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Olaf Schneewind - One of the best experts on this subject based on the ideXlab platform.
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Sortases, Surface Proteins, and Their Roles in Staphylococcus aureus Disease and Vaccine Development
Microbiology spectrum, 2019Co-Authors: Olaf Schneewind, Dominique MissiakasAbstract: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 Staphylococcus aureus, 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. S. aureus 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 S. aureus. Alternatively, small-molecule inhibitors of Sortase may be useful agents for the prevention of S. aureus colonization and invasive disease.
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Total Chemical Synthesis of the Enzyme Sortase AΔN59 with Full Catalytic Activity
Angewandte Chemie, 2014Co-Authors: Fang‐kun Deng, Ya-ting Wang, Olaf Schneewind, Liang Zhang, Stephen B. H. KentAbstract:The enzyme Sortase A is a ligase which catalyzes transpeptidation reactions.1, 2 Surface proteins, including virulence factors, that have a C terminal recognition sequence are attached to Gly5 on the peptidoglycan of bacterial cell walls by Sortase A.1 The enzyme is an important anti-virulence and anti-infective drug target for resistant strains of Gram-positive bacteria.2 In addition, because Sortase A enables the splicing of polypeptide chains, the transpeptidation reaction catalyzed by Sortase A is a potentially valuable tool for protein science.3 Here we describe the total chemical synthesis of enzymatically active Sortase A. The target 148 residue polypeptide chain of Sortase AΔN59 was synthesized by the convergent chemical ligation of four unprotected synthetic peptide segments. The folded protein molecule was isolated by size-exclusion chromatography and had full enzymatic activity in a transpeptidation assay. Total synthesis of Sortase A will enable more sophisticated engineering of this important enzyme molecule.
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Architects at the bacterial surface — Sortases and the assembly of pili with isopeptide bonds
Nature reviews. Microbiology, 2011Co-Authors: Antoni P. A. Hendrickx, Jonathan M. Budzik, Olaf SchneewindAbstract:The cell wall envelope of Gram-positive bacteria can be thought of as a surface organelle for the assembly of macromolecular structures that enable the unique lifestyle of each microorganism. Sortases - enzymes that cleave the sorting signals of secreted proteins to form isopeptide (amide) bonds between the secreted proteins and peptidoglycan or polypeptides - function as the principal architects of the bacterial surface. Acting alone or with other Sortase enzymes, Sortase construction leads to the anchoring of surface proteins at specific sites in the envelope or to the assembly of pili, which are fibrous structures formed from many protein subunits. The catalysis of intermolecular isopeptide bonds between pilin subunits is intertwined with the assembly of intramolecular isopeptide bonds within pilin subunits. Together, these isopeptide bonds endow these Sortase products with adhesive properties and resistance to host proteases.
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Sortase d forms the covalent bond that links bcpb to the tip of bacillus cereus pili
Journal of Biological Chemistry, 2009Co-Authors: Jonathan M. Budzik, So-young Oh, Olaf SchneewindAbstract:Bacillus cereus and other Gram-positive bacteria elaborate pili via a Sortase D-catalyzed transpeptidation mechanism from major and minor pilin precursor substrates. After cleavage of the LPXTG sorting signal of the major pilin, BcpA, Sortase D forms an amide bond between the C-terminal threonine and the amino group of lysine within the YPKN motif of another BcpA subunit. Pilus assembly terminates upon Sortase A cleavage of the BcpA sorting signal, resulting in a covalent bond between BcpA and the cell wall cross-bridge. Here, we show that the IPNTG sorting signal of BcpB, the minor pilin, is cleaved by Sortase D but not by Sortase A. The C-terminal threonine of BcpB is amide-linked to the YPKN motif of BcpA, thereby positioning BcpB at the tip of pili. Thus, unique attributes of the sorting signals of minor pilins provide Gram-positive bacteria with a universal mechanism ordering assembly of pili.
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Activation of Inhibitors by Sortase Triggers Irreversible Modification of the Active Site
Journal of Biological Chemistry, 2007Co-Authors: Anthony William Maresso, Ruiying Wu, Justin W. Kern, Rongguang Zhang, Mark-eugene Duban, Dusan Janik, Dominique Missiakas, Andrzej Joachimiak, Olaf SchneewindAbstract:Sortases anchor surface proteins to the cell wall of Gram-positive pathogens through recognition of specific motif sequences. Loss of Sortase leads to large reductions in virulence, which identifies Sortase as a target for the development of antibacterials. By screening 135,625 small molecules for inhibition, we report here that aryl (β-amino)ethyl ketones inhibit Sortase enzymes from staphylococci and bacilli. Inhibition of Sortases occurs through an irreversible, covalent modification of their active site cysteine. Sortases specifically activate this class of molecules via β-elimination, generating a reactive olefin intermediate that covalently modifies the cysteine thiol. Analysis of the three-dimensional structure of Bacillus anthracis Sortase B with and without inhibitor provides insights into the mechanism of inhibition and reveals binding pockets that can be exploited for drug discovery.
