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1.  Pathogenesis of Staphylococcus aureus Bloodstream Infections 
Annual review of pathology  2016;11:343-364.
Staphylococcus aureus , a Gram-positive bacterium colonizing nares, skin, and the gastrointestinal tract, frequently invades the skin, soft tissues, and bloodstreams of humans. Even with surgical and antibiotic therapy, bloodstream infections are associated with significant mortality. The secretion of coagulases, proteins that associate with and activate the host hemostatic factor prothrombin, and the bacterial surface display of agglutinins, proteins that bind polymerized fibrin, are key virulence strategies for the pathogenesis of S. aureus bloodstream infections, which culminate in the establishment of abscess lesions. Pathogen-controlled processes, involving a wide spectrum of secreted factors, are responsible for the recruitment and destruction of immune cells, transforming abscess lesions into purulent exudate, with which staphylococci disseminate to produce new infectious lesions or to infect new hosts. Research on S. aureus bloodstream infections is a frontier for the characterization of protective vaccine antigens and the development of immune therapeutics aiming to prevent disease or improve outcomes.
doi:10.1146/annurev-pathol-012615-044351
PMCID: PMC5068359  PMID: 26925499
coagulation; agglutination; immune evasion; abscess formation; vaccine
2.  SagB Glucosaminidase Is a Determinant of Staphylococcus aureus Glycan Chain Length, Antibiotic Susceptibility, and Protein Secretion 
Journal of Bacteriology  2016;198(7):1123-1136.
ABSTRACT
The envelope of Staphylococcus aureus is comprised of peptidoglycan and its attached secondary polymers, teichoic acid, capsular polysaccharide, and protein. Peptidoglycan synthesis involves polymerization of lipid II precursors into glycan strands that are cross-linked at wall peptides. It is not clear whether peptidoglycan structure is principally determined during polymerization or whether processive enzymes affect cell wall structure and function, for example, by generating conduits for protein secretion. We show here that S. aureus lacking SagB, a membrane-associated N-acetylglucosaminidase, displays growth and cell-morphological defects caused by the exaggerated length of peptidoglycan strands. SagB cleaves polymerized glycan strands to their physiological length and modulates antibiotic resistance in methicillin-resistant S. aureus (MRSA). Deletion of sagB perturbs protein trafficking into and across the envelope, conferring defects in cell wall anchoring and secretion, as well as aberrant excretion of cytoplasmic proteins.
IMPORTANCE Staphylococcus aureus is thought to secrete proteins across the plasma membrane via the Sec pathway; however, protein transport across the cell wall envelope has heretofore not been studied. We report that S. aureus sagB mutants generate elongated peptidoglycan strands and display defects in protein secretion as well as aberrant excretion of cytoplasmic proteins. These results suggest that the thick peptidoglycan layer of staphylococci presents a barrier for protein secretion and that SagB appears to extend the Sec pathway across the cell wall envelope.
doi:10.1128/JB.00983-15
PMCID: PMC4800868  PMID: 26811319
3.  Antibodies against a secreted product of Staphylococcus aureus trigger phagocytic killing 
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.
doi:10.1084/jem.20150074
PMCID: PMC4813671  PMID: 26880578
4.  Contribution of Staphylococcus aureus Coagulases and Clumping Factor A to Abscess Formation in a Rabbit Model of Skin and Soft Tissue Infection 
PLoS ONE  2016;11(6):e0158293.
Staphylococcus aureus produces numerous factors that facilitate survival in the human host. S. aureus coagulase (Coa) and von Willebrand factor-binding protein (vWbp) are known to clot plasma through activation of prothrombin and conversion of fibrinogen to fibrin. In addition, S. aureus clumping factor A (ClfA) binds fibrinogen and contributes to platelet aggregation via a fibrinogen- or complement-dependent mechanism. Here, we evaluated the contribution of Coa, vWbp and ClfA to S. aureus pathogenesis in a rabbit model of skin and soft tissue infection. Compared to skin abscesses caused by the Newman wild-type strain, those caused by isogenic coa, vwb, or clfA deletion strains, or a strain deficient in coa and vwb, were significantly smaller following subcutaneous inoculation in rabbits. Unexpectedly, we found that fibrin deposition and abscess capsule formation appear to be independent of S. aureus coagulase activity in the rabbit infection model. Similarities notwithstanding, S. aureus strains deficient in coa and vwb elicited reduced levels of several proinflammatory molecules in human blood in vitro. Although a specific mechanism remains to be determined, we conclude that S. aureus Coa, vWbp and ClfA contribute to abscess formation in rabbits.
