Housekeeping Sortase Research Articles

Molecular basis for dual functions in pilus assembly modulated by the lid of a pilus-specific sortase

The biphasic assembly of Gram-positive pili begins with the covalent polymerization of distinct pilins catalyzed by a pilus-specific sortase, followed by the cell wall anchoring of the resulting polymers mediated by the housekeeping sortase. In Actinomyces oris, the pilus-specific sortase SrtC2 not only polymerizes FimA pilins to assemble type 2 fimbriae with CafA at the tip, but it can also act as the anchoring sortase, linking both FimA polymers and SrtC1-catalyzed FimP polymers (type 1 fimbriae) to peptidoglycan when the housekeeping sortase SrtA is inactive. To date, the structure-function determinants governing the unique substrate specificity and dual enzymatic activity of SrtC2 have not been illuminated. Here, we present the crystal structure of SrtC2 solved to 2.10-Å resolution. SrtC2 harbors a canonical sortase fold and a lid typical for class C sortases and additional features specific to SrtC2. Structural, biochemical, and mutational analyses of SrtC2 reveal that the extended lid of SrtC2 modulates its dual activity. Specifically, we demonstrate that the polymerizing activity of SrtC2 is still maintained by alanine-substitution, partial deletion, and replacement of the SrtC2 lid with the SrtC1 lid. Strikingly, pilus incorporation of CafA is significantly reduced by these mutations, leading to compromised polymicrobial interactions mediated by CafA. In a srtA mutant, the partial deletion of the SrtC2 lid reduces surface anchoring of FimP polymers, and the lid-swapping mutation enhances this process, while both mutations diminish surface anchoring of FimA pili. Evidently, the extended lid of SrtC2 enables the enzyme the cell wall-anchoring activity in a substrate-selective fashion.

A conserved signal-peptidase antagonist modulates membrane homeostasis of actinobacterial sortase critical for surface morphogenesis

Most Actinobacteria encode a small transmembrane protein, whose gene lies immediately downstream of the housekeeping sortase coding for a transpeptidase that anchors many extracellular proteins to the Gram-positive bacterial cell wall. Here, we uncover the hitherto unknown function of this class of conserved proteins, which we name SafA, as a topological modulator of sortase in the oral Actinobacterium Actinomyces oris. Genetic deletion of safA induces cleavage and excretion of the otherwise predominantly membrane-bound SrtA in wild-type cells. Strikingly, the safA mutant, although viable, exhibits severe abnormalities in cell morphology, pilus assembly, surface protein localization, and polymicrobial interactions-the phenotypes that are mirrored by srtA depletion. The pleiotropic defect of the safA mutant is rescued by ectopic expression of safA from not only A. oris, but also Corynebacterium diphtheriae or Corynebacterium matruchotii. Importantly, the SrtA N terminus harbors a tripartite-domain feature typical of a bacterial signal peptide, including a cleavage motif AXA, mutations in which prevent SrtA cleavage mediated by the signal peptidase LepB2. Bacterial two-hybrid analysis demonstrates that SafA and SrtA directly interact. This interaction involves a conserved motif FPW within the exoplasmic face of SafA, since mutations of this motif abrogate SafA-SrtA interaction and induce SrtA cleavage and excretion as observed in the safA mutant. Evidently, SafA is a membrane-imbedded antagonist of signal peptidase that safeguards and maintains membrane homeostasis of the housekeeping sortase SrtA, a central player of cell surface assembly.

Deciphering the substrate specificity of housekeeping sortase A and pilus-specific sortase C of probiotic bacterium Lactococcus lactis

Several strains and species of lactic acid bacteria (LAB) are widely used in fermented foods, including dairy products and also as probiotics, because of their contribution to various health benefits in humans. Sortase enzymes decorate the bacterial cell wall with different surface proteins and pili for facilitating the interactions with host and environment for the colonization and beneficial effects. While the sortases and sortase anchored proteins from pathogens have been the prime focus of the research in the past, sortases from many non-pathogenic bacteria, including LAB strains, have attracted attention for their potential applications in vaccine delivery and other clinical interventions. Here, we report the purification and functional characterization of two sortases (housekeeping SrtA and pilus-specific SrtC) from a probiotic Lactococcus lactis. The purified sortases were found to be active against the putative LPXTG motif-based peptide substrates, albeit with differences. The in-silico analysis provides insights into the residues involved in substrate binding and specificity. Overall, this study sheds new light on the aspects of structure, substrate specificity, and function of sortases from non-pathogenic bacteria, which may have physiological ramifications as well as their applications in sortase-mediated protein bioconjugation.

