The role of Staphylococcus aureus sortase A and sortase B in murine arthritis

Gram-positive pathogenic bacteria display proteins on their surface that play important roles during infection. In Staphylococcus aureus, these surface proteins are anchored to the cell wall by two sortase enzymes, SrtA and SrtB, that recognize specific surface protein sorting signals. The role of sortase enzymes in bacterial virulence was examined using a murine septic arthritis model. Intravenous inoculation with any of the Delta(srtA), Delta(srtB) or Delta(srtAB) mutants resulted in significantly increased survival and significantly lower weight loss compared with the parental strain. Mice inoculated with the Delta(srtA) mutant did not express severe arthritis, while arthritis in mice inoculated with the Delta(srtB) mutant was not different from that seen in mice that were infected with the wild-type parent strain. Furthermore, persistence of staphylococci in kidneys and joints following intravenous inoculation of mice was more pronounced for wild-type and Delta(srtB) mutant strains than for Delta(srtA) or Delta(srtAB) variants. Together these results indicate that sortase B (srtB) plays a contributing role during the pathogenesis of staphylococcal infections, whereas sortase A (srtA) is an essential virulence factor for the establishment of septic arthritis.     

Sortase B, a new class of sortase in Listeria monocytogenes

Sortases are transamidases that covalently link proteins to the peptidoglycan of gram-positive bacteria. The genome of the pathogenic bacterium Listeria monocytogenes encodes two sortases genes, srtA and srtB. The srtA gene product anchors internalin and some other LPXTG-containing proteins to the listerial surface. Here, we focus on the role of the second sortase, SrtB. Whereas SrtA acts on most of the proteins in the peptidoglycan fraction, SrtB appears to target minor amounts of surface polypeptides. We identified one of the SrtB-anchored proteins as the virulence factor SvpA, a surface-exposed protein which does not contain the LPXTG motif. Therefore, as in Staphylococcus aureus, the listerial SrtB represents a second class of sortase in L. monocytogenes, involved in the attachment of a subset of proteins to the cell wall, most likely by recognizing an NXZTN sorting motif. The DeltasrtB mutant strain does not have defects in bacterial entry, growth, or motility in tissue-cultured cells and does not show attenuated virulence in mice. SrtB-mediated anchoring could therefore be required to anchor surface proteins involved in the adaptation of this microorganism to different environmental conditions.     

Characterization of Streptococcus suis Genes Encoding Proteins Homologous to Sortase of Gram-Positive Bacteria

Many surface proteins which are covalently linked to the cell wall of gram-positive bacteria have a consensus C-terminal motif, Leu-Pro-X-Thr-Gly (LPXTG). This sequence is cleaved, and the processed protein is attached to an amino group of a cross-bridge in the peptidoglycan by a specific enzyme called sortase. Using the type strain of Streptococcus suis, NCTC 10234, we found five genes encoding proteins that were homologous to sortases of other bacteria and determined the nucleotide sequences of the genetic regions. One gene, designated srtA, was linked to gyrA, as were the sortase and sortase-like genes of other streptococci. Three genes, designated srtB, srtC, and srtD, were tandemly clustered in a different location, where there were three segments of directly repeated sequences of approximately 110 bp in close vicinity. The remaining gene, designated srtE, was located separately on the chromosome with a pseudogene which may encode a transposase. The deduced amino acid sequences of the five Srt proteins showed 18 to 31% identity with the sortases of Streptococcus gordonii and Staphylococcus aureus, except that SrtA of S. suis had 65% identity with that of S. gordonii. Isogenic mutants deficient for srtA, srtBCD, or srtE were generated by allelic exchanges. The protein fraction which was released from partially purified cell walls by digestion with N-acetylmuramidase was profiled by two-dimensional gel electrophoresis. More than 15 of the protein spots were missing in the profile of the srtA mutant compared with that of the parent strain, and this phenotype was completely complemented by srtA cloned from S. suis. Four genes encoding proteins corresponding to such spots were identified and sequenced. The deduced translational products of the four genes possessed the LPXTG motif in their C-terminal regions. On the other hand, the protein spots that were missing in the srtA mutant appeared in the profiles of the srtBCD and srtE mutants. These results provide evidence that the cell wall sorting system involving srtA is also present in S. suis.     

