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

ABSTRACT Staphylococcus aureus causes reiterative and chronic persistent infections. This can be explained by the formidable ability of this pathogen to escape immune surveillance mechanisms. Cells of S. aureus 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 (VH3) idiotype (IgM) B cell receptors, promoting B cell expansion and the secretion of nonspecific VH3-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. IMPORTANCE 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 S. aureus carries a LysM domain that binds glycan strands of peptidoglycan to influence defined B cell responses that divert pathogen-specific adaptive immune responses.

Proteome Mining of Sortase A Dependent Proteins (SDPs) in Lactic Acid Bacteria and Docking Analysis of SDPs Interaction with Sortase A

Background: Lactic acid bacteria (LAB), which are important probiotics, play a fundamental role in ensuring the health of the gastrointestinal tract, maintaining the microbiome balance, and preventing the gastrointestinal (GI) tract disorder. One of the effective mechanisms in the bacterial-host interaction is related to the action of the enzyme sortase A and Sortase Dependent Proteins (SDPs). Sortase plays an important role in the stabilization and retention of the probiotic in the gut by exposing various SDPs on the bacterial surface proteins which is involved in the attachment of bacteria to the host intestine and retention in the gut.Methods: The present study aimes to identify and investigate the abundance of sortase A-dependent proteins (SDPs) in lactic acide bacteria, as well as the frequency analysis of X residue in the sortase recognition and cleavage LPXTG motif and its effect on the interaction between sortase and SDPs. For this purpose, genomic and proteomic sequences of 165 LABs including 119 Lactobacilli, 29 Enterococci, 8 Lactococci, 5 Carnobacteria, and 4 Leuconostocs, were extracted from UniProt and Genome NCBI databases,. for this, we designed ProtScreen software with the ability to recognize a specific motif and domain in the proteome, which is available at http://nigebprotscreen.com/. Also interactions between sortase A and LPXTG motif with 18 different amino acids in X position were determined using in silico approach. The structure of the sortase A enzyme and a SDP in Lactobacillus acidophilus was used for docking using HADDOCK and CABS-dock toolsResults: In this study, out of 165 LABs reference proteomes, there were 25 SDP-free strains. Among the 140 strains with SDPs, 707 proteins were found with the potential to function as SDPs. In this way, ProtScreen software with the ability to recognize a specific motif and domain in the proteome, which is available at http://nigebprotscreen.com/ was designed. Also a database including 707 SDPs in Lactobacillus, Enterococcus, Lactococcus, Carnobacterium, and Leuconostoc strains was designed which is available in the project section at online ProtScreen software. Our results showed that the most abundant amino acid in X position in the LPXTG motif among 165(LABs) is glutamine (Q). Results of SDPs and sortase A docking using HADDOCK and CABS-dock tools, showed that the highest binding energy is related to the glutamine, where a positive relationship between frequency of amino acids and binding energy was observed. Therefore, our data shows that why glutamine in nature and during evolution, has been selected as the best amino acid for X site in LPXTG motif.Conclusions: The results of the present research and similar studies could be useful in better understanding the role of sortase A and SDPs in the studies on the mechanisms related to the interactions between bacteria and the host, including longer probiotic persistence in the gut.

A Type I Signal Peptidase Is Required for Pilus Assembly in the Gram-Positive, Biofilm-Forming Bacterium Actinomycesoris

