Publications by authors named "Dominique Missiakas"

120 Publications

A protein A based Staphylococcus aureus vaccine with improved safety.

Vaccine 2021 Jun 1. Epub 2021 Jun 1.

Howard Taylor Ricketts Laboratory, Argonne National Laboratory, Lemont, IL 60439, United States. Electronic address:

Exposure to Staphylococcus aureus does not lead to immunity as evidenced by the persistent colonization of one third of the human population. S. aureus immune escape is mediated by factors that preempt complement activation, destroy phagocytes, and modify B and T cell responses. One such factor, Staphylococcal protein A (SpA) encompasses five Immunoglobulin binding domains (IgBDs) that associate with the Fcγ domain to block phagocytosis. IgBDs also associate with Fab encoded by V3 clan related genes. SpA binding to V3-IgM that serves as a B cell receptor results in B cell expansion and secretion of antibodies with no specificity for S. aureus. SpA crosslinking of V3-IgG and V3-IgE bound to cognate receptors of mast cells and basophils promotes histamine release and anaphylaxis. Earlier work developed a prototype variant SpA with four amino acid substitutions in each IgBD. When tested in animal models, SpA elicited neutralizing antibodies and protection against infection. We show here that SpA retains crosslinking activity for V3-IgG and V3-IgE. We use a rational approach to design and test 67 new SpA variants for loss of V3 binding and anaphylactic activities. We identify two detoxified candidates that elicit SpA-neutralizing antibodies and protect animals from S. aureus colonization and bloodstream infection. The new detoxified SpA candidates bear three instead of four amino acid substitutions thus increasing the development of SpA-specific antibodies. We propose that detoxified SpA variants unable to crosslink V3-idiotypic immunoglobulin may be suitably developed as clinical-grade vaccines for safety and efficacy testing in humans.
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http://dx.doi.org/10.1016/j.vaccine.2021.05.072DOI Listing
June 2021

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

Staphylococcus aureus vWF-binding protein triggers a strong interaction between clumping factor A and host vWF.

Commun Biol 2021 Apr 12;4(1):453. Epub 2021 Apr 12.

Louvain Institute of Biomolecular Science and Technology, UCLouvain, Louvain-la-Neuve, Belgium.

The Staphylococcus aureus cell wall-anchored adhesin ClfA binds to the very large blood circulating protein, von Willebrand factor (vWF) via vWF-binding protein (vWbp), a secreted protein that does not bind the cell wall covalently. Here we perform force spectroscopy studies on living bacteria to unravel the molecular mechanism of this interaction. We discover that the presence of all three binding partners leads to very high binding forces (2000 pN), largely outperforming other known ternary complexes involving adhesins. Strikingly, our experiments indicate that a direct interaction involving features of the dock, lock and latch mechanism must occur between ClfA and vWF to sustain the extreme tensile strength of the ternary complex. Our results support a previously undescribed mechanism whereby vWbp activates a direct, ultra-strong interaction between ClfA and vWF. This intriguing interaction represents a potential target for therapeutic interventions, including synthetic peptides inhibiting the ultra-strong interactions between ClfA and its ligands.
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http://dx.doi.org/10.1038/s42003-021-01986-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8041789PMC
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
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8092163PMC
February 2021

Selective Host Cell Death by : A Strategy for Bacterial Persistence.

Front Immunol 2020 21;11:621733. Epub 2021 Jan 21.

Research Group Pathogenesis of Bacterial Infections, TWINCORE, Centre for Experimental and Clinical Infection Research, a joint venture between the Hannover Medical School and the Helmholtz Centre for Infection Research, Hannover, Germany.

Host cell death programs are fundamental processes that shape cellular homeostasis, embryonic development, and tissue regeneration. Death signaling and downstream host cell responses are not only critical to guide mammalian development, they often act as terminal responses to invading pathogens. Here, we briefly review and contrast how invading pathogens and specifically manipulate apoptotic, necroptotic, and pyroptotic cell death modes to establish infection. Rather than invading host cells, subverts these cells to produce diffusible molecules that cause death of neighboring hematopoietic cells and thus shapes an immune environment conducive to persistence. The exploitation of cell death pathways by is yet another virulence strategy that must be juxtaposed to mechanisms of immune evasion, autophagy escape, and tolerance to intracellular killing, and brings us closer to the true portrait of this pathogen for the design of effective therapeutics and intervention strategies.
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http://dx.doi.org/10.3389/fimmu.2020.621733DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7859115PMC
January 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

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

Staphylococcus aureus alpha-hemolysin impairs corneal epithelial wound healing and promotes intracellular bacterial invasion.