Bacillus anthracis sortase A (SrtA) anchors LPXTG motif-containing surface proteins to the cell wall envelope

Cell wall-anchored surface proteins of gram-positive pathogens play important roles during the establishment of many infectious diseases, but the contributions of surface proteins to the pathogenesis of anthrax have not yet been revealed. Cell wall anchoring in Staphylococcus aureus occurs by a transpeptidation mechanism requiring surface proteins with C-terminal sorting signals as well as sortase enzymes. The genome sequence of Bacillus anthracis encodes three sortase genes and eleven surface proteins with different types of cell wall sorting signals. Purified B. anthracis sortase A cleaved peptides encompassing LPXTG motif-type sorting signals between the threonine (T) and the glycine (G) residues in vitro. Sortase A activity could be inhibited by thiol-reactive reagents, similar to staphylococcal sortases. B. anthracis parent strain Sterne 34F(2), but not variants lacking the srtA gene, anchored the collagen-binding MSCRAMM (microbial surface components recognizing adhesive matrix molecules) BasC (BA5258/BAS4884) to the bacterial cell wall. These results suggest that B. anthracis SrtA anchors surface proteins bearing LPXTG motif sorting signals to the cell wall envelope of vegetative bacilli.     

Crystal structures of Staphylococcus aureus sortase A and its substrate complex

The cell wall envelope of staphylococci and other Gram-positive pathogens is coated with surface proteins that interact with human host tissues. Surface proteins of Staphylococcus aureus are covalently linked to the cell wall envelope by a mechanism requiring C-terminal sorting signals with an LPXTG motif. Sortase (SrtA) cleaves surface proteins between the threonine (T) and the glycine (G) of the LPXTG motif and catalyzes the formation of an amide bond between threonine at the C-terminal end of polypeptides and cell wall cross-bridges. The active site architecture and catalytic mechanism of sortase A has hitherto not been revealed. Here we present the crystal structures of native SrtA, of an active site mutant of SrtA, and of the mutant SrtA complexed with its substrate LPETG peptide and describe the substrate binding pocket of the enzyme. Highly conserved proline (P) and threonine (T) residues of the LPXTG motif are held in position by hydrophobic contacts, whereas the glutamic acid residue (E) at the X position points out into the solvent. The scissile T-G peptide bond is positioned between the active site Cys(184) and Arg(197) residues and at a greater distance from the imidazolium side chain of His(120). All three residues, His(120), Cys(184), and Arg(197), are conserved in sortase enzymes from Gram-positive bacteria. Comparison of the active sites of S. aureus sortase A and sortase B provides insight into substrate specificity and suggests a universal sortase-catalyzed mechanism of bacterial surface protein anchoring in Gram-positive bacteria.     

Differential recognition of surface proteins in Streptococcus pyogenes by two sortase gene homologs

The interaction of Streptococcus pyogenes (group A streptococcus [GAS]) with its human host requires several surface proteins. In this study, we isolated mutations in a gene required for the surface localization of protein F by transposon mutagenesis of the M6 strain JRS4. This gene (srtA) encodes a protein homologous to Staphylococcus aureus sortase, which covalently links proteins containing an LPXTG motif to the cell wall. The GAS srtA mutant was defective in anchoring the LPXTG-containing proteins M6, protein F, ScpA, and GRAB to the cell surface. This phenotype was complemented when a wild-type srtA gene was provided in trans. The surface localization of T6, however, was unaffected by the srtA mutation. The M1 genome sequence contains a second open reading frame with a motif characteristic of sortase proteins. Inactivation of this gene (designated srtB) in strain JRS4 affected the surface localization of T6 but not M6, protein F, ScpA, or GRAB. This phenotype was complemented by srtB in trans. An srtA probe hybridized with DNA from all GAS strains tested (M types 1, 3, 4, 5, 6, 18, 22, and 50 and nontypeable strain 64/14) and from streptococcal groups C and G, while srtB hybridized with DNA from only a few GAS strains. We conclude that srtA and srtB encode sortase enzymes required for anchoring different subsets of proteins to the cell wall. It seems likely that the multiple sortase homologs in the genomes of other gram-positive bacteria have a similar substrate-specific role.     

