Publications by authors named "Olaf Schneewind"

223 Publications

Peptidoglycan Contribution to the B Cell Superantigen Activity of Staphylococcal Protein A.

mBio 2021 04 20;12(2). Epub 2021 Apr 20.

Howard Taylor Ricketts Laboratory, Argonne National Laboratory, Lemont, Illinois, USA

causes reiterative and chronic persistent infections. This can be explained by the formidable ability of this pathogen to escape immune surveillance mechanisms. Cells of 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 (V3) idiotype (IgM) B cell receptors, promoting B cell expansion and the secretion of nonspecific V3-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. 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. Plants recognize microbe-associated molecular patterns such as chitin via receptors endowed with LysM-containing ectodomains. In plants, such receptors play equally important roles in defense and symbiosis signaling. SpA of carries a LysM domain that binds glycan strands of peptidoglycan to influence defined B cell responses that divert pathogen-specific adaptive immune responses.
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http://dx.doi.org/10.1128/mBio.00039-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8092194PMC
April 2021

Regulated cleavage of glycan strands by the murein hydrolase SagB in involves a direct interaction with LyrA (SpdC).

J Bacteriol 2021 Feb 16. Epub 2021 Feb 16.

Department of Microbiology, University of Chicago, Chicago, Illinois, USA

LyrA (SpdC), a homologue of eukaryotic CAAX proteases that act on prenylated substrates, has been implicated in the assembly of several pathways of the envelope of We described earlier the sostaphin esistance (Lyr) and taphylococcal rotein A isplay (Spd) phenotypes associated with loss of the () gene. However, a direct contribution to the assembly of pentaglycine crossbridges, the target of lysostaphin cleavage in peptidoglycan, or of Staphylococcal protein A attachment to peptidoglycan could not be attributed directly to LyrA (SpdC). These two processes are catalyzed by the Fem factors and Sortase A, respectively. To gain insight into the function of LyrA (SpdC), here we use affinity chromatography and LC-MS/MS analysis and report that LyrA interacts with SagB. SagB cleaves glycan strands of peptidoglycan to achieve physiological length. Similar to peptidoglycan, peptidoglycan contains extended glycan strands. Purified peptidoglycan can still be cleaved to physiological length by SagB LyrA does not modify or cleave peptidoglycan, it also does not modify or stabilize SagB. The membrane bound domain of LyrA is sufficient to support SagB activity but predicted 'CAAX enzyme' catalytic residues in this domain are dispensable. We speculate that LyrA exerts its effect on bacterial prenyl substrates, specifically undecaprenol-bound peptidoglycan substrates of SagB, to help control glycan length. Such an activity also explains the Lyr and Spd phenotypes observed earlier. Peptidoglycan is assembled on the side of the plasma membrane from lipid II precursors into glycan chains that are crosslinked at stem peptides. In , SagB, a membrane-associated N-acetylglucosaminidase, cleaves polymerized glycan chains to their physiological length. Deletion of is associated with longer glycan strands in peptidoglycan, altered protein trafficking and secretion in the envelope, and aberrant excretion of cytosolic proteins. It is not clear whether SagB, with its single transmembrane segment, serves as the molecular ruler of glycan chains or whether other factors modulate its activity. Here, we show that LyrA (SpdC), a protein of the CAAX type II prenyl endopeptidase family, modulates SagB activity via interaction though its transmembrane domain.
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http://dx.doi.org/10.1128/JB.00014-21DOI Listing
February 2021

The Expression of von Willebrand Factor-Binding Protein Determines Joint-Invading Capacity of Staphylococcus aureus, a Core Mechanism of Septic Arthritis.

mBio 2020 11 17;11(6). Epub 2020 Nov 17.

Department of Rheumatology and Inflammation Research, Institution of Medicine, Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden

Septic arthritis, one of the most dangerous joint diseases, is predominantly caused by In contrast, coagulase-negative staphylococci are rarely found in septic arthritis. We hypothesize that coagulases released by , including coagulase (Coa) and von Willebrand factor-binding protein (vWbp), play potent roles in the induction of septic arthritis. Four isogenic strains differing in expression of coagulases (wild-type [WT] Newman, Δ, Δ, and Δ Δ) were used to induce septic arthritis in both wild-type and von Willebrand factor (vWF)-deficient mice. Septic arthritis severity was greatly reduced when wild-type mice were infected with the Δ Δ and Δ variants compared to WT or Δ strains, suggesting that vWbp rather than Coa is a major virulence factor in septic arthritis. vWF-deficient mice were more susceptible to bone damage in septic arthritis, especially when the Δ strain was used. Importantly, no difference in arthritis severity between the Δ and WT strains was observed in vWF-deficient mice. Collectively, we conclude that vWbp production by enhances staphylococcal septic arthritis. Septic arthritis remains one of the most dangerous joint diseases with a rapidly progressive disease character. Despite advances in the use of antibiotics, permanent reductions in joint function due to joint deformation and deleterious contractures occur in up to 50% of patients with septic arthritis. So far, it is still largely unknown how initiates and establishes joint infection. Here, we demonstrate that von Willebrand factor-binding protein expressed by facilitates the initiation of septic arthritis. Such effect might be mediated through its interaction with a host factor (von Willebrand factor). Our finding contributes significantly to the full understanding of septic arthritis etiology and will pave the way for new therapeutic modalities for this devastating disease.
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http://dx.doi.org/10.1128/mBio.02472-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7683397PMC
November 2020

Glycosylation-dependent opsonophagocytic activity of staphylococcal protein A antibodies.

Proc Natl Acad Sci U S A 2020 09 27;117(37):22992-23000. Epub 2020 Aug 27.