A cell wall-anchored glycoprotein confers resistance to cation stress in Actinomyces oris biofilms.

Actinomycesoris plays an important role in oral biofilm development. Like many gram-positive bacteria, A. oris produces a sizable number of surface proteins that are anchored to bacterial peptidoglycan by a conserved transpeptidase named the housekeeping sortase SrtA; however, the biological role of many A. oris surface proteins in biofilm formation is largely unknown. Here, we report that the glycoprotein GspA-a genetic suppressor of srtA deletion lethality-not only promotes biofilm formation but also maintains cell membrane integrity under cation stress. In comparison to wild-type cells, under elevated concentrations of mono- and divalent cations the formation of mono- and multi-species biofilms by mutant cells devoid of gspA was significantly diminished, although planktonic growth of both cell types in the presence of cations was indistinguishable. Because gspA overexpression is lethal to cells lacking gspA and srtA, we performed a genetic screen to identify GspA determinants involving cell viability. DNA sequencing and biochemical characterizations of viable clones revealed that mutations of two critical cysteine residues and a serine residue severely affected GspA glycosylation and biofilm formation. Furthermore, mutant cells lacking gspA were markedly sensitive to sodium dodecyl sulfate, a detergent that solubilizes the cytoplasmic membranes, suggesting the cell envelope of the gspA mutant was altered. Consistent with this observation, the gspA mutant exhibited increased membrane permeability, independent of GspA glycosylation, compared to the wild-type strain. Altogether, the results support the notion that the cell wall-anchored glycoprotein GspA provides a defense mechanism against cation stress in biofilm development promoted by A. oris.

Covalent sortase A inhibitor ML346 prevents Staphylococcus aureus infection of Galleria mellonella.

The housekeeping sortase A (SrtA), a membrane-associated cysteine transpeptidase, is responsible for anchoring surface proteins to the cell wall peptidoglycan in Gram-positive bacteria. This process is essential for the regulation of bacterial virulence and pathogenicity. Therefore, SrtA is considered to be an ideal target for antivirulence therapy. In this study, we report that ML346, a compound with a barbituric acid and cinnamaldehyde scaffold, functions as an irreversible inhibitor of Staphylococcus aureus SrtA (SaSrtA) and Streptococcus pyogenes SrtA (SpSrtA) in vitro at low micromolar concentrations. According to our X-ray crystal structure of the SpSrtAΔN81/ML346 complex (Protein Data Bank ID: 7V6K), ML346 covalently modifies the thiol group of Cys208 in the active site of SpSrtA. Importantly, ML346 significantly attenuated the virulence phenotypes of S. aureus and exhibited inhibitory effects on Galleria mellonella larva infection caused by S. aureus. Collectively, our results indicate that ML346 has potential for development as a covalent antivirulence agent for treating S. aureus infections, including methicillin-resistant S. aureus.

Critical roles of a housekeeping sortase of probiotic Bifidobacterium bifidum in bacterium–host cell crosstalk

SummaryBifidobacterium bifidum YIT 10347 (BF-1) is adhesive in vitro. Here we studied the molecular aspects of the BF-1 adhesion process. We identified and characterized non-adhesive mutants and found that a class E housekeeping sortase was critical for the adhesion to mucin. These mutants were significantly less adhesive to GCIY cells than was the wild type (WT), which protected GCIY cells against acid treatment more than did a non-adhesive mutant. The non-adhesive mutants aberrantly accumulated precursors of putative sortase-dependent proteins (SDPs). Recombinant SDPs bound to mucin. Disruption of the housekeeping sortase influenced expression of SDPs and pilus components. Mutants defective in a pilin or in an SDP showed the same adhesion properties as WT. Therefore, multiple SDPs and pili seem to work cooperatively to achieve adhesion, and the housekeeping sortase is responsible for cell wall anchoring of its substrates to ensure their proper biological function.