Analysis of the substrate specificity of the Staphylococcus aureus sortase transpeptidase SrtA

The Staphylococcus aureus sortase transpeptidase SrtA isoform is responsible for the covalent attachment of virulence and colonization-associated proteins to the bacterial peptidoglycan. SrtA utilizes two substrates, undecaprenol-pyrophosphoryl-MurNAc(GlcNAc)-Ala-D-isoGlu-Lys(epsilon-Gly(5))-D-Ala-D-Ala (branched Lipid II) and secreted proteins containing a highly conserved C-terminal LPXTG sequence. SrtA simultaneously cleaves the Thr-Gly bond of the LPXTG-containing protein and forms a new amide bond with the nucleophilic amino group of the Gly(5) portion of branched Lipid II, anchoring the protein to this key intermediate that is subsequently polymerized into peptidoglycan. Here we describe the development of a general in vitro method for elucidating the substrate specificity of sortase enzymes. In addition, using immunofluorescence, cell adhesion assays, and transmission electron microscopy, we establish links between in vitro substrate specificity and in vivo function of the S. aureus sortase isoforms. Results from these studies provide strong supporting evidence of a primary role of the SrtA isoform in S. aureus adhesion and host colonization, illustrate a lack of specificity cross talk between SrtA and SrtB isoforms, and highlight the potential of SrtA as a target for the development of antivirulence chemotherapeutics against Gram-positive bacterial pathogens.     

葡萄球菌生物膜形成机制与ica之间的关系

ica位点编码的胞外多糖(PIA/PNAG)对理解葡萄球菌生物膜相关感染病理学方面具有重要的意义.关于ica位点与PIA/PNAG之间如何调节的研究还不全面,另外一种独立于ica的生物膜形成机制存在于表皮葡萄球菌和金黄色葡萄球菌中;细胞表面相关蛋白也能调节生物膜的形成,这些发现为探究它们在生物膜形成机制的潜在作用提供了重要基础.     

ica and beyond: biofilm mechanisms and regulation in Staphylococcus epidermidis and Staphylococcus aureus

Recent progress in elucidating the role of the icaADBC-encoded polysaccharide intercellular adhesin (PIA) or polymeric N-acetyl-glucosamine (PNAG) in staphylococcal biofilm development has in turn contributed significantly to our understanding of the pathogenesis of device-related infections. Nevertheless, our understanding of how the ica locus and PIA/PNAG biosynthesis are regulated is far from complete and many questions remain. Moreover, beyond ica, evidence is now emerging for the existence of ica-independent biofilm mechanisms in both Staphylococcus aureus and Staphylococcus epidermidis. Teichoic acids, which are a major carbohydrate component of the S. epidermidis biofilm matrix and the major cell wall autolysin, play an important role in the primary attachment phase of biofilm development, whereas the cell surface biofilm-associated protein and accumulation-associated protein are capable of mediating intercellular accumulation. These findings raise the exciting prospect that other surface proteins, which typically function as antigenic determinants or in binding to extracellular matrix proteins, may also act as biofilm adhesins. Given the impressive array of surface proteins expressed by S. aureus and S. epidermidis, future research into their potential role in biofilm development either independent of PIA/PNAG or in cooperation with PIA/PNAG will be of particular interest.     

Sortase-catalysed anchoring of surface proteins to the cell wall of Staphylococcus aureus

Many surface proteins of Gram-positive bacteria are anchored to the cell wall envelope by a transpeptidation mechanism, requiring a C-terminal sorting signal with a conserved LPXTG motif. Sortase, a membrane protein of Staphylococcus aureus, cleaves polypeptides between the threonine and the glycine of the LPXTG motif and catalyses the formation of an amide bond between the carboxyl-group of threonine and the amino-group of peptidoglycan cross-bridges. S. aureus mutants lacking the srtA gene fail to anchor and display some surface proteins and are impaired in the ability to cause animal infections. Sortase acts on surface proteins that are initiated into the secretion (Sec) pathway and have their signal peptide removed by signal peptidase. The S. aureus genome encodes two sets of sortase and secretion genes. It is conceivable that S. aureus has evolved more than one pathway for the transport of 20 surface proteins to the cell wall envelope.     