Howard Taylor Ricketts Laboratory, Argonne National Laboratory, Lemont, IL 60439;

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 , secrete numerous toxins and evolved multiple mechanisms to escape opsonization and complement killing. Several vaccine candidates targeting defined surface antigens of 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.
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http://dx.doi.org/10.1073/pnas.2003621117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7502815PMC
September 2020

FmhA and FmhC of incorporate serine residues into peptidoglycan cross-bridges.

J Biol Chem 2020 09 5;295(39):13664-13676. Epub 2020 Aug 5.

Department of Microbiology, University of Chicago, Chicago, Illinois, USA; Howard Taylor Ricketts Laboratory, Argonne National Laboratory, Lemont, Illinois, USA. Electronic address:

Staphylococcal peptidoglycan is characterized by pentaglycine cross-bridges that are cross-linked between adjacent wall peptides by penicillin-binding proteins to confer robustness and flexibility. In , pentaglycine cross-bridges are synthesized by three proteins: FemX adds the first glycine, and the homodimers FemA and FemB sequentially add two Gly-Gly dipeptides. Occasionally, serine residues are also incorporated into the cross-bridges by enzymes that have heretofore not been identified. Here, we show that the FemA/FemB homologues FmhA and FmhC pair with FemA and FemB to incorporate Gly-Ser dipeptides into cross-bridges and to confer resistance to lysostaphin, a secreted bacteriocin that cleaves the pentaglycine cross-bridge. FmhA incorporates serine residues at positions 3 and 5 of the cross-bridge. In contrast, FmhC incorporates a single serine at position 5. Serine incorporation also lowers resistance toward oxacillin, an antibiotic that targets penicillin-binding proteins, in both methicillin-sensitive and methicillin-resistant strains of FmhC is encoded by a gene immediately adjacent to lytN, which specifies a hydrolase that cleaves the bond between the fifth glycine of cross-bridges and the alanine of the adjacent stem peptide. In this manner, LytN facilitates the separation of daughter cells. Cell wall damage induced upon lytN overexpression can be alleviated by overexpression of fmhC. Together, these observations suggest that FmhA and FmhC generate peptidoglycan cross-bridges with unique serine patterns that provide protection from endogenous murein hydrolases governing cell division and from bacteriocins produced by microbial competitors.
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http://dx.doi.org/10.1074/jbc.RA120.014371DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7521636PMC
September 2020

Distinct Pathways Carry Out α and β Galactosylation of Secondary Cell Wall Polysaccharide in Bacillus anthracis.

J Bacteriol 2020 07 9;202(15). Epub 2020 Jul 9.

Howard Taylor Ricketts Laboratory, Argonne National Laboratory, Lemont, Illinois, USA

, the causative agent of anthrax disease, elaborates a secondary cell wall polysaccharide (SCWP) that is required for the retention of surface layer (S-layer) and S-layer homology (SLH) domain proteins. Genetic disruption of the SCWP biosynthetic pathway impairs growth and cell division. SCWP is comprised of trisaccharide repeats composed of one ManNAc and two GlcNAc residues with O-3-α-Gal and O-4-β-Gal substitutions. UDP-Gal, synthesized by GalE1, is the substrate of galactosyltransferases that modify the SCWP repeat. Here, we show that the gene, which encodes a predicted glycosyltransferase with a GT-A fold, is required for O-4-β-Gal modification of trisaccharide repeats. We identify a DXD motif critical for GtsE activity. Three distinct genes, , , and , are required for O-3-α-Gal modification of trisaccharide repeats. Based on the similarity with other three-component glycosyltransferase systems, we propose that GtsA transfers Gal from cytosolic UDP-Gal to undecaprenyl phosphate (C-P), GtsB flips the C-P-Gal intermediate to the side of the membrane, and GtsC transfers Gal onto trisaccharide repeats. The deletion of does not affect growth , suggesting that galactosyl modifications are dispensable for the function of SCWP. The deletion of , , or leads to a loss of viability, yet and can be deleted in strains lacking or We propose that the loss of viability is caused by the accumulation of undecaprenol-bound precursors and present an updated model for SCWP assembly in to account for the galactosylation of repeat units. Peptidoglycan is a conserved extracellular macromolecule that protects bacterial cells from turgor pressure. Peptidoglycan of Gram-positive bacteria serves as a scaffold for the attachment of polymers that provide defined bacterial interactions with their environment. One such polymer, SCWP, is pyruvylated at its distal end to serve as a receptor for secreted proteins bearing the S-layer homology domain. Repeat units of SCWP carry three galactoses in Glycosylation is a recurring theme in nature and often represents a means to mask or alter conserved molecular signatures from intruders such as bacteriophages. Several glycosyltransferase families have been described based on bioinformatics prediction, but few have been studied. Here, we describe the glycosyltransferases that mediate the galactosylation of SCWP.
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http://dx.doi.org/10.1128/JB.00191-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7348550PMC
July 2020

Rapid pathogen identification and antimicrobial susceptibility testing in in vitro endophthalmitis with matrix assisted laser desorption-ionization Time-of-Flight Mass Spectrometry and VITEK 2 without prior culture.

PLoS One 2019 30;14(12):e0227071. Epub 2019 Dec 30.

Department of Ophthalmology and Visual Science, The University of Chicago Hospitals and Health System, Chicago, Illinois, United States of America.

