Anchor structure of staphylococcal surface proteins (original) (raw)
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Bioconjugate Chemistry, 2022
Endogenous Staphylococcus aureus sortase A (SrtA) covalently incorporates cell wall anchored proteins equipped with a SrtA recognition motif (LPXTG) via a lipid II-dependent pathway into the staphylococcal peptidoglycan layer. Previously, we found that the endogenous S. aureus SrtA is able to recognize and process a variety of exogenously added synthetic SrtA substrates, including K(FITC)LPMTG-amide and K(FITC)-K-vancomycin-LPMTGamide. These synthetic substrates are covalently incorporated into the bacterial peptidoglycan (PG) of S. aureus with varying efficiencies. In this study, we examined if native and synthetic substrates are processed by SrtA via the same pathway. Therefore, the effect of the lipid II inhibiting antibiotic bacitracin on the incorporation of native and synthetic SrtA substrates was assessed. Treatment of S. aureus with bacitracin resulted in a decreased incorporation of protein A in the bacterial cell wall, whereas incorporation of exogenous synthetic substrates was increased. These results suggest that natural and exogenous synthetic substrates are processed by S. aureus via different pathways.
ChemBioChem
The surfaces of most Gram-positive bacterial cells, including that of Staphylococcus aureus (S. aureus), are heavily decorated with proteins that coordinate cellular interactions with the extracellular space. In S. aureus, sortase A is the principal enzyme responsible for covalently anchoring proteins, which display the sorting signal LPXTG, onto the peptidoglycan (PG) matrix. Considerable efforts have been made to understand the role of this signal peptide in the sortase-mediated reaction. In contrast, much less is known about how the primary structure of the other substrate involved in the reaction (PG stem peptide) could impact sortase activity. To assess the sortase activity, a library of synthetic analogs of the stem peptide that mimic naturally existing variations found in the S. aureus PG primary sequence were evaluated. Using a combination of two unique assays, we showed that there is broad tolerability of substrate variations that are effectively processed by sortase A. While some of these stem peptide derivatives are naturally found in mature PG, they are not known to be present in the PG precursor, lipid II. These results suggest that sortase A could process both lipid II and mature PG as acyl-acceptor strands that might reside near the membrane, which has not been previously described.
Structure, 2004
zymes contain a C-terminal segment of hydrophobic amino acids which serves as a membrane anchor, in Argonne National Laboratory 9700 South Cass Avenue addition to an N-terminal signal peptide. Class I sortases adopt a type 2 transmembrane topology (N terminus Building 202 Argonne, Illinois 60439 inside, C terminus outside the cytoplasm); class II enzymes are thought to represent type 1 transmembrane 2 Committee on Microbiology 3 Department of Molecular Genetics and proteins (N terminus outside, C terminus inside). S. aureus and B. anthracis sortases are anchored to the Cell Biology 4 Department of Biochemistry and cell membrane via their N-terminal hydrophobic leader sequence, and they appear to belong to class I trans-Molecular Biology University of Chicago membrane proteins, and therefore their C terminus containing the catalytic domain is located outside the cell. 920 East 58 th Street Chicago, Illinois 60637 During sorting process, the C-terminal peptide signal is removed and the mature protein is covalently attached to a specific component of the cell wall (Schneewind et al., 1995; Navarre and Schneewind, 1994; Ton-That et Summary al., 1999; Navarre et al., 1998; Dhar et al., 2000). In S. aureus, the cleavage occurs after a Thr residue within Surface proteins attached by sortases to the cell wall envelope of bacterial pathogens play important roles a conserved 5 amino acid motif, either LPXTG (X, any amino acid) or NPQTN, and the Thr carboxyl group is during infection. Sorting and attachment of these proteins is directed by C-terminal signals. Sortase B of amide linked to the amino group of pentaglycine crossbridges of the staphylococcal cell wall peptidoglycan S. aureus recognizes a motif NPQTN, cleaves the polypeptide after the Thr residue, and attaches the protein (Ton-That et al., 1997). It has been proposed that lipid II [undecaprenyl pyrophosphate-MurNAc(-L-Ala-D-iGln-to pentaglycine cross-bridges. Sortase B of B. anthracis is thought to recognize the NPKTG motif, and L-Lys(NH 2-Gly 5)-D-Ala-D-Ala)-()1-4-GlcNAc], a membrane-anchored precursor of cell wall synthesis (Higashi attaches surface proteins to m-diaminopimelic acid cross-bridges. We have determined crystal structure et al., 1967, 1970), serves as the substrate for the protein anchoring (Perry et al., 2002; Ruzin et al., 2002). Surface of sortase B from B. anthracis and S. aureus at 1.6 and 2.0 Å resolutions, respectively. These structures proteins tethered to lipid II are subsequently incorporated into the peptidoglycan by means of the transglyco-show a -barrel fold with ␣-helical elements on its outside, a structure thus far exclusive to the sortase sylation and transpeptidation reactions of bacterial cell wall synthesis (Strominger et al., 1967; Ton-That et al., family. A putative active site located on the edge of the -barrel is comprised of a Cys-His-Asp catalytic 1998, 1999). Genes encoding sortases have been identified in B. anthracis, and these transpeptidases are triad and presumably faces the bacterial cell surface. A putative binding site for the sorting signal is located thought to cleave surface protein sorting signals with LPXTG or NPKTG motifs (Read et al., 2003; Pallen et nearby. al., 2001; Mazmanian and Schneewind, 2004). Because the cell wall peptidoglycan of B. anthracis is synthesized Introduction from precursors with different lipid II structure [undecaprenyl pyrophosphate-MurNAc(-L-Ala-D-iGln-(NH 2)-m-Surface proteins of Gram-positive bacteria perform criti-Dpm-D-Ala-D-Ala)-()1-4-GlcNAc], surface proteins are cal biological functions that are required for the colonithought to be attached to the m-diaminopimelic acid zation of host tissues, the evasion of immune defenses, cross-bridges within the cell wall (Dhar et al., 2000; and the acquisition of nutrients; they promote bacterial Schleifer and Kandler, 1972). adhesion to specific tissues and invasion of host cells; Several sortase genes have been characterized in and provide resistance to phagocytic killing (Foster and Gram-positive bacteria (
Surface Proteins of Staphylococcus aureus
Clinical Infectious Diseases, 1988
Staphylococcus aureus is a commensal bacterium that causes infections such as sepsis, endocarditis, and pneumonia. S. aureus can express a variety of virulence factors, including surface proteins. Surface proteins are characterized by presence of a Sec-dependent signal sequence at the amino terminal, and the sorting signal domain. Surface proteins are covalently attached to peptidoglycan and they are commonly known as cell wallanchored (CWA) proteins. CWA proteins have many functions and participate in the pathogenesis of S. aureus. Furthermore, these proteins have been proposed as therapeutic targets for the generation of vaccines. In this chapter, different topics related to CWA proteins of S. aureus are addressed. The molecular structure of CWA proteins and their role as virulence factors of S. aureus are described. Furthermore, the involvement of CWA proteins in the processes of adhesion, invasion of host cells and tissues, evasion of the immune response, and the formation of biofilm is discussed. In addition, the role of CWA proteins in skin infection and the proposal to use them as potential vaccine antigens are described. The information contained in this chapter will help the readers to understand the biology of CWA proteins and to recognize the importance of surface molecules of S. aureus.
Crystal Structure of Streptococcus pyogenes Sortase A: IMPLICATIONS FOR SORTASE MECHANISM
Journal of Biological Chemistry, 2009
Sortases are a family of Gram-positive bacterial transpeptidases that anchor secreted proteins to bacterial cell surfaces. These include many proteins that play critical roles in the virulence of Gram-positive bacterial pathogens such that sortases are attractive targets for development of novel antimicrobial agents. All Gram-positive pathogens express a "housekeeping" sortase that recognizes the majority of secreted proteins containing an LPXTG wall-sorting motif and covalently attaches these to bacterial cell wall peptidoglycan. Many Gram-positive pathogens also express additional sortases that link a small number of proteins, often with variant wall-sorting motifs, to either other surface proteins or peptidoglycan. To better understand the mechanisms of catalysis and substrate recognition by the housekeeping sortase produced by the important human pathogen Streptococcus pyogenes, the crystal structure of this protein has been solved and its transpeptidase activity established in vitro. The structure reveals a novel arrangement of key catalytic residues in the active site of a sortase, the first that is consistent with kinetic analysis. The structure also provides a complete description of residue positions surrounding the active site, overcoming the limitation of localized disorder in previous structures of sortase A-type proteins. Modification of the active site Cys through oxidation to its sulfenic acid form or by an alkylating reagent supports a role for a reactive thiol/ thiolate in the catalytic mechanism. These new insights into sortase structure and function could have important consequences for inhibitor design.
Cell wall sorting of lipoproteins in Staphylococcus aureus
Journal of bacteriology, 1996
Many surface proteins are thought to be anchored to the cell wall of gram-positive organisms via their C termini, while the N-terminal domains of these molecules are displayed on the bacterial surface. Cell wall anchoring of surface proteins in Staphylococcus aureus requires both an N-terminal leader peptide and a C-terminal cell wall sorting signal. By fusing the cell wall sorting of protein A to the C terminus of staphylococcal beta-lactamase, we demonstrate here that lipoproteins can also be anchored to the cell wall of S. aureus. The topology of cell wall-anchored beta-lactamase is reminiscent of that described for Braun's murein lipoprotein in that the N terminus of the polypeptide chain is membrane anchored whereas the C-terminal end is tethered to the bacterial cell wall.
