphylococcal abscess formation in mice. Here, we isolated monoclonal antibodies (MAbs) against SpA KKAA that, by binding to the triple-helical bundle fold of its immunoglobulin binding domains (IgBDs), neutralize the Fcγ and Fab binding activities of SpA. SpA KKAA MAbs promoted opsonophagocytic killing of MRSA in mouse and human blood, provided protection from abscess formation, and stimulated pathogen-specific immune responses in a mouse model of staphylococcal disease. Thus, SpA KKAA MAbs may be useful for the prevention and therapy of staphylococcal disease in humans.

Bacteremia and septic shock account for nearly 80% of invasive disease caused by methicillin-resistant Staphylococcus aureus [1]. The vascular endothelium functions as a potent barrier between the bloodstream and the tissues, preventing bacterial dissemination and contributing to homeostatic regulation of blood flow and coagulation. In the context of sepsis, both proinflammatory mediators and pathogen-induced endothelial injury impair vascular integrity, permitting extravasation of fluid, proteins, and cells into the tissues. Injured endothelial cells contribute to systemic inflammatory responses and serve as a nidus for microvascular thrombosis. Together, the triad of endothelial barrier disruption, profound inflammation, and impaired microvascular blood flow with coagulopathy culminate in impaired tissue oxygen delivery, a hallmark of sepsis [2]. As mortality from bacterial sepsis remains high, it is essential to better understand the molecular pathogenesis of this disease. Staphylococcal α-hemolysin (Hla), a pore-forming cytotoxin secreted by almost all strains of S. aureus, directly contributes to endothelial injury [3, 4]. Recently, we defined A-disintegrin and metalloprotease 10 (ADAM10) as a eukaryotic receptor for Hla [5]. ADAM10 is a widely expressed zinc metalloprotease that principally regulates cellular adhesion and migration [6]. In the endothelium, ADAM10-mediated proteolysis of the extracellular domain of vascular endothelial (VE)–cadherin disrupts cadherin-dependent homotypic intercellular interactions [7]. As VE-cadherin provides the structural framework of the adherens junction, ADAM10 plays an important role in the regulation of endothelial permeability. The physiologic importance of VE-cadherin in endothelial barrier integrity during infection is highlighted by London et al [8] wherein enhanced expression of VE-cadherin conferred protection against lethal polymicrobial or influenza A–induced sepsis. Previous studies have shown that Hla induces loss of VE-cadherin expression on the endothelial surface and compromises endothelial barrier function in vitro [4, 9, 10]. The molecular mechanism by which this occurs has yet to be determined. We hypothesized that Hla may contribute to lethal S. aureus sepsis through its ability to directly injure the endothelium, and that ADAM10 may provide the molecular link by which the toxin causes barrier disruption.

Staphylococcus aureus is a commensal of the human skin or nares and a pathogen that frequently causes skin and soft tissue infections as well as bacteremia and sepsis. Recent efforts in understanding the molecular mechanisms of pathogenesis revealed key virulence strategies of S. aureus in host tissues: bacterial scavenging of iron, induction of coagulation pathways to promote staphylococcal agglutination in the vasculature, and suppression of innate and adaptive immune responses. Advances in all three areas have been explored for opportunities in vaccine design in an effort to identify the critical protective antigens of S. aureus. Human clinical trials with specific subunit vaccines have failed, yet provide important insights for the design of future trials that must address the current epidemic of S. aureus infections with drug-resistant isolates (MRSA, methicillin-resistant S. aureus).

Staphylococcus aureus infections result in abscesses as well as septicemia. Even with therapy, abscesses can persist or even reoccur, as staphylococcal infections fail to induce protective immune responses. Here, we show that prior infection with certain attenuated strains may elicit protective immunity. A closer examination reveals that protection correlates with antibody responses elicited on exposure to particular attenuated variants. Linear regression analysis was used to compare reduction in staphylococcal disease and antibody responses to infection with wild-type and attenuated variants. This analysis identified protective antigens that, when tested as vaccines in mice, elicited disease protection. Protection afforded by attenuated strains correlates in part with the ability of Staphylococcus aureus to modulate B cell responses via protein A (spa encoded). We designate this approach “genetic vaccinology,” since it exploits genetic variants to draw a correlation between disease protection and humoral immune responses for the deduction of vaccine antigens. Genetic vaccinology is particularly useful for microbes that do not elicit natural protective immunity during infection.—Kim, H. K., Kim, H. -Y., Schneewind, O., Missiakas, D. Identifying protective antigens of Staphylococcus aureus, a pathogen that suppresses host immune responses.