Selection and characterization of a SpaCBA pilus-secreting food-grade derivative of Lacticaseibacillus rhamnosus GG

Many studies have established the functional properties of Lacticaseibacillus rhamnosus GG, previously known as Lactobacillus rhamnosus GG, marketed worldwide as a probiotic. The extraordinary capacity of L. rhamnosus GG to bind to human mucus and influence the immune system especially stand out. Earlier, we have shown the key role of its SpaCBA sortase-dependent pili encoded by the spaCBA-srtC1 gene cluster herein. These heterotrimeric pili consist of a shaft pilin SpaA, a basal pilin SpaB, and tip pilin SpaC that contains a mucus-binding domain. Here, we set out to characterize a food-grade non-GMO mutant of L. rhamnosus GG, strain PA11, which secretes its pilins, rather than coupling them to the cell surface, due to a defect in the housekeeping sortase A. The sortase-negative strain PA11 was extensively characterized using functional genomics and biochemical approaches and found to secrete the SpaCBA pili into the supernatant. Given the functional importance and uniqueness of the mucus-binding pili of L. rhamnosus GG, strain PA11 offers novel opportunities towards the characterization and further therapeutic application of SpaCBA pili and their low-cost, large-scale production.Key points•Creation of pilus-secreting mutant (PA11) of the key probiotic LGG.•Strain PA11 is defective in a functional housekeeping sortase SrtA.•Strain PA11 opens novel biotherapeutic application avenues.Graphical abstract

Interrogation of 3D-swapped structure and functional attributes of quintessential Sortase A from Streptococcus pneumoniae.

The anchoring of the surface proteins to the cell wall in gram-positive bacteria involves a peptide ligation reaction catalyzed by transpeptidase sortase. Most bacterial genomes encode multiple sortases with dedicated functions. Streptococcus pneumoniae (Sp) carries four sortases; a housekeeping sortase (SrtA), and three pilin specific sortases (SrtC1, C2, C3) dedicated to the biosynthesis of covalent pilus. Interestingly, SrtA, meant for performing housekeeping roles, is also implicated in pilus assembly of Sp. The allegiance of SpSrtA to the pathogenic pilus assembly makes it an ideal target for clinical inhibitor development. In this paper, we describe biochemical characterization, crystal structure and peptide substrate preference of SpSrtA. Transpeptidation reaction with a variety of substrates revealed that the enzyme preferred elongated LPXTG sequences and transferred them equally well to both Ala- and Gly-terminated peptides. Curiously, crystal structure of both wild type and an active site (Cys to Ala) mutant of SpSrtA displayed inter-twined 3D-swapped dimers in which each protomer generated a classic eight stranded beta-barrel "sortase fold". Size-exclusion chromatography and sedimentation equilibrium measurements revealed predominant presence of a dimer in equilibrium with its monomer. The crystal structure-based Cys-Cys distance mapping with defined chemical cross-linkers established the existence of 3D-swapped structure in solution. The swapping in SpSrtA, unprecedented for sortase family, may be physiologically relevant and meant to perform regulatory functions.

Role of SrtA in Pathogenicity of Staphylococcus lugdunensis.