doi:10.1371/journal.pone.0158293
PMCID: PMC4918888  PMID: 27336691
5.  Bacillus anthracis lcp Genes Support Vegetative Growth, Envelope Assembly, and Spore Formation 
Journal of Bacteriology  2015;197(23):3731-3741.
ABSTRACT
Bacillus anthracis, a spore-forming pathogen, replicates as chains of vegetative cells by regulating the separation of septal peptidoglycan. Surface (S)-layer proteins and B. anthracis S-layer-associated proteins (BSLs) function as chain length determinants and are assembled in the envelope by binding to the secondary cell wall polysaccharide (SCWP). B. anthracis expresses six different genes encoding LytR-CpsA-Psr (LCP) enzymes (lcpB1 to -4, lcpC, and lcpD), which when expressed in Staphylococcus aureus promote attachment of wall teichoic acid to peptidoglycan. Mutations in B. anthracis lcpB3 and lcpD cause aberrations in cell size and chain length that can be explained as discrete defects in SCWP assembly; however, the function of the other lcp genes is not known. By deleting combinations of lcp genes from the B. anthracis genome, we generated variants with single lcp genes. B. anthracis expressing lcpB3 alone displayed physiological cell size, vegetative growth, spore formation, and S-layer assembly. Strains expressing lcpB1 or lcpB4 displayed defects in cell size and shape, S-layer assembly, and spore formation yet sustained vegetative growth. In contrast, the lcpB2 strain was unable to grow unless the gene was expressed from a multicopy plasmid (lcpB2++), and variants expressing lcpC or lcpD displayed severe defects in growth and cell shape. The lcpB2++, lcpC, or lcpD strains supported neither S-layer assembly nor spore formation. We propose a model whereby LCP enzymes fulfill partially overlapping functions in transferring SCWP molecules to discrete sites within the bacterial envelope.
IMPORTANCE Products of genes essential for bacterial envelope assembly represent targets for antibiotic development. The LytR-CpsA-Psr (LCP) enzymes tether bactoprenol-linked intermediates of secondary cell wall polymers to the C6 hydroxyl of N-acetylmuramic acid in peptidoglycan; however, the role of LCPs as a target for antibiotic therapy is not defined. We show here that LCP enzymes are essential for the cell cycle, vegetative growth, and spore formation of Bacillus anthracis, the causative agent of anthrax disease. Furthermore, we assign functions for each of the six LCP enzymes, including cell size and shape, vegetative growth and sporulation, and S-layer and S-layer-associated protein assembly.
doi:10.1128/JB.00656-15
PMCID: PMC4626891  PMID: 26391207
6.  Bacillus anthracis tagO Is Required for Vegetative Growth and Secondary Cell Wall Polysaccharide Synthesis 
Journal of Bacteriology  2015;197(22):3511-3520.
ABSTRACT
Bacillus anthracis elaborates a linear secondary cell wall polysaccharide (SCWP) that retains surface (S)-layer and associated proteins via their S-layer homology (SLH) domains. The SCWP is comprised of trisaccharide repeats [→4)-β-ManNAc-(1→4)-β-GlcNAc-(1→6)-α-GlcNAc-(1→] and tethered via acid-labile phosphodiester bonds to peptidoglycan. Earlier work identified UDP-GlcNAc 2-epimerases GneY (BAS5048) and GneZ (BAS5117), which act as catalysts of ManNAc synthesis, as well as a polysaccharide deacetylase (BAS5051), as factors contributing to SCWP synthesis. Here, we show that tagO (BAS5050), which encodes a UDP-N-acetylglucosamine:undecaprenyl-P N-acetylglucosaminyl 1-P transferase, the enzyme that initiates the synthesis of murein linkage units, is required for B. anthracis SCWP synthesis and S-layer assembly. Similar to gneY-gneZ mutants, B. anthracis strains lacking tagO cannot maintain cell shape or support vegetative growth. In contrast, mutations in BAS5051 do not affect B. anthracis cell shape, vegetative growth, SCWP synthesis, or S-layer assembly. These data suggest that TagO-mediated murein linkage unit assembly supports SCWP synthesis and attachment to the peptidoglycan via acid-labile phosphodiester bonds. Further, B. anthracis variants unable to synthesize SCWP trisaccharide repeats cannot sustain cell shape and vegetative growth.