Purpose: Prompt clinical diagnosis and initiation of treatment are critical in the management of infectious endophthalmitis. Current methods used to identify causative agents of infectious endophthalmitis are mostly inefficient, owing to suboptimal sensitivity, length, and cost. Matrix Assisted Laser Desorption-Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) can be used to rapidly identity pathogens without a need for culture. Similarly, automated antimicrobial susceptibility test systems (AST, VITEK 2) provide accurate antimicrobial susceptibility profiles. In this proof-of-concept study, we apply these technologies for the direct identification and characterization of pathogens in vitreous samples, without culture, as an in vitro model of infectious endophthalmitis.

Methods: Vitreous humor aspirated from freshly enucleated porcine eyes was inoculated with different inocula of Staphylococcus aureus (S. aureus) and incubated at 37°C. Vitreous endophthalmitis samples were centrifuged and pellets were directly analyzed with MALDI-TOF MS and VITEK 2 without prior culture. S. aureus colonies that were conventionally grown on culture medium were used as control samples. Time-to-identification, minimum concentration of bacteria required for identification, and accuracy of results compared to standard methods were determined.

Results: MALDI-TOF MS achieved accurate pathogen identification from direct analysis of intraocular samples with confidence values of up to 99.9%. Time from sample processing to pathogen identification was <30 minutes. The minimum number of bacteria needed for positive identification was 7.889x106 colony forming units (cfu/μl). Direct analysis of intraocular samples with VITEK 2 gave AST profiles that were up to 94.4% identical to the positive control S. aureus analyzed per standard protocol.

Conclusion: Our findings demonstrate that the direct analysis of vitreous samples with MALDI-TOF MS and VITEK 2 without prior culture could serve as new, improved methods for rapid, accurate pathogen identification and targeted treatment design in infectious endophthalmitis. In vivo models and standardized comparisons against other microbiological methods are needed to determine the value of direct analysis of intraocular samples from infectious endophthalmitis with MALDI-TOF MS and VITEK 2.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0227071PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6936829PMC
April 2020

Staphylococcus aureus Exploits the Host Apoptotic Pathway To Persist during Infection.

mBio 2019 11 12;10(6). Epub 2019 Nov 12.

Department of Microbiology, University of Chicago, Chicago, Illinois, USA

is a deadly pathogen that causes fatal diseases in humans. During infection, secretes nuclease (Nuc) and adenosine synthase A (AdsA) to generate cytotoxic deoxyadenosine (dAdo) from neutrophil extracellular traps which triggers noninflammatory apoptosis in macrophages. In this manner, replicating staphylococci escape phagocytic killing without alerting the immune system. Here, we show that mice lacking caspase-3 in immune cells exhibit increased resistance toward Caspase-3-deficient macrophages are resistant to staphylococcal dAdo and gain access to abscess lesions to promote bacterial clearance in infected animals. We identify specific single nucleotide polymorphisms in as candidate human resistance alleles that protect macrophages from -derived dAdo, raising the possibility that the allelic repertoire of caspase-3 may contribute to the outcome of infections in humans. Caspase-3 controls the apoptotic pathway, a form of programmed cell death designed to be immunologically silent. Polymorphisms leading to reduced caspase-3 activity are associated with variable effects on tumorigenesis and yet arise frequently. is a human commensal and a frequent cause of soft tissue and bloodstream infections. Successful commensalism and virulence can be explained by the secretion of a plethora of immune evasion factors. One such factor, AdsA, destroys phagocytic cells by exploiting the apoptotic pathway. However, human variants with loss-of-function alleles shield phagocytes from AdsA-mediated killing. This finding raises the possibility that some caspase-3 alleles may arise from exposure to and other human pathogens that exploit the apoptotic pathway for infection.
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http://dx.doi.org/10.1128/mBio.02270-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6851280PMC
November 2019

FPR1 is the plague receptor on host immune cells.

Nature 2019 10 18;574(7776):57-62. Epub 2019 Sep 18.

Howard Taylor Ricketts Laboratory, Argonne National Laboratory, Lemont, IL, USA.

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 FPR1 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.
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http://dx.doi.org/10.1038/s41586-019-1570-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6776691PMC
October 2019

B cell superantigens in the human intestinal microbiota.

Sci Transl Med 2019 08;11(507)

Committee on Immunology, University of Chicago, Chicago, IL 60637, USA.

IgA is prominently secreted at mucosal surfaces and coats a fraction of the commensal microbiota, a process that is critical for intestinal homeostasis. However, the mechanisms of IgA induction and the molecular targets of these antibodies remain poorly understood, particularly in humans. Here, we demonstrate that microbiota from a subset of human individuals encode two protein "superantigens" expressed on the surface of commensal bacteria of the family Lachnospiraceae such as that bind IgA variable regions and stimulate potent IgA responses in mice. These superantigens stimulate B cells expressing human VH3 or murine VH5/6/7 variable regions and subsequently bind their antibodies, allowing these microbial organisms to become highly coated with IgA in vivo. These findings demonstrate a previously unappreciated role for commensal superantigens in host-microbiota interactions. Furthermore, as superantigen-expressing strains show an uneven distribution across human populations, they should be systematically considered in studies evaluating human B cell responses and microbiota during homeostasis and disease.
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http://dx.doi.org/10.1126/scitranslmed.aau9356DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6758550PMC
August 2019

O antigen is the target of bactericidal Weil-Felix antibodies.

Proc Natl Acad Sci U S A 2019 09 14;116(39):19659-19664. Epub 2019 Aug 14.

Howard Taylor Ricketts Laboratory, Argonne National Laboratory, Lemont, IL 60649.