Among coagulase-negative staphylococci (CoNS), Staphylococcus lugdunensis has a special position as causative agent of aggressive courses of infectious endocarditis (IE) more reminiscent of IEs caused by Staphylococcus aureus than those by CoNS. To initiate colonization and invasion, bacterial cell surface proteins are required; however, only little is known about adhesion of S. lugdunensis to biotic surfaces. Cell surface proteins containing the LPXTG anchor motif are covalently attached to the cell wall by sortases. Here, we report the functionality of Staphylococcus lugdunensis sortase A (SrtA) to link LPXTG substrates to the cell wall. To determine the role of SrtA dependent surface proteins in biofilm formation and binding eukaryotic cells, we generated SrtA-deficient mutants (ΔsrtA). These mutants formed a smaller amount of biofilm and bound less to immobilized fibronectin, fibrinogen, and vitronectin. Furthermore, SrtA absence affected the gene expression of two different adhesins on transcription level. Surprisingly, we found no decreased adherence and invasion in human cell lines, probably caused by the upregulation of further adhesins in ΔsrtA mutant strains. In conclusion, the functionality of S. lugdunensis SrtA in anchoring LPXTG substrates to the cell wall let us define it as the pathogen’s housekeeping sortase.

Development and validation of a high-throughput whole cell assay to investigate Staphylococcus aureus adhesion to host ligands

Staphylococcus aureus adhesion to the host's skin and mucosae enables asymptomatic colonization and the establishment of infection. This process is facilitated by cell wall-anchored adhesins that bind to host ligands. Therapeutics targeting this process could provide significant clinical benefits; however, the development of anti-adhesives requires an in-depth knowledge of adhesion-associated factors and an assay amenable to high-throughput applications. Here, we describe the development of a sensitive and robust whole cell assay to enable the large-scale profiling of S. aureus adhesion to host ligands. To validate the assay, and to gain insight into cellular factors contributing to adhesion, we profiled a sequence-defined S. aureus transposon mutant library, identifying mutants with attenuated adhesion to human-derived fibronectin, keratin, and fibrinogen. Our screening approach was validated by the identification of known adhesion-related proteins, such as the housekeeping sortase responsible for covalently linking adhesins to the cell wall. In addition, we also identified genetic loci that could represent undescribed anti-adhesive targets. To compare and contrast the genetic requirements of adhesion to each host ligand, we generated a S. aureus Genetic Adhesion Network, which identified a core gene set involved in adhesion to all three host ligands, and unique genetic signatures. In summary, this assay will enable high-throughput chemical screens to identify anti-adhesives and our findings provide insight into the target space of such an approach.

New Paradigms of Pilus Assembly Mechanisms in Gram-Positive Actinobacteria.

Adhesive pili in Gram-positive bacteria represent a variety of extracellular multiprotein polymers that mediate bacterial colonization of specific host tissues and associated pathogenesis. Pili are assembled in two distinct but coupled steps, an orderly crosslinking of pilin monomers and subsequent anchoring of the polymer to peptidoglycan, catalyzed by two transpeptidase enzymes - the pilus-specific sortase and the housekeeping sortase. Here, we review this biphasic assembly mechanism based on studies of two prototypical models, the heterotrimeric pili in Corynebacterium diphtheriae and the heterodimeric pili in Actinomyces oris, highlighting some newly emerged basic paradigms. The disparate mechanisms of protein ligation mediated by the pilus-specific sortase and the spatial positioning of adhesive pili on the cell surface modulated by the housekeeping sortase are among the notable highlights.

Role of Sortase A in Lactobacillus gasseri Kx110A1 Adhesion to Gastric Epithelial Cells and Competitive Exclusion of Helicobacter pylori.

We have previously shown that Lactobacillus gasseri Kx110A1, a human stomach isolate, can colonize mouse stomach and reduce the initial colonization of Helicobacter pylori. Here, we investigated the role of sortase-dependent proteins (SDPs) involved in these functions by the construction of a mutant for srtA, the gene encoding the housekeeping sortase that covalently anchors SDPs to the cell surface. The srtA mutant showed a decrease in hydrophobicity and autoaggregation under acidic conditions, indicating the effect of SDPs on cell surface properties. Correspondingly, the srtA mutant lost the capacity to adhere to gastric epithelial cells, thus resulting in an inability to provide a physical barrier to prevent H. pylori adherence. These results indicate that sortase A is a key determinant of the cell surface properties of L. gasseri Kx110A1 and contributes to Lactobacillus-mediated exclusion of H. pylori. Understanding the molecular mechanisms by which lactobacilli antagonize H. pylori might contribute to the development of novel therapeutic strategies that take advantage of health-promoting bacteria and reduce the burden of antibiotic resistance.