一种抗革兰氏阳性致病菌新型靶酶——分选酶

许多革兰氏阳性菌的表面蛋白是经过一种被称为分选酶的半胱氨酸转肽酶的作用而锚定到细胞壁上,由于表面蛋白在病原菌的致病性方面起关键作用,分选酶有可能成为降低革兰氏阳性菌致病性的药物靶点.目前,通过对金黄色葡萄球菌中分选酶A(SrtA)的研究,已初步阐明分选酶的作用机制及其活性位点,与此同时,一些SrtA抑制剂的初步鉴定为今后抑制剂更深层次的筛选提供了基础.     

Interaction of sortase A and lipase 2 in the inhibition of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> biofilm formation

Recombinant sortase A (SrtA) was used to immune rabbit, and the inhibitory activity of anti-SrtA serum on Staphylococcus aureus biofilm formation was tested. Biofilm formation was inhibited by anti-SrtA rabbit serum in S. aureus ATCC25923 and two clinical isolated strains. The antiserum was separated into two fractions, and the main component with the inhibitory activity was demonstrated to be the IgG fraction. Two proteins interact with the IgG fraction were identified by using an in vitro pull-down assay and were confirmed to be lipase 2 and γ-hemolysin by mass spectrometry. Cross-interaction between SrtA and lipase 2 was further confirmed by Western blotting. Addition of anti-lipase 2 serum in the culture medium also showed inhibitory effect against biofilm formation. Together, our study suggests anti-SrtA serum inhibits S. aureus biofilm formation and lipase 2 is one of the targets of anti-SrtA serum in this inhibition process. This is the first study to demonstrate the roles of antisera against SrtA and lipase 2 in the inhibition of biofilm formation in S. aureus.     

Induction of Staphylococcus epidermidis biofilm formation via proteolytic processing of the accumulation-associated protein by staphylococcal and host proteases

Because of its biofilm forming potential Staphylococcus epidermidis has evolved as a leading cause of device-related infections. The polysaccharide intercellular adhesin (PIA) is significantly involved in biofilm accumulation. However, infections because of PIA-negative strains are not uncommon, suggesting the existence of PIA-independent biofilm accumulation mechanisms. Here we found that biofilm formation in the clinically significant S. epidermidis 5179 depended on the expression of a truncated 140 kDa isoform of the 220 kDa accumulation-associated protein Aap. As expression of the truncated Aap isoform leads to biofilm formation in aap-negative S. epidermidis 1585, this domain mediates intercellular adhesion in a polysaccharide-independent manner. In contrast, expression of full-length Aap did not lead to a biofilm-positive phenotype. Obviously, to gain adhesive function, full-length Aap has to be proteolytically processed through staphylococcal proteases as demonstrated by inhibition of biofilm formation by alpha(2)-macroglobulin. Importantly, also exogenously added granulocyte proteases activated Aap, thereby inducing biofilm formation in S. epidermidis 5179 and four additional, independent clinical S. epidermidis strains. It is therefore reasonable to assume that in vivo effector mechanisms of the innate immunity can directly induce protein-dependent S. epidermidis cell aggregation and biofilm formation, thereby enabling the pathogen to evade clearance by phagocytes.     

Role of Streptococcus sanguinis sortase A in bacterial colonization

Streptococcus sanguinis, a normal inhabitant of the human oral cavity, has low cariogenicity, though colonization on tooth surfaces by this bacterium initiates aggregation by other oral bacteria and maturation of dental plaque. Additionally, S. sanguinis is frequently isolated from infective endocarditis patients. We investigated the functions of sortase A (SrtA), which cleaves LPXTG-containing proteins and anchors them to the bacterial cell wall, as a possible virulence factor of S. sanguinis. We identified the srtA gene of S. sanguinis by searching a homologous gene of Streptococcus mutans in genome databases. Next, we constructed an srtA-deficient mutant strain of S. sanguinis by insertional inactivation and compared it to the wild type strain. In the case of the mutant strain, some surface proteins could not anchor to the cell wall and were partially released into the culture supernatant. Furthermore, adherence to saliva-coated hydroxyapatite beads and polystyrene plates, as well as adherence to and invasion of human epithelial cells were reduced significantly in the srtA-deficient strain when compared to the wild type. In addition, antiopsonization levels and bacterial survival of the srtA-deficient mutant were decreased in human whole blood. This is the first known study to report that SrtA contributes to antiopsonization in streptococci. Our results suggest that SrtA anchors surface adhesins as well as some proteins that function as antiopsonic molecules as a means of evading the human immune system. Furthermore, they demonstrate that SrtA of S. sanguinis plays important roles in bacterial colonization.     