Region X, the cell-wall-attachment part of staphylococcal protein A
European Journal of Biochemistry, 1984
The sequence of region X of staphylococcal protein A has been determined. The hypothesis has been put forward that this region spans the Staphylococcus aureus cell wall and is responsible for the binding to the peptidoglycan. The primary amino acid sequence of region X was determined for two strains exhibiting cell-wall-bound protein A, Cowan I and 8325 -4. The sequence determination of the Cowan I material is partial and was performed by Edman degradation, in contrast to the sequence of the 8325 -4 material which was completely analyzed by nucleotide sequencing of the corresponding gene. The region consists of two structurally different domains, a highly repetitive region (X,), with an octapeptide structure repeated approximately 12 times, and a C-terminal domain (X,) with an unique sequence. A comparison between the two strains reveals a high mutual homology as well as a high internal homology between the octapeptide structures. Six out of eight amino acids are identical in the repetition of this structure throughout region X, in both proteins and the other two are changed in a rather regular pattern.
Frontiers in Microbiology, 2020
Staphylococcus aureus is one of the most notorious pathogens and is frequently associated with nosocomial infections imposing serious risk to immune-compromised patients. This is in part due to its ability to colonize at the surface of indwelling medical devices and biofilm formation. Combating the biofilm formation with antibiotics has its own challenges like higher values of minimum inhibitory concentrations. Here, we describe a new approach to target biofilm formation by Gram positive bacteria. Sortase A is a transpeptidase enzyme which is responsible for tagging of around ∼22 cell surface proteins onto the outer surface. These proteins play a major role in the bacterial virulence. Sortase A recognizes its substrate through LPXTG motif. Here, we use this approach to install the synthetic peptide substrates on S. aureus. Sortase A substrate mimic, 6His-LPETG peptide was synthesized using solid phase peptide chemistry. Incorporation of the peptide on the cell surface was measured using ELISA. Effect of peptide incubation on Staphylococcus aureus biofilm was also studied. 71.1% biofilm inhibition was observed with 100 µM peptide while on silicon coated rubber latex catheter, 45.82% inhibition was observed. The present work demonstrates the inability of surface modified S. aureus to establish biofilm formation thereby presenting a novel method for attenuating its virulence.
Monofunctional Transglycosylases Are Not Essential for Staphylococcus aureus Cell Wall Synthesis
The polymerization of peptidoglycan is the result of two types of enzymatic activities: transglycosylation, the formation of linear glycan chains, and transpeptidation, the formation of peptide cross-bridges between the glycan strands. Staphylococcus aureus has four penicillin binding proteins (PBP1 to PBP4) with transpeptidation activity, one of which, PBP2, is a bifunctional enzyme that is also capable of catalyzing transglycosylation reactions. Additionally, two monofunctional transglycosylases have been reported in S. aureus: MGT, which has been shown to have in vitro transglycosylase activity, and a second putative transglycosylase, SgtA, identified only by sequence analysis. We have now shown that purified SgtA has in vitro transglycosylase activity and that both MGT and SgtA are not essential in S. aureus. However, in the absence of PBP2 transglycosylase activity, MGT but not SgtA becomes essential for cell viability. This indicates that S. aureus cells require one transglycosylase for survival, either PBP2 or MGT, both of which can act as the sole synthetic transglycosylase for cell wall synthesis. We have also shown that both MGT and SgtA interact with PBP2 and other enzymes involved in cell wall synthesis in a bacterial two-hybrid assay, suggesting that these enzymes may work in collaboration as part of a larger, as-yet-uncharacterized cell wall-synthetic complex.
Crystal Structure of Streptococcus Pyogenes Sortase A: IMPLICATIONS FOR SORTASE MECHANISM* S⃞
The Journal of …, 2009
Sortases are a family of Gram-positive bacterial transpeptidases that anchor secreted proteins to bacterial cell surfaces. These include many proteins that play critical roles in the virulence of Gram-positive bacterial pathogens such that sortases are attractive targets for development of novel antimicrobial agents. All Gram-positive pathogens express a "housekeeping" sortase that recognizes the majority of secreted proteins containing an LPXTG wall-sorting motif and covalently attaches these to bacterial cell wall peptidoglycan. Many Gram-positive pathogens also express additional sortases that link a small number of proteins, often with variant wall-sorting motifs, to either other surface proteins or peptidoglycan. To better understand the mechanisms of catalysis and substrate recognition by the housekeeping sortase produced by the important human pathogen Streptococcus pyogenes, the crystal structure of this protein has been solved and its transpeptidase activity established in vitro. The structure reveals a novel arrangement of key catalytic residues in the active site of a sortase, the first that is consistent with kinetic analysis. The structure also provides a complete description of residue positions surrounding the active site, overcoming the limitation of localized disorder in previous structures of sortase A-type proteins. Modification of the active site Cys through oxidation to its sulfenic acid form or by an alkylating reagent supports a role for a reactive thiol/ thiolate in the catalytic mechanism. These new insights into sortase structure and function could have important consequences for inhibitor design. Cell wall-anchored proteins play critical roles in the virulence of most Gram-positive bacterial pathogens by acting as