Cell-to-cell interaction requires optimal positioning of a pilus tip adhesin modulated by gram-positive transpeptidase enzymes

Assembly of pili on the gram-positive bacterial cell wall involves 2 conserved transpeptidase enzymes named sortases: One for polymerization of pilin subunits and another for anchoring pili to peptidoglycan. How this machine controls pilus length and whether pilus length is critical for cell-to-cell interactions remain unknown. We report here in Actinomyces oris, a key colonizer in the development of oral biofilms, that genetic disruption of its housekeeping sortase SrtA generates exceedingly long pili, catalyzed by its pilus-specific sortase SrtC2 that possesses both pilus polymerization and cell wall anchoring functions. Remarkably, the srtA-deficient mutant fails to mediate interspecies interactions, or coaggregation, even though the coaggregation factor CafA is present at the pilus tip. Increasing ectopic expression of srtA in the mutant progressively shortens pilus length and restores coaggregation accordingly, while elevated levels of shaft pilins and SrtC2 produce long pili and block coaggregation by SrtA+ bacteria. With structural studies, we uncovered 2 key structural elements in SrtA that partake in recognition of pilin substrates and regulate pilus length by inducing the capture and transfer of pilus polymers to the cell wall. Evidently, coaggregation requires proper positioning of the tip adhesin CafA via modulation of pilus length by the housekeeping sortase SrtA.

Sortase-Dependent Proteins Promote Gastrointestinal Colonization by Enterococci.

The human gastrointestinal tract (GIT) is inhabited by a dense microbial community of symbionts. Enterococci are among the earliest members of this community and remain core members of the GIT microbiota throughout life. Enterococci have also recently emerged as opportunistic pathogens and major causes of nosocomial infections. Although recognized as a prerequisite for infection, colonization of the GIT by enterococci remains poorly understood. One way that bacteria adapt to dynamic ecosystems like the GIT is through the use of their surface proteins to sense and interact with components of their immediate environment. In Gram-positive bacteria, a subset of surface proteins relies on an enzyme called sortase for covalent attachment to the cell wall. Here, we show that the housekeeping sortase A (SrtA) enzyme promotes intestinal colonization by enterococci. Furthermore, we show that the enzymatic activity of SrtA is key to the ability of Enterococcus faecalis to bind mucin (a major component of the GIT mucus). We also report the GIT colonization phenotypes of E. faecalis mutants lacking selected sortase-dependent proteins (SDPs). Further examination of the mucin binding ability of these mutants suggests that adhesion to mucin contributes to intestinal colonization by E. faecalis.

Characterization of the housekeeping sortase from the human pathogen Propionibacterium acnes: first investigation of a class F sortase.

Sortase enzymes play an important role in Gram-positive bacteria. They are responsible for the covalent attachment of proteins to the surface of the bacteria and perform this task via a highly sequence-specific transpeptidation reaction. Since these immobilized proteins are often involved in pathogenicity of Gram-positive bacteria, characterization of this type of enzyme is also of medical relevance. Different classes of sortases (A-F) have been found, which recognize characteristic recognition sequences present in substrate proteins. Up to date, sortase A from Staphylococcus aureus, a housekeeping class A sortase, is the most thoroughly studied representative of the sortase family of enzymes. Here we report the in-depth characterization of the class F sortase from Propionibacterium acnes, a class of sortases that has not been investigated before. As Sortase F is the only transpeptidase found in the P. acnes genome, it is the housekeeping sortase of this organism. Sortase F from P. acnes shows a behavior similar to sortases from class A in terms of pH dependence, recognition sequence and catalytic activity; furthermore, its activity is independent of bivalent ions, which contrasts to sortase A from S. aureus We demonstrate that sortase F is useful for protein engineering applications, by producing a site-specifically conjugated homogenous antibody-drug conjugate with a potency similar to that of a conjugate prepared with sortase A. Thus, the detailed characterization presented here will not only enable the development of anti-virulence agents targeting P. acnes but also provides a powerful alternative to sortase A for protein engineering applications.