Staphylococcus aureus causes purulent skin and soft tissue infections (SSTIs) that frequently reoccur. Staphylococal SSTIs can lead to invasive disease and sepsis, which are among the most significant causes of infectious disease mortality in both developed and developing countries. Human or animal infections with S. aureus do not elicit protective immunity against staphylococcal diseases. Here we review what is known about the immune evasive strategies of S. aureus that enable the pathogen's escape from protective immune responses. Three secreted products are discussed in detail, staphylococcal protein A (SpA), staphylococcal binder of immunoglobulin (Sbi) and adenosine synthase A (AdsA). By forming a complex with V(H)3-type IgM on the surface of B cells, SpA functions as a superantigen to modulate antibody responses to staphylococcal infection. SpA also captures pathogen-specific antibodies by binding their Fcγ portion. The latter activity of SpA is shared by Sbi, which also associates with complement factors 3d and factor H to promote the depletion of complement. AdsA synthesizes the immune signaling molecule adenosine, thereby dampening innate and adaptive immune responses during infection. We discuss strategies how the three secreted products of staphylococci may be exploited for the development of vaccines and therapeutics.

Staphylococcus aureus infection is a frequent cause of sepsis in humans, a disease associated with high mortality and without specific intervention. When suspended in human or animal plasma, staphylococci are known to agglutinate, however the bacterial factors responsible for agglutination and their possible contribution to disease pathogenesis have not yet been revealed. Using a mouse model for S. aureus sepsis, we report here that staphylococcal agglutination in blood was associated with a lethal outcome of this disease. Three secreted products of staphylococci - coagulase (Coa), von Willebrand factor binding protein (vWbp) and clumping factor (ClfA) – were required for agglutination. Coa and vWbp activate prothrombin to cleave fibrinogen, whereas ClfA allowed staphylococci to associate with the resulting fibrin cables. All three virulence genes promoted the formation of thromboembolic lesions in heart tissues. S. aureus agglutination could be disrupted and the lethal outcome of sepsis could be prevented by combining dabigatran-etexilate treatment, which blocked Coa and vWbp activity, with antibodies specific for ClfA. Together these results suggest that the combined administration of direct thrombin inhibitors and ClfA-antibodies that block S. aureus agglutination with fibrin may be useful for the prevention of staphylococcal sepsis in humans., Author Summary Staphylococcus aureus secretes factors that perturb blood coagulation in infected hosts. We report here that three bacterial products – coagulase (Coa), von Willebrand factor binding protein (vWbp) and clumping factor (ClfA) - act together and promote agglutination, the association of staphylococci with polymerized fibrin cables. Staphylococcal agglutination was associated with thromboembolic lesions in heart tissues and a lethal outcome of S. aureus sepsis in mice. Inhibition of Coa and vWbp with direct thrombin inhibitors, drugs already approved for the prevention of stroke, as well as passive transfer of antibodies specific for Coa, vWbp and ClfA could prevent the pathogenesis of S. aureus sepsis. These results suggest new preventive and/or therapeutic strategies that may improve the outcome of S. aureus sepsis in humans, a disease that is otherwise associated with high mortality.

The current epidemic of hospital- and community-acquired methicillin-resistant Staphylococcus aureus (MRSA) infections has caused significant human morbidity, but a protective vaccine is not yet available. Prior infection with S. aureus is not associated with protective immunity. This phenomenon involves staphylococcal protein A (SpA), an S. aureus surface molecule that binds to Fcgamma of immunoglobulin (Ig) and to the Fab portion of V(H)3-type B cell receptors, thereby interfering with opsonophagocytic clearance of the pathogen and ablating adaptive immune responses. We show that mutation of each of the five Ig-binding domains of SpA with amino acid substitutions abolished the ability of the resulting variant SpA(KKAA) to bind Fcgamma or Fab V(H)3 and promote B cell apoptosis. Immunization of mice with SpA(KKAA) raised antibodies that blocked the virulence of staphylococci, promoted opsonophagocytic clearance, and protected mice against challenge with highly virulent MRSA strains. Furthermore, SpA(KKAA) immunization enabled MRSA-challenged mice to mount antibody responses to many different staphylococcal antigens.