IMPORTANCE Bacillus anthracis elaborates an SCWP to support vegetative growth and envelope assembly. Here, we show that some, but not all, SCWP synthesis is dependent on tagO-derived murein linkage units and subsequent attachment of SCWP to peptidoglycan. The data implicate secondary polymer modifications of peptidoglycan and subcellular distributions as a key feature of the cell cycle in Gram-positive bacteria and establish foundations for work on the molecular functions of the SCWP and on inhibitors with antibiotic attributes.
doi:10.1128/JB.00494-15
PMCID: PMC4621081  PMID: 26324447
7.  Bacillus anthracis SlaQ Promotes S-Layer Protein Assembly 
Journal of Bacteriology  2015;197(19):3216-3227.
ABSTRACT
Bacillus anthracis vegetative forms assemble an S-layer comprised of two S-layer proteins, Sap and EA1. A hallmark of S-layer proteins are their C-terminal crystallization domains, which assemble into a crystalline lattice once these polypeptides are deposited on the bacterial surface via association between their N-terminal S-layer homology domains and the secondary cell wall polysaccharide. Here we show that slaQ, encoding a small cytoplasmic protein conserved among pathogenic bacilli elaborating S-layers, is required for the efficient secretion and assembly of Sap and EA1. S-layer protein precursors cosediment with SlaQ, and SlaQ appears to facilitate Sap assembly. Purified SlaQ polymerizes and when mixed with purified Sap promotes the in vitro formation of tubular S-layer structures. A model is discussed whereby SlaQ, in conjunction with S-layer secretion factors SecA2 and SlaP, promotes localized secretion and S-layer assembly in B. anthracis.
IMPORTANCE S-layer proteins are endowed with the propensity for self-assembly into crystalline arrays. Factors promoting S-layer protein assembly have heretofore not been reported. We identified Bacillus anthracis SlaQ, a small cytoplasmic protein that facilitates S-layer protein assembly in vivo and in vitro.
doi:10.1128/JB.00492-15
PMCID: PMC4560277  PMID: 26216847
8.  EsxB, a secreted protein from B acillus anthracis forms two distinct helical bundles 
Abstract
The EsxB protein from Bacillus anthracis belongs to the WXG100 family, a group of proteins secreted by a specialized secretion system. We have determined the crystal structures of recombinant EsxB and discovered that the small protein (∼10 kDa), comprised of a helix‐loop‐helix (HLH) hairpin, is capable of associating into two different helical bundles. The two basic quaternary assemblies of EsxB are an antiparallel (AP) dimer and a rarely observed bisecting U (BU) dimer. This structural duality of EsxB is believed to originate from the heptad repeat sequence diversity of the first helix of its HLH hairpin, which allows for two alternative helix packing. The flexibility of EsxB and the ability to form alternative helical bundles underscore the possibility that this protein can serve as an adaptor in secretion and can form hetero‐oligomeric helix bundle(s) with other secreted members of the WXG100 family, such as EsxW. The highly conserved WXG motif is located within the loop of the HLH hairpin and is mostly buried within the helix bundle suggesting that its role is mainly structural. The exact functions of the motif, including a proposed role as a secretion signal, remain unknown.
PDB Code(s): 4J10
PDB Code(s): 4IYI
PDB Code(s): 4J42
PDB Code(s): 4J41
doi:10.1002/pro.2715
PMCID: PMC4570534  PMID: 26032645
type VII secretion system; ESAT‐6 like secretion system; WXG family; EsxB; helix bundle; antiparallel dimer; bisecting U dimer; tetramer
9.  Protein A-neutralizing monoclonal antibody protects neonatal mice against Staphylococcus aureus 
Vaccine  2014;33(4):523-526.