Rickettsial diseases have long been diagnosed with serum antibodies cross-reactive against (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 , isolating variants defective for replication in cultured cells and in spotted fever pathogenesis. Mutations in the () abolish lipopolysaccharide O-antigen synthesis and Weil-Felix serology and alter outer-membrane protein assembly. Unlike wild-type , mutants cannot elicit bactericidal antibodies that bind O antigen. The 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.
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http://dx.doi.org/10.1073/pnas.1911922116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6765297PMC
September 2019

Staphylococcal Protein Secretion and Envelope Assembly.

Microbiol Spectr 2019 07;7(4)

Department of Microbiology, University of Chicago, Chicago, IL 60637.

The highly cross-linked peptidoglycan represents the rigid layer of the bacterial envelope and protects bacteria from osmotic lysis. In Gram-positive bacteria, peptidoglycan also functions as a scaffold for the immobilization of capsular polysaccharide, wall teichoic acid (WTA), and surface proteins. This chapter captures recent development on the assembly of the envelope of including mechanisms accounting for immobilization of molecules to peptidoglycan as well as hydrolysis of peptidoglycan for the specific release of bound molecules, facilitation of protein secretion across the envelope and cell division. Peptidoglycan, WTA and capsular polysaccharide are directly synthesized onto undecaprenol. Surface proteins are anchored by Sortase A, a membrane-embedded transpeptidase that scans secreted polypeptides for the C-terminal LPXTG motif of sorting signals. The resulting acyl enzyme intermediate is resolved by lipid II, the undecaprenol-bound peptidoglycan precursor. While these pathways share membrane diffusible undecaprenol, assembly of these molecules occurs either at the cross-walls or the cell poles. In , the cross-wall represents the site of peptidoglycan synthesis which is eventually split to complete the cell cycle yielding newly divided daughter cells. Peptidoglycan synthesized at the cross-wall is initially devoid of WTA. Conversely, lipoteichoic acid (LTA) synthesis which does not require bactoprenol is seemingly restricted to septal membranes. Similarly, distinguishes two types of surface protein precursors. Polypeptides with canonical signal peptides are deposited at the cell poles, whereas precursors with conserved YSIRK-GXXS motif signal peptides traffic to the cross-wall. A model for protein trafficking in the envelope and uneven distribution of teichoic acids is discussed.
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http://dx.doi.org/10.1128/microbiolspec.GPP3-0070-2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7028390PMC
July 2019

Staphylococcus aureus endocarditis: distinct mechanisms of bacterial adhesion to damaged and inflamed heart valves.

Eur Heart J 2019 10;40(39):3248-3259

Department of Cardiovascular Sciences, Center for Molecular and Vascular Biology, KU Leuven, Herestraat 49, Leuven, Belgium.

Aims: The pathogenesis of endocarditis is not well understood resulting in unsuccessful attempts at prevention. Clinical observations suggest that Staphylococcus aureus infects either damaged or inflamed heart valves. Using a newly developed endocarditis mouse model, we therefore studied the initial adhesion of S. aureus in both risk states.

Methods And Results: Using 3D confocal microscopy, we examined the adhesion of fluorescent S. aureus to murine aortic valves. To mimic different risk states we either damaged the valves with a surgically placed catheter or simulated valve inflammation by local endothelium activation. We used von Willebrand factor (VWF) gene-deficient mice, induced platelet and fibrinogen depletion and used several S. aureus mutant strains to investigate the contribution of both host and bacterial factors in early bacterial adhesion. Both cardiac valve damage and inflammation predisposed to endocarditis, but by distinct mechanisms. Following valve damage, S. aureus adhered directly to VWF and fibrin, deposited on the damaged valve. This was mediated by Sortase A-dependent adhesins such as VWF-binding protein and Clumping factor A. Platelets did not contribute. In contrast, upon cardiac valve inflammation, widespread endothelial activation led to endothelial cell-bound VWF release. This recruited large amounts of platelets, capturing S. aureus to the valve surface. Here, neither fibrinogen, nor Sortase A were essential.

Conclusion: Cardiac valve damage and inflammation predispose to S. aureus endocarditis via distinct mechanisms. These findings may have important implications for the development of new preventive strategies, as some interventions might be effective in one risk state, but not in the other.
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http://dx.doi.org/10.1093/eurheartj/ehz175DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7963134PMC
October 2019

Extraction and Purification of Wall-Bound Polymers of Gram-Positive Bacteria.

Methods Mol Biol 2019 ;1954:47-57

Howard Taylor Ricketts Laboratory, Argonne National Laboratory, Lemont, IL, USA.

The envelope of gram-positive bacteria encompasses the cell wall, a rigid exoskeleton comprised of peptidoglycan that provides protection against lysis and governs bacterial cell shapes. Peptidoglycan also serves as the site of attachment for proteins and nonproteinaceous polymers that interact with the bacterial environment. Nonproteinaceous molecules include teichoic acids, capsular polysaccharides, and secondary cell wall polysaccharides (SCWP). Treatment of gram-positive bacterial cells with proteases, nucleases, and detergents results in the isolation of "murein sacculi" (i.e., peptidoglycan with bound carbohydrate polymers). Incubation of sacculi with acid or base releases carbohydrate polymers that can be purified for further biochemical characterization. This protocol describes the hydrofluoric acid extraction and purification of the secondary cell wall polymer of Bacillus anthracis that is also found in the envelope of the other members of the Bacillus cereus sensu lato group of bacteria.
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http://dx.doi.org/10.1007/978-1-4939-9154-9_5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7206762PMC
July 2019

Sortases, Surface Proteins, and Their Roles in Disease and Vaccine Development.

Microbiol Spectr 2019 01;7(1)

Department of Microbiology, University of Chicago, Chicago, IL 60637.