Exp Eye Res 2019 04 27;181:263-270. Epub 2019 Feb 27.

Stem Cell Therapy and Corneal Tissue Engineering Laboratory, Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, 1905 W. Taylor St., L-213, Chicago, IL, 60612, United States. Electronic address:

Colonization by Staphylococcus aureus (S. aureus) has been implicated in many infectious and wound healing disorders. This study was performed to characterize the pathogenic role of S. aureus alpha-hemolysin (alpha-toxin) in corneal epithelial wound healing and infectious keratitis in the setting of a corneal wound. The effect of wild-type and isogenic Hla mutant (α-hemolysin gene deleted) S. aureus bacteria and conditioned media on corneal epithelial wound healing was tested in vitro using a scratch assay and in vivo using a murine epithelial debridement model. The invasiveness of wild-type and Hla mutant S. aureus was evaluated in vitro in human corneal epithelial cells and in vivo in a murine model of infectious keratitis following total epithelial debridement. S. aureus and its conditioned media significantly delayed epithelial wound closure both in vitro (P < 0.05) and in vivo (P < 0.05). The effect of S. aureus on wound healing was significantly diminished with the Hla mutant strain (P < 0.05). Likewise, compared to the wild-type strain, the Hla mutant strain demonstrated significantly reduced ability to invade corneal epithelial cells in vitro (P < 0.05) and infect murine corneas following total epithelial debridement in vivo (P < 0.05). In conclusion, S. aureus alpha-hemolysin plays a major role in the pathologic modulation of corneal epithelial wound healing and the intracellular invasion of the bacteria. Limiting colonization by S. aureus and/or blocking alpha-hemolysin may provide a therapeutic approach for corneal wound healing and infectious disorders.
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http://dx.doi.org/10.1016/j.exer.2019.02.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6447303PMC
April 2019

Staphylococcus aureus TarP: A Brick in the Wall or Rosetta Stone?

Cell Host Microbe 2019 02;25(2):182-183

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

Staphylococcus aureus infection elicits antibodies against wall teichoic acid (WTA). Several glycosyltransferases modify WTA to generate anomeric heterogeneity. In recent work, Gerlach et al. (2018) show that modification by prophage-encoded TarP diminishes WTA immunogenicity, allowing staphylococci to evade host adaptive immune responses, and propose to exploit these insights for vaccines.
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http://dx.doi.org/10.1016/j.chom.2019.01.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6724547PMC
February 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

Development of a Computational Model of Abscess Formation.

Front Microbiol 2018 26;9:1355. Epub 2018 Jun 26.

Graduate Program in Computational Modeling, Federal University of Juiz de Fora, Juiz de Fora, Brazil.

In some bacterial infections, the immune system cannot eliminate the invading pathogen. In these cases, the invading pathogen is successful in establishing a favorable environment to survive and persist in the host organism. For example, bacteria survive in organ tissues employing a set of mechanisms that work in a coordinated and highly regulated way allowing: (1) efficient impairment of the immune response; and (2) protection from the immune cells and molecules. secretes several proteins including coagulases and toxins that drive abscess formation and persistence. Unless staphylococcal abscesses are surgically drained and treated with antibiotics, disseminated infection and septicemia produce a lethal outcome. Within this context, this paper develops a simple mathematical model of abscess formation incorporating characteristics that we judge important for an abscess to be formed. Our aim is to build a mathematical model that reproduces some characteristics and behaviors that are observed in the process of abscess formation.
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http://dx.doi.org/10.3389/fmicb.2018.01355DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6029511PMC
June 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

The transmembrane domain of the Staphylococcus aureus ESAT-6 component EssB mediates interaction with the integral membrane protein EsaA, facilitating partially regulated secretion in a heterologous host.

Arch Microbiol 2018 Sep 8;200(7):1075-1086. Epub 2018 May 8.

Department of Microbiology and Immunology, Faculty of Pharmacy and Pharmaceutical Industries, Sinai University, El-Masaeed, El-Arish, North Sinai, Egypt.