Structure and Mechanism of LcpA, a Phosphotransferase That Mediates Glycosylation of a Gram-Positive Bacterial Cell Wall-Anchored Protein.

The widely conserved LytR-CpsA-Psr (LCP) family of enzymes in Gram-positive bacteria is known to attach glycopolymers, including wall teichoic acid, to the cell envelope. However, it is undetermined if these enzymes are capable of catalyzing glycan attachment to surface proteins. In the actinobacterium Actinomyces oris, an LCP homolog here named LcpA is genetically linked to GspA, a glycoprotein that is covalently attached to the bacterial peptidoglycan by the housekeeping sortase SrtA. Here we show by X-ray crystallography that LcpA adopts an α-β-α structural fold, akin to the conserved LCP domain, which harbors characteristic catalytic arginine residues. Consistently, alanine substitution for these residues, R149 and R266, abrogates GspA glycosylation, leading to accumulation of an intermediate form termed GspALMM, which is also observed in the lcpA mutant. Unlike other LCP proteins characterized to date, LcpA contains a stabilizing disulfide bond, mutations of which severely affect LcpA stability. In line with the established role of disulfide bond formation in oxidative protein folding in A. oris, deletion of vkor, coding for the thiol-disulfide oxidoreductase VKOR, also significantly reduces LcpA stability. Biochemical studies demonstrated that the recombinant LcpA enzyme possesses pyrophosphatase activity, enabling hydrolysis of diphosphate bonds. Furthermore, this recombinant enzyme, which weakly interacts with GspA in solution, catalyzes phosphotransfer to GspALMM Altogether, the findings support that A. oris LcpA is an archetypal LCP enzyme that glycosylates a cell wall-anchored protein, a process that may be conserved in Actinobacteria, given the conservation of LcpA and GspA in these high-GC-content organisms.IMPORTANCE In Gram-positive bacteria, the conserved LCP family enzymes studied to date are known to attach glycopolymers, including wall teichoic acid, to the cell envelope. It is unknown if these enzymes catalyze glycosylation of surface proteins. We show here in the actinobacterium Actinomyces oris by X-ray crystallography and biochemical analyses that A. oris LcpA is an LCP homolog, possessing pyrophosphatase and phosphotransferase activities known to belong to LCP enzymes that require conserved catalytic Arg residues, while harboring a unique disulfide bond critical for protein stability. Importantly, LcpA mediates glycosylation of the surface protein GspA via phosphotransferase activity. Our studies provide the first experimental evidence of an archetypal LCP enzyme that promotes glycosylation of a cell wall-anchored protein in Gram-positive bacteria.

The Streptococcos suis sortases SrtB and SrtF are essential for disease in pigs.

The porcine pathogen Streptococcus suis colonizes the upper respiratory tracts of pigs, potentially causing septicaemia, meningitis and death, thus placing a severe burden on the agricultural industry worldwide. It is also a zoonotic pathogen that is known to cause systemic infections and meningitis in humans. Understanding how S. suis colonizes and interacts with its hosts is relevant for future strategies of drug and vaccine development. As with other Gram-positive bacteria, S. suis utilizes enzymes known as sortases to attach specific proteins bearing cell wall sorting signals to its surface, where they can play a role in host-pathogen interactions. The surface proteins of bacteria are often important in adhesion to and invasion of host cells. In this study, markerless in-frame deletion mutants of the housekeeping sortase srtA and the two pilus-associated sortases, srtB and srtF, were generated and their importance in S. suis infections was investigated. We found that all three of these sortases are essential to disease in pigs, concluding that their cognate-sorted proteins may also be useful in protecting pigs against infection.

Rapid and Direct VHH and Target Identification by Staphylococcal Surface Display Libraries.