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.
doi:10.1016/j.vaccine.2014.11.051
PMCID: PMC4378561  PMID: 25488332
Staphylococcus aureus; Staphylococcal protein A; Monoclonal antibody; Neonatal bacteremia and meningitis; Protective immunity; Recurrent infection
10.  Glutamate Racemase Mutants of Bacillus anthracis 
Journal of Bacteriology  2015;197(11):1854-1861.
ABSTRACT
d-Glutamate is an essential component of bacterial peptidoglycan and a building block of the poly-γ-d-glutamic acid (PDGA) capsule of Bacillus anthracis, the causative agent of anthrax. Earlier work suggested that two glutamate racemases, encoded by racE1 and racE2, are each essential for growth of B. anthracis, supplying d-glutamic acid for the synthesis of peptidoglycan and PDGA capsule. Earlier work could not explain, however, why two enzymes that catalyze the same reaction may be needed for bacterial growth. Here, we report that deletion of racE1 or racE2 did not prevent growth of B. anthracis Sterne (pXO1+ pXO2−), the noncapsulating vaccine strain, or of B. anthracis Ames (pXO1+ pXO2+), a fully virulent, capsulating isolate. While mutants with deletions in racE1 and racE2 were not viable, racE2 deletion delayed vegetative growth of B. anthracis following spore germination and caused aberrant cell shapes, phenotypes that were partially restored by exogenous d-glutamate. Deletion of racE1 or racE2 from B. anthracis Ames did not affect the production or stereochemical composition of the PDGA capsule. A model is presented whereby B. anthracis, similar to Bacillus subtilis, utilizes two functionally redundant racemase enzymes to synthesize d-glutamic acid for peptidoglycan synthesis.
IMPORTANCE Glutamate racemases, enzymes that convert l-glutamate to d-glutamate, are targeted for antibiotic development. Glutamate racemase inhibitors may be useful for the treatment of bacterial infections such as anthrax, where the causative agent, B. anthracis, requires d-glutamate for the synthesis of peptidoglycan and poly-γ-d-glutamic acid (PDGA) capsule. Here we show that B. anthracis possesses two glutamate racemase genes that can be deleted without abolishing either bacterial growth or PDGA synthesis. These data indicate that drug candidates must inhibit both glutamate racemases, RacE1 and RacE2, in order to block B. anthracis growth and achieve therapeutic efficacy.
doi:10.1128/JB.00070-15
PMCID: PMC4420906  PMID: 25777674
11.  Staphylococcal manipulation of host immune responses 
Nature reviews. Microbiology  2015;13(9):529-543.
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 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.
doi:10.1038/nrmicro3521
PMCID: PMC4625792  PMID: 26272408
12.  Sec-Secretion and Sortase-Mediated Anchoring of Proteins in Gram-Postive Bacteria 
Biochimica et biophysica acta  2013;1843(8):1687-1697.
Signal peptide-driven secretion of precursor proteins directs polypeptides across the plasma membrane of bacteria. Two pathways, Sec- and SRP-dependent, converge at the SecYEG translocon to thread unfolded precursor proteins across the membrane, whereas folded preproteins are routed via the Tat secretion pathway. Gram-positive bacteria lack an outer membrane and are surrounded by a rigid layer of peptidoglycan. Interactions with their environment are mediated by proteins that are retained in the cell wall, often through covalent attachment to the peptidoglycan. In this review, we describe the mechanisms for both Sec-dependent secretion and sortase-dependent assembly of proteins in the envelope of Gram-positive bacteria.
doi:10.1016/j.bbamcr.2013.11.009
PMCID: PMC4031296  PMID: 24269844
Sec; leader peptide; LPXTG; sortase; cell wall; peptidoglycan
13.  LytR-CpsA-Psr Enzymes as Determinants of Bacillus anthracis Secondary Cell Wall Polysaccharide Assembly 
Journal of Bacteriology  2014;197(2):343-353.