Sortases cleave short peptide motif sequences at the C-terminal end of secreted surface protein precursors and either attach these polypeptides to the peptidoglycan of Gram-positive bacteria or promote their assembly into pilus structures that are also attached to peptidoglycan. Sortase A, the enzyme first identified in the human pathogen , binds LPXTG motif sorting signals, cleaves between threonine (T) and glycine (G) residues, and forms an acyl enzyme between its active-site cysteine thiol and the carboxyl group of threonine (T). Sortase A acyl enzyme is relieved by the nucleophilic attack of the cross bridge amino group within lipid II, thereby generating surface protein linked to peptidoglycan precursor. Such products are subsequently incorporated into the cell wall envelope by enzymes of the peptidoglycan synthesis pathway. Surface proteins linked to peptidoglycan may be released from the bacterial envelope to diffuse into host tissues and fulfill specific biological functions. sortase A is essential for host colonization and for the pathogenesis of invasive diseases. Staphylococcal sortase-anchored surface proteins fulfill key functions during the infectious process, and vaccine-induced antibodies targeting surface proteins may provide protection against . Alternatively, small-molecule inhibitors of sortase may be useful agents for the prevention of colonization and invasive disease.
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http://dx.doi.org/10.1128/microbiolspec.PSIB-0004-2018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6386163PMC
January 2019

Staphylococcus aureus Decolonization of Mice With Monoclonal Antibody Neutralizing Protein A.

J Infect Dis 2019 02;219(6):884-888

Department of Microbiology, University of Chicago, Illinois.

Staphylococcus aureus persistently colonizes the nasopharynx of about one-third of the human population, a key risk factor for community- and hospital-acquired invasive infections. Current strategies for S. aureus decolonization include topical and systemic administration of antibiotics, which is associated with selection for antibiotic resistance and posttreatment recolonization. Using a mouse model for S. aureus colonization, we show here that systemic administration of a recombinant monoclonal antibody neutralizing staphylococcal protein A (SpA) can stimulate antibacterial immunoglobulin G and immunoglobulin A responses and promote S. aureus decolonization. These results suggest that antibody neutralizing SpA, a B-cell superantigen, may also be useful for S. aureus decolonization in humans.
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http://dx.doi.org/10.1093/infdis/jiy597DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7184913PMC
February 2019

Marginal role of von Willebrand factor-binding protein and coagulase in the initiation of endocarditis in rats with catheter-induced aortic vegetations.

Virulence 2018 ;9(1):1615-1624

a Department of Fundamental Microbiology , University of Lausanne , Lausanne , Switzerland.

Staphylococcus aureus is the leading cause of infective endocarditis (IE). While the role of S. aureus cell-wall associated protein clumping factor A (ClfA) in promoting IE has been already demonstrated, that of the secreted plasma-clotting factors staphylocoagulase (Coa) and von Willebrand factor-binding protein (vWbp) has not yet been elucidated. We investigated the role of Coa and vWbp in IE initiation in rats with catheter-induced aortic vegetations, using Lactococcus lactis expressing coa, vWbp, clfA or vWbp/clfA, and S. aureus Newman Δcoa, ΔvWbp, ΔclfA or Δcoa/ΔvWbp/ΔclfA mutants. vWbp-expression increased L. lactis valve infection compared to parent and coa-expressing strains (incidence: 62%, versus 0% and 13%, respectively; P < 0.01). Likewise, expression of clfA increased L. lactis infectivity (incidence: 80%), which was not further affected by co-expression of vWbp. In symmetry, deletion of the coa or vWbp genes in S. aureus did not decrease infectivity (incidence: 68 and 64%, respectively) whereas deletion of clfA did decrease valve infection (incidence: 45%; P = 0.03 versus parent), which was not further affected by the triple deletion Δcoa/ΔvWbp/ΔclfA (incidence: 36%; P > 0.05 versus ΔclfA mutant). Coa does not support the initial colonization of IE (in L. lactis) without other key virulence factors and vWbp contributes to initiation of IE (in L. lactis) but is marginal in the present of ClfA.
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http://dx.doi.org/10.1080/21505594.2018.1528845DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7000203PMC
April 2019

EssH Peptidoglycan Hydrolase Enables Staphylococcus aureus Type VII Secretion across the Bacterial Cell Wall Envelope.

J Bacteriol 2018 10 24;200(20). Epub 2018 Sep 24.

Department of Microbiology, University of Chicago, Chicago, Illinois, USA

The ESAT-6-like secretion system (ESS) of is assembled in the bacterial membrane from core components that promote the secretion of WXG-like proteins (EsxA, EsxB, EsxC, and EsxD) and the EssD effector. Genes encoding the ESS secretion machinery components, effector, and WXG-like proteins are located in the locus. Here, we identify , a heretofore uncharacterized gene of the locus, whose product is secreted via an N-terminal signal peptide into the extracellular medium of staphylococcal cultures. EssH exhibits two peptidoglycan hydrolase activities, cleaving the pentaglycine cross bridge and the amide bond of -acetylmuramyl-l-alanine, thereby separating glycan chains and wall peptides with cleaved cross bridges. Unlike other peptidoglycan hydrolases, EssH does not promote the lysis of staphylococci. EssH residues Cys and His, which are conserved in other CHAP domain enzymes, are required for peptidoglycan hydrolase activity and for ESS secretion. These data suggest that EssH and its murein hydrolase activity are required for protein secretion by the ESS pathway. Gene clusters encoding WXG-like proteins and FtsK/SpoIIIE-like P loop ATPases in encode type 7b secretion systems (T7bSS) for the transport of select protein substrates. The T7bSS assembles in the bacterial membrane and promotes the secretion of WXG-like proteins and effectors. The mechanisms whereby staphylococci extend the T7SS across the bacterial cell wall envelope are not known. Here, we show that staphylococci secrete EssH to cleave their peptidoglycan, thereby enabling T7bSS transport of proteins across the bacterial cell wall envelope.
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http://dx.doi.org/10.1128/JB.00268-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6153663PMC
October 2018

targets the purine salvage pathway to kill phagocytes.