ABSTRACTThe Gram-positive bacteriumActinomycesoris, a key colonizer in the development of oral biofilms, contains 18 LPXTG motif-containing proteins, including fimbrillins that constitute two fimbrial types critical for adherence, biofilm formation, and polymicrobial interactions. Export of these protein precursors, which harbor a signal peptide, is thought to be mediated by the Sec machine and require cleavage of the signal peptide by type I signal peptidases (SPases). Like many Gram-positive bacteria,A. orisexpresses two SPases, named LepB1 and LepB2. The latter has been linked to suppression of lethal “glyco-stress,” caused by membrane accumulation of the LPXTG motif-containing glycoprotein GspA when the housekeeping sortasesrtAis genetically disrupted. Consistent with this finding, we show here that a mutant lackinglepB2andsrtAwas unable to produce high levels of glycosylated GspA and hence was viable. However, deletion of neitherlepB1norlepB2abrogated the signal peptide cleavage and glycosylation of GspA, indicating redundancy of SPases for GspA. In contrast, thelepB2deletion mutant failed to assemble the wild-type levels of type 1 and 2 fimbriae, which are built by the shaft fimbrillins FimP and FimA, respectively; this phenotype was attributed to aberrant cleavage of the fimbrillin signal peptides. Furthermore, thelepB2mutants, including the catalytically inactive S101A and K169A variants, exhibited significant defects in polymicrobial interactions and biofilm formation. Conversely,lepB1was dispensable for the aforementioned processes. These results support the idea that LepB2 is specifically utilized for processing of fimbrial proteins, thus providing an experimental model with which to study the basis of type I SPase specificity.IMPORTANCESec-mediated translocation of bacterial protein precursors across the cytoplasmic membrane involves cleavage of their signal peptide by a signal peptidase (SPase). Like many Gram-positive bacteria,A. orisexpresses two SPases, LepB1 and LepB2. The latter is a genetic suppressor of lethal “glyco-stress” caused by membrane accumulation of glycosylated GspA when the housekeeping sortasesrtAis genetically disrupted. We show here that LepB1 and LepB2 are capable of processing GspA, whereas only LepB2 is required for cleavage of fimbrial signal peptides. This is the first example of a type I SPase dedicated to LPXTG motif-containing fimbrial proteins. Thus,A. orisprovides an experimental model with which to investigate the specificity mechanism of type I SPases.

Acyl Enzyme Intermediates in Sortase-Catalyzed Pilus Morphogenesis in Gram-Positive Bacteria

ABSTRACT In gram-positive bacteria, covalently linked pilus polymers are assembled by a specific transpeptidase enzyme called pilus-specific sortase. This sortase is postulated to cleave the LPXTG motif of a pilin precursor between threonine and glycine and to form an acyl enzyme intermediate with the substrate. Pilus polymerization is believed to occur through the resolution of this intermediate upon specific nucleophilic attack by the conserved lysine located within the pilin motif of another pilin monomer, which joins two pilins with an isopeptide bond formed between threonine and lysine. Here, we present evidence for sortase reaction intermediates in Corynebacterium diphtheriae. We show that truncated SrtA mutants that are loosely bound to the cytoplasmic membrane form high-molecular-weight complexes with SpaA polymers secreted into the extracellular milieu. These complexes are not formed with SpaA pilin mutants that have alanine substitutions in place of threonine in the LPXTG motif or lysine in the pilin motif. The same phenotype is observed with alanine substitutions of either the conserved cysteine or histidine residue of SrtA known to be required for catalysis. Remarkably, the assembly of SpaA pili, or the formation of intermediates, is abolished with a SrtA mutant missing the membrane-anchoring domain. We infer that pilus polymerization involves the formation of covalent pilin-sortase intermediates, which occurs within a molecular platform on the exoplasmic face of the cytoplasmic membrane that brings together both sortase and its cognate substrates in close proximity to each other, likely surrounding a secretion apparatus. We present electron microscopic data in support of this picture.

Inhibition of Staphylococcus epidermidis Biofilm Formation by Rabbit Polyclonal Antibodies against the SesC Protein

ABSTRACT Several well-studied proteins with defined roles in Staphylococcus epidermidis biofilm formation are LPXTG motif-containing proteins. Here, we investigate the possible use of the LPXTG motif-containing protein SesC (S . epidermidis surface protein C; accession no. NP_765787) as a target for antibodies to prevent biofilm formation. In vitro and in a in vivo rat model of catheter infection, gene and protein expression analysis showed that SesC is expressed more strongly in biofilm-associated cells than in planktonic cells and is expressed particularly during the late phase of in vivo biofilm formation. Polyclonal rabbit antibodies raised against SesC reduced the fibrinogen-binding ability of S. epidermidis RP62A and Staphylococcus aureus RN4220 transformants expressing SesC, inhibited in vitro biofilm formation by S. epidermidis strains 10b and 1457, and significantly reduced the numbers of bacteria in a 1-day-old in vivo biofilm (P < 0.001, one-way analysis of variance). Our findings revealed that SesC is a promising target for prevention and treatment of S. epidermidis biofilms because it affects both the primary attachment and biofilm accumulation phases. The precise role of SesC in biofilm formation remains to be identified.