Bacillus anthracis, the causative agent of anthrax, replicates as chains of vegetative cells by regulating the separation of septal peptidoglycan. Surface (S)-layer proteins and associated proteins (BSLs) function as chain length determinants and bind to the secondary cell wall polysaccharide (SCWP). In this study, we identified the B. anthracis lcpD mutant, which displays increased chain length and S-layer assembly defects due to diminished SCWP attachment to peptidoglycan. In contrast, the B. anthracis lcpB3 variant displayed reduced cell size and chain length, which could be attributed to increased deposition of BSLs. In other bacteria, LytR-CpsA-Psr (LCP) proteins attach wall teichoic acid (WTA) and polysaccharide capsule to peptidoglycan. B. anthracis does not synthesize these polymers, yet its genome encodes six LCP homologues, which, when expressed in S. aureus, promote WTA attachment. We propose a model whereby B. anthracis LCPs promote attachment of SCWP precursors to discrete locations in the peptidoglycan, enabling BSL assembly and regulated separation of septal peptidoglycan.
doi:10.1128/JB.02364-14
PMCID: PMC4272586  PMID: 25384480
14.  EsxB, a secreted protein from Bacillus anthracis forms two distinct helical bundles 
The EsxB protein from Bacillus anthracis belongs to the WXG100 family, a group of proteins secreted by a specialized secretion system. We have determined the crystal structures of recombinant EsxB and discovered that the small protein (∼10 kDa), comprised of a helix-loop-helix (HLH) hairpin, is capable of associating into two different helical bundles. The two basic quaternary assemblies of EsxB are an antiparallel (AP) dimer and a rarely observed bisecting U (BU) dimer. This structural duality of EsxB is believed to originate from the heptad repeat sequence diversity of the first helix of its HLH hairpin, which allows for two alternative helix packing. The flexibility of EsxB and the ability to form alternative helical bundles underscore the possibility that this protein can serve as an adaptor in secretion and can form hetero-oligomeric helix bundle(s) with other secreted members of the WXG100 family, such as EsxW. The highly conserved WXG motif is located within the loop of the HLH hairpin and is mostly buried within the helix bundle suggesting that its role is mainly structural. The exact functions of the motif, including a proposed role as a secretion signal, remain unknown.
doi:10.1002/pro.2715
PMCID: PMC4570534  PMID: 26032645
type VII secretion system; ESAT-6 like secretion system; WXG family; EsxB; helix bundle; antiparallel dimer; bisecting U dimer; tetramer
15.  Vaccine Protection of Leukopenic Mice against Staphylococcus aureus Bloodstream Infection 
Infection and Immunity  2014;82(11):4889-4898.
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 SpAKKAA), which was identified by genetic vaccinology using sortase A mutants, raised antigen-specific immune responses that protected leukopenic mice against staphylococcal BSI.
doi:10.1128/IAI.02328-14
PMCID: PMC4249340  PMID: 25183728
16.  GneZ, a UDP-GlcNAc 2-Epimerase, Is Required for S-Layer Assembly and Vegetative Growth of Bacillus anthracis 
Journal of Bacteriology  2014;196(16):2969-2978.
Bacillus anthracis, the causative agent of anthrax, forms an S-layer atop its peptidoglycan envelope and displays S-layer proteins and Bacillus S-layer-associated (BSL) proteins with specific functions to support cell separation of vegetative bacilli and growth in infected mammalian hosts. S-layer and BSL proteins bind via the S-layer homology (SLH) domain to the pyruvylated secondary cell wall polysaccharide (SCWP) with the repeat structure [→4)-β-ManNAc-(1→4)-β-GlcNAc-(1→6)-α-GlcNAc-(1→]n, where α-GlcNAc and β-GlcNAc are substituted with two and one galactosyl residues, respectively. B. anthracis gneY (BAS5048) and gneZ (BAS5117) encode nearly identical UDP-GlcNAc 2-epimerase enzymes that catalyze the reversible conversion of UDP-GlcNAc and UDP-ManNAc. UDP-GlcNAc 2-epimerase enzymes have been shown to be required for the attachment of the phage lysin PlyG with the bacterial envelope and for bacterial growth. Here, we asked whether gneY and gneZ are required for the synthesis of the pyruvylated SCWP and for S-layer assembly. We show that gneZ, but not gneY, is required for B. anthracis vegetative growth, rod cell shape, S-layer assembly, and synthesis of pyruvylated SCWP. Nevertheless, inducible expression of gneY alleviated all the defects associated with the gneZ mutant. In contrast to vegetative growth, neither germination of B. anthracis spores nor the formation of spores in mother cells required UDP-GlcNAc 2-epimerase activity.