Proc Natl Acad Sci U S A 2018 06 11;115(26):6846-6851. Epub 2018 Jun 11.

Department of Microbiology, University of Chicago, Chicago, IL 60637

colonizes large segments of the human population and causes invasive infections due to its ability to escape phagocytic clearance. During infection, staphylococcal nuclease and adenosine synthase A convert neutrophil extracellular traps to deoxyadenosine (dAdo), which kills phagocytes. The mechanism whereby staphylococcal dAdo intoxicates phagocytes is not known. Here we used CRISPR-Cas9 mutagenesis to show that phagocyte intoxication involves uptake of dAdo via the human equilibrative nucleoside transporter 1, dAdo conversion to dAMP by deoxycytidine kinase and adenosine kinase, and signaling via subsequent dATP formation to activate caspase-3-induced cell death. Disruption of this signaling cascade confers resistance to dAdo-induced intoxication of phagocytes and may provide therapeutic opportunities for the treatment of infections caused by antibiotic-resistant strains.
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http://dx.doi.org/10.1073/pnas.1805622115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6042115PMC
June 2018

Septal secretion of protein A in requires SecA and lipoteichoic acid synthesis.

Elife 2018 05 14;7. Epub 2018 May 14.

Department of Microbiology, University of Chicago, Chicago, United States.

Surface proteins of are secreted across septal membranes for assembly into the bacterial cross-wall. This localized secretion requires the YSIRK/GXXS motif signal peptide, however the mechanisms supporting precursor trafficking are not known. We show here that the signal peptide of staphylococcal protein A (SpA) is cleaved at the YSIRK/GXXS motif. A SpA signal peptide mutant defective for YSIRK/GXXS cleavage is also impaired for septal secretion and co-purifies with SecA, SecDF and LtaS. SecA depletion blocks precursor targeting to septal membranes, whereas deletion of diminishes SpA secretion into the cross-wall. Depletion of LtaS blocks lipoteichoic acid synthesis and abolishes SpA precursor trafficking to septal membranes. We propose a model whereby SecA directs SpA precursors to lipoteichoic acid-rich septal membranes for YSIRK/GXXS motif cleavage and secretion into the cross-wall.
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http://dx.doi.org/10.7554/eLife.34092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5962339PMC
May 2018

Staphylococcal Protein A Contributes to Persistent Colonization of Mice with Staphylococcus aureus.

J Bacteriol 2018 05 9;200(9). Epub 2018 Apr 9.

Department of Microbiology, University of Chicago, Chicago, Illinois, USA

persistently colonizes the nasopharynx in humans, which increases the risk for invasive diseases, such as skin infection and bacteremia. Nasal colonization triggers IgG responses against staphylococcal surface antigens; however, these antibodies cannot prevent subsequent colonization or disease. Here, we describe WU1, a multilocus sequence type 88 (ST88) isolate that persistently colonizes the nasopharynx in mice. We report that staphylococcal protein A (SpA) is required for persistence of WU1 in the nasopharynx. Compared to animals colonized by wild-type , mice colonized with the Δ variant mount increased IgG responses against staphylococcal colonization determinants. Immunization of mice with a nontoxigenic SpA variant, which cannot cross-link B cell receptors and divert antibody responses, elicits protein A-neutralizing antibodies that promote IgG responses against colonizing and diminish pathogen persistence. persistently colonizes the nasopharynx in about one-third of the human population, thereby promoting community- and hospital-acquired infections. Antibiotics are currently used for decolonization of individuals at increased risk of infection. However, the efficacy of antibiotics is limited by recolonization and selection for drug-resistant strains. Here, we propose a model of how staphylococcal protein A (SpA), a B cell superantigen, modifies host immune responses during colonization to support continued persistence of in the nasopharynx. We show that this mechanism can be thwarted by vaccine-induced anti-SpA antibodies that promote IgG responses against staphylococcal antigens and diminish colonization.
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http://dx.doi.org/10.1128/JB.00735-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5892116PMC
May 2018

Galactosylation of the Secondary Cell Wall Polysaccharide of Bacillus anthracis and Its Contribution to Anthrax Pathogenesis.

J Bacteriol 2018 03 7;200(5). Epub 2018 Feb 7.

Howard Taylor Ricketts Laboratory, Argonne National Laboratory, Lemont, Illinois, USA