Evaluation of Staphylococcus aureus virulence factors using a silkworm model

Previous studies have indicated that the silkworm model is useful for identifying virulence genes of Staphylococcus aureus, a human pathogenic bacterium. Here we examined the scope of S.?aureus virulence factors that can be evaluated using the silkworm model. Gene-disrupted mutants of the agr locus, arlS gene and saeS gene, which regulate the expression of cell surface adhesins and hemolysins, exhibited attenuated virulence in silkworms. Mutants of the hla gene encoding α-hemolysin, the hlb gene encoding β-hemolysin, and the psmα and psmβ operons encoding cytolysins, however, showed virulence in silkworms indistinguishable from that of the parent strain. Thus, these S.?aureus cytolysins are not required for virulence in silkworms. In contrast, the gene-disrupted mutants of clfB, fnbB and sdrC, which encode cell-wall-anchored proteins, attenuated S.?aureus virulence in silkworms. In addition, the mutant of the srtA gene encoding sortase A, which anchors cell-wall proteins, showed attenuated virulence in silkworms. These findings suggest that the silkworm model can be used to evaluate S.?aureus cell-wall proteins and regulatory proteins as virulence factors.     

Staphylococcus aureus sortase A contributes to the Trojan horse mechanism of immune defense evasion with its intrinsic resistance to Cys184 oxidation

Staphylococcus aureus is a Gram-positive bacterial pathogen that causes serious infections which have become increasingly difficult to treat due to antimicrobial resistance and natural virulence strategies. Bacterial sortase enzymes are important virulence factors and good targets for future antibiotic development. It has recently been shown that sortase enzymes are integral to bacterial survival of phagocytosis, an underappreciated, but vital, step in S. aureus pathogenesis. Of note, the reaction mechanism of sortases relies on a solvent-accessible cysteine for transpeptidation. Because of the common strategy of oxidative damage employed by professional phagocytes to kill pathogens, it is possible that this cysteine may be oxidized inside the phagosome, thereby inhibiting the enzyme. This study addresses this apparent paradox by assessing the ability of physiological reactive oxygen species, hydrogen peroxide and hypochlorite, to inhibit sortase A (SrtA) from S. aureus. Surprisingly, we found that SrtA is highly resistant to oxidative inhibition, both in vitro and in vivo. The mechanism of resistance to oxidative damage is likely mediated by maintaining a high reduction potential of the catalytic cysteine residue, Cys184. This is due to the unusual active site utilized by S. aureus SrtA, which employs a reverse protonation mechanism for transpeptidation, resulting in a high pK(a) as well as reduction potential for Cys184. The results of this study suggest that S. aureus SrtA is able to withstand the extreme conditions encountered in the phagosome and maintain function, contributing to survival of phagocytotic killing.     

Kinetic mechanism of Staphylococcus aureus sortase SrtA

Staphylococcus aureus sortase (SrtA) is a thiol transpeptidase. The enzyme catalyzes a cell wall sorting reaction in which a surface protein with a sorting signal containing a LPXTG motif is cleaved between the threonine and glycine residues. The resulting threonine carboxyl end of this protein is covalently attached to a pentaglycine cross-bridge of peptidoglycan. The transpeptidase activity of sortase has been demonstrated in in vitro reactions between a LPETG-containing peptide and triglycine. When a nucleophile is not available, sortase slowly hydrolyzes the LPETG peptide at the same site. In this study, we have analyzed the steady-state kinetics of these two types of reactions catalyzed by sortase. The kinetic results fully support a ping-pong mechanism in which a common acyl-enzyme intermediate is formed in transpeptidation and hydrolysis. However, each reaction has a distinct rate-limiting step: the formation of the acyl-enzyme in transpeptidation and the hydrolysis of the same acyl-enzyme in the hydrolysis reaction. We have also demonstrated in this study that the nucleophile binding site of S. aureus sortase SrtA is specific for diglycine. While S1' and S2' sites of the enzyme both prefer a glycine residue, the S1' site is exclusively selective for glycine. Lengthening of the polyglycine acceptor nucleophile beyond diglycine does not further enhance the binding and catalysis.