doi:10.1128/JB.01829-14
PMCID: PMC4135639  PMID: 24914184
17.  Protein A Suppresses Immune Responses during Staphylococcus aureus Bloodstream Infection in Guinea Pigs 
mBio  2015;6(1):e02369-14.
ABSTRACT  
Staphylococcus aureus infection is not associated with the development of protective immunity, and disease relapses occur frequently. We hypothesize that protein A, a factor that binds immunoglobulin Fcγ and cross-links VH3 clan B cell receptors (IgM), is the staphylococcal determinant for host immune suppression. To test this, vertebrate IgM was examined for protein A cross-linking. High VH3 binding activity occurred with human and guinea immunoglobulin, whereas mouse and rabbit immunoglobulins displayed little and no binding, respectively. Establishing a guinea pig model of S. aureus bloodstream infection, we show that protein A functions as a virulence determinant and suppresses host B cell responses. Immunization with SpAKKAA, which cannot bind immunoglobulin, elicits neutralizing antibodies that enable guinea pigs to develop protective immunity.
Importance  Staphylococcus aureus is the leading cause of soft tissue and bloodstream infections; however, a vaccine with clinical efficacy is not available. Using mice to model staphylococcal infection, earlier work identified protective antigens; however, corresponding human clinical trials did not reach their endpoints. We show that B cell receptor (IgM) cross-linking by protein A is an important immune evasion strategy of S. aureus that can be monitored in a guinea pig model of bloodstream infection. Further, immunization with nontoxigenic protein A enables infected guinea pigs to elicit antibody responses that are protective against S. aureus. Thus, the guinea pig model may support preclinical development of staphylococcal vaccines.
IMPORTANCE 
Staphylococcus aureus is the leading cause of soft tissue and bloodstream infections; however, a vaccine with clinical efficacy is not available. Using mice to model staphylococcal infection, earlier work identified protective antigens; however, corresponding human clinical trials did not reach their endpoints. We show that B cell receptor (IgM) cross-linking by protein A is an important immune evasion strategy of S. aureus that can be monitored in a guinea pig model of bloodstream infection. Further, immunization with nontoxigenic protein A enables infected guinea pigs to elicit antibody responses that are protective against S. aureus. Thus, the guinea pig model may support preclinical development of staphylococcal vaccines.
doi:10.1128/mBio.02369-14
PMCID: PMC4313907  PMID: 25564466
18.  Secretion of atypical protein subtrates by the ESAT-6 Secretion System of Staphylococcus aureus 
Molecular microbiology  2013;90(4):734-743.
SUMMARY
Staphylococcus aureus encodes the specialized ESAT-6 Secretion System (ESS). EsxA and EsxB are secreted by the ESS pathway, and share sequence features of ESAT-6 and CFP-10 of the Type VII Secretion System (T7SS) of Mycobacterium tuberculosis. Unlike ESAT-6 and CFP-10, EsxA and EsxB do not interact. Instead, EsxB associates with a novel substrate, EsxD, and EsxA dimerizes with itself or EsxC (EsaC). Unlike EsxA and EsxB, EsxC and EsxD do not share obvious sequence features of WXG100 proteins nor PE/PPE and Esp families of proteins, all of which belong to the pfam EsxAB clan of mycobacterial T7SS. EsxD carries the C terminal motif YxxxD/E that has been proposed to target T7 substrates for secretion in mycobacteria. Here, we find that deletion, but not amino acid substitutions, in this motif prevent secretion of EsxA and EsxC but not EsxB or EsxD. This is unlike the genetic inactivation of esxA, esxB, esxC or esxD that leads to loss of secretion of all four substrates. Thus, substrate secretion can be uncoupled by deleting the last six amino acids of EsxD. The physical association of EsxC and EsxD with canonical WXG100 proteins suggests that these proteins belong to the EsxAB clan.
doi:10.1111/mmi.12395
PMCID: PMC3951145  PMID: 24033479
19.  The Giant Protein Ebh Is a Determinant of Staphylococcus aureus Cell Size and Complement Resistance 
Journal of Bacteriology  2014;196(5):971-981.