, the causative agent of anthrax disease, elaborates a secondary cell wall polysaccharide (SCWP) that is essential for bacterial growth and cell division. SCWP is comprised of trisaccharide repeats with the structure, [→4)-β-ManNAc-(1→4)-β-GlcNAc(3-α-Gal)-(1→6)-α-GlcNAc(3-α-Gal, 4-β-Gal)-(1→] The genes whose products promote the galactosylation of SCWP are not yet known. We show here that the expression of , encoding a UDP-glucose 4-epimerase necessary for the synthesis of UDP-galactose, is required for SCWP galactosylation. The mutant assembles surface (S) layer and S layer-associated proteins that associate with ketal-pyruvylated SCWP via their S layer homology domains similarly to wild-type , but the mutant displays a defect in γ-phage murein hydrolase binding to SCWP. Furthermore, deletion of diminishes the capsulation of with poly-d-γ-glutamic acid (PDGA) and causes a reduction in bacterial virulence. These data suggest that SCWP galactosylation is required for the physiologic assembly of the cell wall envelope and for the pathogenesis of anthrax disease. Unlike virulent isolates, strain CDC684 synthesizes secondary cell wall polysaccharide (SCWP) trisaccharide repeats without galactosyl modification, exhibits diminished growth in broth cultures, and is severely attenuated in an animal model of anthrax. To examine whether SCWP galactosylation is a requirement for anthrax disease, we generated variants of strains Sterne 34F2 and Ames lacking UDP-glucose 4-epimerase by mutating the genes and We identified as necessary for SCWP galactosylation. Deletion of decreased the poly-d-γ-glutamic acid (PDGA) capsulation of the vegetative form of and increased the bacterial inoculum required to produce lethal disease in mice, indicating that SCWP galactosylation is indeed a determinant of anthrax disease.
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http://dx.doi.org/10.1128/JB.00562-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5809694PMC
March 2018

Assembly and Function of the Bacillus anthracis S-Layer.

Annu Rev Microbiol 2017 09;71:79-98

Howard Taylor Ricketts Laboratory, Argonne National Laboratory, Lemont, Illinois 60649.

Bacillus anthracis, the anthrax agent, is a member of the Bacillus cereus sensu lato group, which includes invasive pathogens of mammals or insects as well as nonpathogenic environmental strains. The genes for anthrax pathogenesis are located on two large virulence plasmids. Similar virulence plasmids have been acquired by other B. cereus strains and enable the pathogenesis of anthrax-like diseases. Among the virulence factors of B. anthracis is the S-layer-associated protein BslA, which endows bacilli with invasive attributes for mammalian hosts. BslA surface display and function are dependent on the bacterial S-layer, whose constituents assemble by binding to the secondary cell wall polysaccharide (SCWP) via S-layer homology (SLH) domains. B. anthracis and other pathogenic B. cereus isolates harbor genes for the secretion of S-layer proteins, for S-layer assembly, and for synthesis of the SCWP. We review here recent insights into the assembly and function of the S-layer and the SCWP.
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http://dx.doi.org/10.1146/annurev-micro-090816-093512DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6190687PMC
September 2017

Glutathionylation of LcrV and Its Effects on Plague Pathogenesis.

mBio 2017 05 16;8(3). Epub 2017 May 16.

Howard T. Ricketts Laboratory, Argonne National Laboratory, Lemont, lllinois, USA

Glutathionylation, the formation of reversible mixed disulfides between glutathione and protein cysteine residues, is a posttranslational modification previously observed for intracellular proteins of bacteria. Here we show that LcrV, a secreted protein capping the type III secretion machine, is glutathionylated at Cys and that this modification promotes association with host ribosomal protein S3 (RPS3), moderates type III effector transport and killing of macrophages, and enhances bubonic plague pathogenesis in mice and rats. Secreted LcrV was purified and analyzed by mass spectrometry to reveal glutathionylation, a modification that is abolished by the codon substitution CysAla in Moreover, the mutation enhanced the survival of animals in models of bubonic plague. Investigating the molecular mechanism responsible for these virulence attributes, we identified macrophage RPS3 as a ligand of LcrV, an association that is perturbed by the CysAla substitution. Furthermore, macrophages infected by the variant displayed accelerated apoptotic death and diminished proinflammatory cytokine release. Deletion of , which encodes glutathione synthetase of , resulted in undetectable levels of intracellular glutathione, and we used a Δ mutant to characterize the biochemical pathway of LcrV glutathionylation, establishing that LcrV is modified after its transport to the type III needle via disulfide bond formation with extracellular oxidized glutathione., the causative agent of plague, has killed large segments of the human population; however, the molecular bases for the extraordinary virulence attributes of this pathogen are not well understood. We show here that LcrV, the cap protein of bacterial type III secretion needles, is modified by host glutathione and that this modification contributes to the high virulence of in mouse and rat models for bubonic plague. These data suggest that exploits glutathione in host tissues to activate a virulence strategy, thereby accelerating plague pathogenesis.
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http://dx.doi.org/10.1128/mBio.00646-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5433101PMC
May 2017

The role of pili in Bacillus cereus intraocular infection.

Exp Eye Res 2017 06 20;159:69-76. Epub 2017 Mar 20.

Department of Microbiology, University of Chicago, 920 East 58th Street, Chicago, IL, 60637, USA. Electronic address:

Bacterial endophthalmitis is a potentially blinding intraocular infection. The bacterium Bacillus cereus causes a devastating form of this disease which progresses rapidly, resulting in significant inflammation and loss of vision within a few days. The outer surface of B. cereus incites the intraocular inflammatory response, likely through interactions with innate immune receptors such as TLRs. This study analyzed the role of B. cereus pili, adhesion appendages located on the bacterial surface, in experimental endophthalmitis. To test the hypothesis that the presence of pili contributed to intraocular inflammation and virulence, we analyzed the progress of experimental endophthalmitis in mouse eyes infected with wild type B. cereus (ATCC 14579) or its isogenic pilus-deficient mutant (ΔbcpA-srtD-bcpB or ΔPil). One hundred CFU were injected into the mid-vitreous of one eye of each mouse. Infections were analyzed by quantifying intraocular bacilli and retinal function loss, and by histology from 0 to 12 h postinfection. In vitro growth and hemolytic phenotypes of the infecting strains were also compared. There was no difference in hemolytic activity (1:8 titer), motility, or in vitro growth (p > 0.05, every 2 h, 0-18 h) between wild type B. cereus and the ΔPil mutant. However, infected eyes contained greater numbers of wild type B. cereus than ΔPil during the infection course (p ≤ 0.05, 3-12 h). Eyes infected with wild type B. cereus experienced greater losses in retinal function than eyes infected with the ΔPil mutant, but the differences were not always significant. Eyes infected with ΔPil or wild type B. cereus achieved similar degrees of severe inflammation. The results indicated that the intraocular growth of pilus-deficient B. cereus may have been better controlled, leading to a trend of greater retinal function in eyes infected with the pilus-deficient strain. Although this difference was not enough to significantly alter the severity of the inflammatory response, these results suggest a potential role for pili in protecting B. cereus from clearance during the early stages of endophthalmitis, which is a newly described virulence mechanism for this organism and this infection.
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http://dx.doi.org/10.1016/j.exer.2017.03.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5492386PMC
June 2017