The ESAT-6-like secretion system (ESS) of Staphylococcus aureus plays a significant role in persistent infections. EssB is a highly conserved bitopic ESS protein comprising a cytosolic N-terminus, single transmembrane helix and a C-terminus located on the trans-side of the membrane. Six systematic truncations covering various domains of EssB were constructed, followed by bacterial two-hybrid screening of their interaction with EsaA, another conserved integral membrane component of the ESS pathway. Results show that the transmembrane domain of EssB is critical for heterodimerization with EsaA. In vivo crosslinking followed by Western blot analysis revealed high molecular weight species when wild-type EssB and EsaA were crosslinked, but this band was not detected in the absence of the transmembrane domain of EssB. Heterologous overproduction of EssB, EsaA and five other components of the ESS pathway in Escherichia coli BL21(DE3), followed by fractionation experiments led to a remarkable increase in the periplasmic protein content, suggesting the assembly of partially regulated secretion mechanism. These data identify the transmembrane domain of EssB as indispensable for interaction with EsaA, thereby facilitating protein secretion across bacterial membranes in a fashion that requires other components of the ESS pathway.
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http://dx.doi.org/10.1007/s00203-018-1519-xDOI Listing
September 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

Isolation of a Membrane Protein Complex for Type VII Secretion in Staphylococcus aureus.

J Bacteriol 2017 12 31;199(23). Epub 2017 Oct 31.

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

The ESAT6-like secretion system (ESS) of promotes effector protein transport across the bacterial envelope. Genes in the ESS cluster are required for establishment of persistent abscess lesions and the modulation of immune responses during bloodstream infections. However, the biochemical functions of most of the ESS gene products, specifically the identity of secretion machine components, are unknown. Earlier work demonstrated that deletion of , which encodes a membrane protein, abolishes ESS secretion. Loss-of-function mutations truncating the gene product cause dominant-negative phenotypes on ESS secretion, suggesting that EssB is a central component of the secretion machinery. To test this prediction, we purified native and affinity-tagged EssB from staphylococcal membranes via dodecyl-maltoside extraction, identifying a complex assembled from five proteins, EsaA, EssA, EssB, EssD, and EsxA. All five proteins are essential for secretion, as knockout mutations in the corresponding genes abolish ESS transport. Biochemical and bacterial two-hybrid analyses revealed a direct interaction between EssB and EsaA that, by engaging a mobile machine component, EsxA, may also recruit EssA and EssD. Type VII secretion systems support the lifestyle of Gram-positive bacteria, including important human pathogens such as , , and Genes encoding SpoIIIE-FtsK-like ATPases and WXG100-secreted products are conserved features of type VII secretion pathways; however, most of the genes in T7SS clusters are not conserved between different bacterial species. Here, we isolate a complex of proteins from the membranes of that appears to represent the core secretion machinery, designated ESS. These results suggest that three membrane proteins, EsaA, EssB, and EssA, form a secretion complex that associates with EssC, the SpoIIIE-FtsK-like ATPase, and with EsxA, a mobile machine component and member of the WXG100 protein family.
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http://dx.doi.org/10.1128/JB.00482-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5686593PMC
December 2017

A novel STK1-targeted small-molecule as an "antibiotic resistance breaker" against multidrug-resistant Staphylococcus aureus.

Sci Rep 2017 07 11;7(1):5067. Epub 2017 Jul 11.

Department of Pathology, The Ohio State University College of Medicine, Columbus, Ohio, USA.

Ser/Thr protein kinase (STK1) plays a critical role in cell wall biosynthesis of and drug resistance in methicillin-resistant Staphylococcus aureus (MRSA). MRSA strains lacking STK1 become susceptible to failing cephalosporins, such as Ceftriaxone and Cefotaxime. STK1, despite being nonessential protein for MRSA survival, it can serve as an important therapeutic agent for combination therapy. Here, we report a novel small molecule quinazoline compound, Inh2-B1, which specifically inhibits STK1 activity by directly binding to its ATP-binding catalytic domain. Functional analyses encompassing in vitro growth inhibition of MRSA, and in vivo protection studies in mice against the lethal MRSA challenge indicated that at high concentration neither Inh2-B1 nor Ceftriaxone or Cefotaxime alone was able to inhibit the growth of bacteria or protect the challenged mice. However, the growth of MRSA was inhibited, and a significant protection in mice against the bacterial challenge was observed at a micromolar concentration of Ceftriaxone or Cefotaxime in the presence of Inh2-B1. Cell-dependent minimal to no toxicity of Inh2-B1, and its abilities to down-regulate cell wall hydrolase genes and disrupt the biofilm formation of MRSA clearly indicated that Inh2-B1 serves as a therapeutically important "antibiotic-resistance-breaker," which enhances the bactericidal activity of Ceftriaxone/Cefotaxime against highly pathogenic MRSA infection.
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http://dx.doi.org/10.1038/s41598-017-05314-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5505960PMC
July 2017