Staphylococcus aureus USA300, the clonal type associated with epidemic community-acquired methicillin-resistant S. aureus (MRSA) infections, displays the giant protein Ebh on its surface. Mutations that disrupt the ebh reading frame increase the volume of staphylococcal cells and alter the cross wall, a membrane-enclosed peptidoglycan synthesis and assembly compartment. S. aureus ebh variants display increased sensitivity to oxacillin (methicillin) as well as susceptibility to complement-mediated killing. Mutations in ebh are associated with reduced survival of mutant staphylococci in blood and diminished virulence in mice. We propose that Ebh, following its secretion into the cross wall, contributes to the characteristic cell growth and envelope assembly pathways of S. aureus, thereby enabling complement resistance and the pathogenesis of staphylococcal infections.
doi:10.1128/JB.01366-13
PMCID: PMC3957702  PMID: 24363342
20.  Lipoteichoic Acids, Phosphate-Containing Polymers in the Envelope of Gram-Positive Bacteria 
Journal of Bacteriology  2014;196(6):1133-1142.
Lipoteichoic acids (LTA) are polymers of alternating units of a polyhydroxy alkane, including glycerol and ribitol, and phosphoric acid, joined to form phosphodiester units that are found in the envelope of Gram-positive bacteria. Here we review four different types of LTA that can be distinguished on the basis of their chemical structure and describe recent advances in the biosynthesis pathway for type I LTA, d-alanylated polyglycerol-phosphate linked to di-glucosyl-diacylglycerol. The physiological functions of type I LTA are discussed in the context of inhibitors that block their synthesis and of mutants with discrete synthesis defects. Research on LTA structure and function represents a large frontier that has been investigated in only few Gram-positive bacteria.
doi:10.1128/JB.01155-13
PMCID: PMC3957714  PMID: 24415723
21.  Staphylococcus aureus degrades neutrophil extracellular traps to promote immune cell death 
Science (New York, N.Y.)  2013;342(6160):863-866.
Bacterial invasion of host tissues triggers polymorphonuclear leukocytes to release DNA (NETs, neutrophil extracellular traps), thereby immobilizing microbes for subsequent clearance by innate defenses including macrophage phagocytosis. We report here that Staphylococcus aureus escapes these defenses by converting NETs to deoxyadenosine, which triggers the caspase-3 mediated death of immune cells. Conversion of NETs to deoxyadenosine requires two enzymes, nuclease and adenosine synthase, that are secreted by S. aureus and necessary for the exclusion of macrophages from staphylococcal abscesses. Thus, the pathogenesis of S. aureus infections has evolved to anticipate host defenses and to repurpose them for the destruction of the immune system.
doi:10.1126/science.1242255
PMCID: PMC4026193  PMID: 24233725
22.  Staphylococcus aureus Mutants Lacking the LytR-CpsA-Psr Family of Enzymes Release Cell Wall Teichoic Acids into the Extracellular Medium 
Journal of Bacteriology  2013;195(20):4650-4659.