Pathogenic conversion of coagulase-negative staphylococci.

Microbes Infect 2017 02 21;19(2):101-109. Epub 2016 Dec 21.

Department of Microbiology, University of Chicago, Chicago, IL, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.micinf.2016.12.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5274588PMC
February 2017

Genes Required for Bacillus anthracis Secondary Cell Wall Polysaccharide Synthesis.

J Bacteriol 2017 01 13;199(1). Epub 2016 Dec 13.

Howard Taylor Ricketts Laboratory, Argonne National Laboratory, Lemont, Illinois, USA, and Department of Microbiology, University of Chicago, Chicago, Illinois, USA

The secondary cell wall polysaccharide (SCWP) is thought to be essential for vegetative growth and surface (S)-layer assembly in Bacillus anthracis; however, the genetic determinants for the assembly of its trisaccharide repeat structure are not known. Here, we report that WpaA (BAS0847) and WpaB (BAS5274) share features with membrane proteins involved in the assembly of O-antigen lipopolysaccharide in Gram-negative bacteria and propose that WpaA and WpaB contribute to the assembly of the SCWP in B. anthracis Vegetative forms of the B. anthracis wpaA mutant displayed increased lengths of cell chains, a cell separation defect that was attributed to mislocalization of the S-layer-associated murein hydrolases BslO, BslS, and BslT. The wpaB mutant was defective in vegetative replication during early logarithmic growth and formed smaller colonies. Deletion of both genes, wpaA and wpaB, did not yield viable bacilli, and when depleted of both wpaA and wpaB, B. anthracis could not maintain cell shape, support vegetative growth, or assemble SCWP. We propose that WpaA and WpaB fulfill overlapping glycosyltransferase functions of either polymerizing repeat units or transferring SCWP polymers to linkage units prior to LCP-mediated anchoring of the polysaccharide to peptidoglycan.

Importance: The secondary cell wall polysaccharide (SCWP) is essential for Bacillus anthracis growth, cell shape, and division. SCWP is comprised of trisaccharide repeats (→4)-β-ManNAc-(1→4)-β-GlcNAc-(1→6)-α-GlcNAc-(1→) with α-Gal and β-Gal substitutions; however, the genetic determinants and enzymes for SCWP synthesis are not known. Here, we identify WpaA and WpaB and report that depletion of these factors affects vegetative growth, cell shape, and S-layer assembly. We hypothesize that WpaA and WpaB are involved in the assembly of SCWP prior to transfer of this polymer onto peptidoglycan.
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http://dx.doi.org/10.1128/JB.00613-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5165087PMC
January 2017

EssD, a Nuclease Effector of the Staphylococcus aureus ESS Pathway.

J Bacteriol 2017 01 13;199(1). Epub 2016 Dec 13.

Department of Microbiology, University of Chicago, Chicago, Illinois, USA

Specialized secretion systems of bacteria evolved for selective advantage, either killing microbial competitors or implementing effector functions during parasitism. Earlier work characterized the ESAT-6 secretion system (ESS) of Staphylococcus aureus and demonstrated its contribution to persistent staphylococcal infection of vertebrate hosts. Here, we identify a novel secreted effector of the ESS pathway, EssD, that functions as a nuclease and cleaves DNA but not RNA. EssI, a protein of the DUF600 family, binds EssD to block its nuclease activity in the staphylococcal cytoplasm. An essD knockout mutant or a variant lacking nuclease activity, essD, elicited a diminished interleukin-12 (IL-12) cytokine response following bloodstream infection of mice, suggesting that the effector function of EssD stimulates immune signaling to support the pathogenesis of S. aureus infections.

Importance: Bacterial type VII or ESAT-6-like secretion systems (ESS) may have evolved to modulate host immune responses during infection, thereby contributing to the pathogenesis of important diseases such as tuberculosis and methicillin-resistant S. aureus (MRSA) infection. The molecular mechanisms whereby type VII secretion systems achieve their goals are not fully elucidated as secreted effectors with biochemical functions have heretofore not been identified. We show here that MRSA infection relies on the secretion of a nuclease effector that cleaves DNA and contributes to the stimulation of IL-12 signaling during infection. These results identify a biological mechanism for the contribution of the ESS pathway toward the establishment of MRSA disease.
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http://dx.doi.org/10.1128/JB.00528-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5165103PMC
January 2017

EssE Promotes Staphylococcus aureus ESS-Dependent Protein Secretion To Modify Host Immune Responses during Infection.

J Bacteriol 2017 01 13;199(1). Epub 2016 Dec 13.

Department of Microbiology, University of Chicago, Chicago, Illinois, USA

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 T1 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 (EsxA/ESAT-6-like secretion system) 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.
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http://dx.doi.org/10.1128/JB.00527-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5165088PMC
January 2017