Unbiased and simultaneous identification of a specific antibody and its target antigen has been difficult without prior knowledge of at least one interaction partner. Immunization with complex mixtures of antigens such as whole organisms and tissue extracts including tumoral ones evokes a highly diverse immune response. During such a response, antibodies are generated against a variety of epitopes in the mixture. Here, we propose a surface display design that is suited to simultaneously identify camelid single domain antibodies and their targets. Immune libraries of single-domain antigen recognition fragments from camelid heavy chain-only antibodies (VHH) were attached to the peptidoglycan of Gram-positive Staphylococcus aureus employing its endogenous housekeeping sortase enzyme. The sortase transpeptidation reaction covalently attached the VHH to the bacterial peptidoglycan. The reversible nature of the reaction allowed the recovery of the VHH from the bacterial surface and the use of the VHH in downstream applications. These staphylococcal surface display libraries were used to rapidly identify VHH as well as their targets by immunoprecipitation (IP). Our novel bacterial surface display platform was stable under harsh screening conditions, allowed fast target identification, and readily permitted the recovery of the displayed VHH for downstream analysis.

Pilus biogenesis of Gram-positive bacteria: Roles of sortases and implications for assembly.

Successful adherence, colonization, and survival of Gram-positive bacteria require surface proteins, and multiprotein assemblies called pili. These surface appendages are attractive pharmacotherapeutic targets and understanding their assembly mechanisms is essential for identifying a new class of 'anti-infectives' that do not elicit microbial resistance. Molecular details of the Gram-negative pilus assembly are available indepth, but the Gram-positive pilus biogenesis is still an emerging field and investigations continue to reveal novel insights into this process. Pilus biogenesis in Gram-positive bacteria is a biphasic process that requires enzymes called pilus-sortases for assembly and a housekeeping sortase for covalent attachment of the assembled pilus to the peptidoglycan cell wall. Emerging structural and functional data indicate that there are at least two groups of Gram-positive pili, which require either the Class C sortase or Class B sortase in conjunction with LepA/SipA protein for major pilin polymerization. This observation suggests two distinct modes of sortase-mediated pilus biogenesis in Gram-positive bacteria. Here we review the structural and functional biology of the pilus-sortases from select streptococcal pilus systems and their role in Gram-positive pilus assembly.

Structure and specificity of a new class of Ca2+-independent housekeeping sortase from Streptomyces avermitilis provide insights into its non-canonical substrate preference

Surface proteins in Gram-positive bacteria are incorporated into the cell wall through a peptide ligation reaction catalyzed by transpeptidase sortase. Six main classes (A-F) of sortase have been identified of which class A sortase is meant for housekeeping functions. The prototypic housekeeping sortase A (SaSrtA) from Staphylococcus aureus cleaves LPXTG-containing proteins at the scissile T-G peptide bond and ligates protein-LPXT to the terminal Gly residue of the nascent cross-bridge of peptidoglycan lipid II precursor. Sortase-mediated ligation ("sortagging") of LPXTG-containing substrates and Gly-terminated nucleophiles occurs in vitro as well as in cellulo in the presence of Ca2+ and has been applied extensively for protein conjugations. Although the majority of applications emanate from SaSrtA, low catalytic efficiency, LPXTG specificity restriction, and Ca2+ requirement (particularly for in cellulo applications) remain a drawback. Given that Gram-positive bacteria genomes encode a variety of sortases, natural sortase mining can be a viable complementary approach akin to engineering of wild-type SaSrtA. Here, we describe the structure and specificity of a new class E sortase (SavSrtE) annotated to perform housekeeping roles in Streptomyces avermitilis Biochemical experiments define the attributes of an optimum peptide substrate, demonstrate Ca2+-independent activity, and provide insights about contrasting functional characteristics of SavSrtE and SaSrtA. Crystal structure, substrate docking, and mutagenesis experiments have identified a critical residue that dictates the preference for a non-canonical LAXTG recognition motif over LPXTG. These results have implications for rational tailoring of substrate tolerance in sortases. Besides, Ca2+-independent orthogonal specificity of SavSrtE is likely to expand the sortagging toolkit.