The LytR-CpsA-Psr (LCP) proteins are thought to transfer bactoprenol-linked biosynthetic intermediates of wall teichoic acid (WTA) to the peptidoglycan of Gram-positive bacteria. In Bacillus subtilis, mutants lacking all three LCP enzymes do not deposit WTA in the envelope, while Staphylococcus aureus Δlcp mutants display impaired growth and reduced levels of envelope phosphate. We show here that the S. aureus Δlcp mutant synthesized WTA yet released ribitol phosphate polymers into the extracellular medium. Further, Δlcp mutant staphylococci no longer restricted the deposition of LysM-type murein hydrolases to cell division sites, which was associated with defects in cell shape and increased autolysis. Mutations in S. aureus WTA synthesis genes (tagB, tarF, or tarJ2) inhibit growth, which is attributed to the depletion of bactoprenol, an essential component of peptidoglycan synthesis (lipid II). The growth defect of S. aureus tagB and tarFJ mutants was alleviated by inhibition of WTA synthesis with tunicamycin, whereas the growth defect of the Δlcp mutant was not relieved by tunicamycin treatment or by mutation of tagO, whose product catalyzes the first committed step of WTA synthesis. Further, sortase A-mediated anchoring of proteins to peptidoglycan, which also involves bactoprenol and lipid II, was not impaired in the Δlcp mutant. We propose a model whereby the S. aureus Δlcp mutant, defective in tethering WTA to the cell wall, cleaves WTA synthesis intermediates, releasing ribitol phosphate into the medium and recycling bactoprenol for peptidoglycan synthesis.
doi:10.1128/JB.00544-13
PMCID: PMC3807444  PMID: 23935043
23.  Bacillus anthracis Acetyltransferases PatA1 and PatA2 Modify the Secondary Cell Wall Polysaccharide and Affect the Assembly of S-Layer Proteins 
Journal of Bacteriology  2013;195(5):977-989.
The envelope of Bacillus anthracis encompasses a proteinaceous S-layer with two S-layer proteins (Sap and EA1). Protein assembly in the envelope of B. anthracis requires S-layer homology domains (SLH) within S-layer proteins and S-layer-associated proteins (BSLs), which associate with the secondary cell wall polysaccharide (SCWP), an acetylated carbohydrate that is tethered to peptidoglycan. Here, we investigated the contributions of two putative acetyltransferases, PatA1 and PatA2, on SCWP acetylation and S-layer assembly. We show that mutations in patA1 and patA2 affect the chain lengths of B. anthracis vegetative forms and perturb the deposition of the BslO murein hydrolase at cell division septa. The patA1 and patA2 mutants are defective for the assembly of EA1 in the envelope but retain the ability of S-layer formation with Sap. SCWP isolated from the patA1 patA2 mutant lacked acetyl moieties identified in wild-type polysaccharide and failed to associate with the SLH domains of EA1. A model is discussed whereby patA1- and patA2-mediated acetylation of SCWP enables the deposition of EA1 as well as BslO near the septal region of the B. anthracis envelope.
doi:10.1128/JB.01274-12
PMCID: PMC3571321  PMID: 23243307
24.  Vaccine Protection against Bacillus cereus-Mediated Respiratory Anthrax-Like Disease in Mice 
Infection and Immunity  2013;81(3):1008-1017.
Bacillus cereus strains harboring a pXO1-like virulence plasmid cause respiratory anthrax-like disease in humans, particularly in welders. We developed mouse models for intraperitoneal as well as aerosol challenge with spores of B. cereus G9241, harboring pBCXO1 and pBC218 virulence plasmids. Compared to wild-type B. cereus G9241, spores with a deletion of the pBCXO1-carried protective antigen gene (pagA1) were severely attenuated, whereas spores with a deletion of the pBC218-carried protective antigen homologue (pagA2) were not. Anthrax vaccine adsorbed (AVA) immunization raised antibodies that bound and neutralized the pagA1-encoded protective antigen (PA1) but not the PA2 orthologue encoded by pagA2. AVA immunization protected mice against a lethal challenge with spores from B. cereus G9241 or B. cereus Elc4, a strain that had been isolated from a fatal case of anthrax-like disease. As the pathogenesis of B. cereus anthrax-like disease in mice is dependent on pagA1 and PA-neutralizing antibodies provide protection, AVA immunization may also protect humans from respiratory anthrax-like death.
doi:10.1128/IAI.01346-12
PMCID: PMC3584855  PMID: 23319564
25.  Role of Protein A in the Evasion of Host Adaptive Immune Responses by Staphylococcus aureus 
mBio  2013;4(5):e00575-13.
ABSTRACT
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 VH3-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 spaKKAA 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 spaKKAA 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 VH3-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.
doi:10.1128/mBio.00575-13
PMCID: PMC3760252  PMID: 23982075

Results 1-25 (58)