Variable expression of immunoreactive surface proteins of Propionibacterium acnes

Despite accumulating data implicating Propionibacterium acnes in a variety of diseases, its precise role in infection remains to be determined. P. acnes antigen-specific CD4+ T cells are present in early inflamed acne lesions and may be involved in the inflammatory response; however, little is known about the specific antigens involved. In this study, B cell and T cell antigens from P. acnes expression libraries were cloned and evaluated and the four predominant proteins identified were investigated. Two of these antigens share some homology with an M-like protein of Streptococcus equi and have dermatan-sulphate-binding activity (PA-25957 and 5541). The remaining two antigens, PA-21693 and 4687, are similar to the product of the Corynebacterium diphtheriae htaA gene from the hmu ABC transport locus, although only one of these (PA-21693) is encoded within an hmu-like operon and conserved amongst a range of clinical isolates. All four proteins contain an LPXTG motif, although only PA-21693 contains a characteristic sortase-sorting signal. Variation in the expression of PA-4687, 25957 and 5541 is evident amongst clinical isolates and is generated both by frameshifts associated with the putative signal peptide and by variable numbers of repeat regions toward the carboxy-terminus, potentially generating heterogeneity of molecular mass and antigenic variation. In addition, in the case of PA-25957, a frameshift in a C-rich region at the extreme carboxy-terminus eliminates the LPXTG motif in some isolates. For the dermatan-sulphate-binding PA-25957, IgG1 antibody in serum from acne-positive donors was shown to be specific for the amino-terminal region of the protein, which also contains a CD4+ T cell epitope. In contrast, serum from acne-negative donors shows an IgG2 and IgG3 antibody subclass response to the carboxy-terminal region. These data have implications for the potential role of P. acnes in inflammatory acne and other diseases.

Bacillus anthracis Sortase A (SrtA) Anchors LPXTG Motif-Containing Surface Proteins to the Cell Wall Envelope

ABSTRACT 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 34F2, 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.

The SrtA Sortase of Streptococcus agalactiae Is Required for Cell Wall Anchoring of Proteins Containing the LPXTG Motif, for Adhesion to Epithelial Cells, and for Colonization of the Mouse Intestine

ABSTRACT Streptococcus agalactiae (group B streptococcus [GBS]) is the leading cause of neonatal pneumonia, sepsis, and meningitis. An in silico genome analysis indicated that GBS strain NEM316 encodes 35 proteins containing an LPXTG motif which are thought to be covalently linked to the peptidoglycan by an enzyme called sortase. The role of these cell wall-anchored proteins in GBS pathogenesis was evaluated on a global level by inactivating the srtA gene. This gene encodes the major sortase SrtA that anchors most of the LPXTG-containing proteins. We chose the C5a peptidase (ScpB) and Alp2, an abundant immunogenic protein, as prototypical LPXTG-containing proteins. As expected, the SrtA knockout mutant was unable to anchor the C5a peptidase (ScpB) and Alp2 to the cell wall. Complementation with plasmid-borne srtA inserted into the chromosome restored the correct surface localization of both ScpB and Alp2. Interestingly, the SrtA mutant was impaired for binding to the major extracellular matrix components fibronectin and fibrinogen and displayed a significant reduction in adherence to human (A549, HeLa, and Caco-2) and murine (L2) epithelial cells compared to the parental wild-type strain. Surprisingly, the inactivation of srtA had no effect on the virulence of the type III strain of GBS in a neonatal rat model (measured by the 50% lethal dose and lung colonization) but strongly impaired the capacity of the strain to colonize the intestines of gnotobiotic mice in a competition assay. These results demonstrate that LPXTG-containing proteins are involved in cell adhesion and GBS persistence in vivo.