The bacterial pathogen Staphylococcus aureus seeds abscesses in host tissues to replicate at the center of these lesions, protected from host immune cells via a pseudocapsule. Using histochemical staining, we identified prothrombin and fibrin within abscesses and pseudocapsules. S. aureus secretes two clotting factors, coagulase (Coa) and von Willebrand factor binding protein (vWbp). We report here that Coa and vWbp together are required for the formation of abscesses. Coa and vWbp promote the non-proteolytic activation of prothrombin and cleavage of fibrinogen, reactions that are inhibited with specific antibody against each of these molecules. Coa and vWbp specific antibodies confer protection against abscess formation and S. aureus lethal bacteremia, suggesting that coagulases function as protective antigens for a staphylococcal vaccine., Author Summary Clinical isolates of the human pathogen Staphylococcus aureus secrete coagulase (Coa), a polypeptide that binds to and activates prothrombin, thereby converting fibrinogen to fibrin and promoting clotting of plasma or blood. Another secreted coagulase, designated von-Willebrand factor binding protein (vWbp), catalyzes a similar reaction. Staphylococcal binding to fibrinogen or fibrin is an important attribute of disease pathogenesis, which leads to the formation of abscesses and bacterial persistence in host tissues. We report here that Coa and vWbp are essential for S. aureus strain Newman abscess formation and persistence in host tissues. Antibodies directed against Coa or vWbp prevent coagulase binding to prothrombin or fibrinogen and confer protection against challenge with S. aureus Newman or the methicillin-resistant S. aureus isolate USA300 LAC in mouse models of abscess formation or lethal sepsis. These results suggest that coagulases may be used as vaccine antigens to elicit antibodies that protect humans against S. aureus infections.

Staphylococcus aureus is the most frequent cause of bacteremia and hospital-acquired infection, however a vaccine that prevents staphylococcal disease is currently not available. Two sortase-anchored surface proteins, IsdA and IsdB, have been identified as subunit vaccines that, following active immunization, protect experimental animals against intravenous challenge with staphylococci. Here we investigate the molecular basis of this immunity and report that, when passively transferred to naive mice, purified antibodies directed against IsdA or IsdB protected against staphylococcal abscess formation and lethal intravenous challenge. When added to mouse blood, IsdA- or IsdB-specific antibodies did not promote rapid opsonophagocytic killing of wild-type staphylococci. Antibodies directed against IsdA interfered with heme-binding and IsdB antibodies perturbed the ability of this surface protein to bind hemoglobin. As the structural genes for isdA and isdB are required for heme-iron scavenging during the pathogenesis of infection, we hypothesize that IsdA and IsdB antibodies may at least in part provide protection against staphylococci by interfering with the pathogen's heme-iron scavenging mechanisms.

Staphylococcus aureus infections are associated with abscess formation and bacterial persistence; however, the genes that enable this lifestyle are not known. We show here that following intravenous infection of mice, S. aureus disseminates rapidly into organ tissues and elicits abscess lesions that develop over weeks but cannot be cleared by the host. Staphylococci grow as communities at the center of abscess lesions and are enclosed by pseudocapsules, separating the pathogen from immune cells. By testing insertional variants in genes for cell wall-anchored surface proteins, we are able to infer the stage at which these molecules function. Fibrinogen-binding proteins ClfA and ClfB are required during the early phase of staphylococcal dissemination. The heme scavenging factors IsdA and IsdB, as well as SdrD and protein A, are necessary for abscess formation. Envelope-associated proteins, Emp and Eap, are either required for abscess formation or contribute to persistence. Fluorescence microscopy revealed Eap deposition within the pseudocapsule, whereas Emp was localized within staphylococcal abscess communities. Antibodies directed against envelope-associated proteins generated vaccine protection against staphylococcal abscess formation. Thus, staphylococci employ envelope proteins at discrete stages of a developmental program that enables abscess formation and bacterial persistence in host tissues.—Cheng, A. G., Kim, H. K., Burts, M. L., Krausz, T., Schneewind, O., Missiakas, D. M. Genetic requirements for Staphylococcus aureus abscess formation and persistence in host tissues.

Human or animal infections with S. aureus do not elicit protective immunity against staphylococcal diseases. Staphylococci are notorious to deploy a wide spectrum of strategies to avoid host immune system. Previous work revealed that both innate and adaptive immune systems are hampered by the expression of two immunoglobulin binding proteins; staphylococcal protein A (SpA) and staphylococcal binder of immunoglobulins (Sbi). Staphylococcal protein A, a molecule that associates with both Fc and Fab portions of immunoglobulins, is necessary 1) to neutralize the antibody mediated opsonophagocytosis and 2) to trigger the expansion and apoptotic demise of B cell populations. The second immunoglobulin binding protein, Sbi, can associate with Fc portion of immunoglobulin and complement factor C3 to disable a critical juncture between the innate and adaptive immunity. Although much of biochemical analysis revealed how the two staphylococcal proteins engage with immunoglobulins for many years, their role as immuno modulatory virulence factors in vivo has not been fully understood. We have investigated the significance of immunoglobulin association with staphylococci during acute and chronic infection in animal model and characterized the molecular mechanisms to avert host immune defense system. Strategies are also discussed on how the two secreted products of staphylococci may be exploited for the development of vaccines and therapeutics.