Hidde L Ploegh - One of the best experts on this subject based on the ideXlab platform.
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Site-Specific Protein Labeling via Sortase-Mediated Transpeptidation.
Current protocols in protein science, 2017Co-Authors: John M. Antos, Jessica Ingram, Matthias C Truttmann, Novalia Pishesha, Tao Fang, Hidde L PloeghAbstract:Strategies for site-specific protein modification are highly desirable for the construction of conjugates containing non-genetically-encoded functional groups. Ideally, these strategies should proceed under mild conditions, and be compatible with a wide range of protein targets and non-natural moieties. The transpeptidation reaction catalyzed by bacterial Sortases is a prominent strategy for protein derivatization that possesses these features. Naturally occurring or engineered variants of Sortase A from Staphylococcus aureus catalyze a ligation reaction between a five-amino-acid substrate motif (LPXTG) and oligoglycine nucleophiles. By pairing proteins and synthetic peptides that possess these ligation handles, it is possible to install modifications onto the protein N- or C-terminus in site-specific fashion. As described in this unit, the successful implementation of Sortase-mediated labeling involves straightforward solid-phase synthesis and molecular biology techniques, and this method is compatible with proteins in solution or on the surface of live cells. © 2017 by John Wiley & Sons, Inc.
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Recent advances in Sortase-catalyzed ligation methodology.
Current Opinion in Structural Biology, 2016Co-Authors: John M. Antos, Matthias C Truttmann, Hidde L PloeghAbstract:The transpeptidation reaction catalyzed by bacterial Sortases continues to see increasing use in the construction of novel protein derivatives. In addition to growth in the number of applications that rely on Sortase, this field has also seen methodology improvements that enhance reaction performance and scope. In this opinion, we present an overview of key developments in the practice and implementation of Sortase-based strategies, including applications relevant to structural biology. Topics include the use of engineered Sortases to increase reaction rates, the use of redesigned acyl donors and acceptors to mitigate reaction reversibility, and strategies for expanding the range of substrates that are compatible with a Sortase-based approach.
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site specific n terminal labeling of proteins using Sortase mediated reactions
Nature Protocols, 2013Co-Authors: Christopher S Theile, Annet E M Blom, Martin D. Witte, Lenka Kundrat, Hidde L Ploegh, Carla P. GuimaraesAbstract:This protocol describes the use of Sortase-mediated reactions to label the N terminus of any given protein of interest. The Sortase recognition sequence, LPXTG (for Streptococcus aureus Sortase A) or LPXTA (for Staphylococcus pyogenes Sortase A), can be appended to a variety of probes such as fluorophores, biotin or even to other proteins. The protein to be labeled acts as a nucleophile by attacking the intermediate formed between the probe containing the LPXTG/A motif and the Sortase enzyme. If Sortase, the protein of interest and a suitably functionalized label are available, the reactions usually require less than 3 h.
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site specific c terminal and internal loop labeling of proteins using Sortase mediated reactions
Nature Protocols, 2013Co-Authors: Carla P. Guimaraes, Annet E M Blom, Martin D. Witte, Christopher S Theile, Lenka Kundrat, Gunes Bozkurt, Hidde L PloeghAbstract:Methods for site-specific modification of proteins should be quantitative and versatile with respect to the nature and size of the biological or chemical targets involved. They should require minimal modification of the target, and the underlying reactions should be completed in a reasonable amount of time under physiological conditions. Sortase-mediated transpeptidation reactions meet these criteria and are compatible with other labeling methods. Here we describe the expression and purification conditions for two Sortase A enzymes that have different recognition sequences. We also provide a protocol that allows the functionalization of any given protein at its C terminus, or, for select proteins, at an internal site. The target protein is engineered with a Sortase-recognition motif (LPXTG) at the place where modification is desired. Upon recognition, Sortase cleaves the protein between the threonine and glycine residues, facilitating the attachment of an exogenously added oligoglycine peptide modified with the functional group of choice (e.g., fluorophore, biotin, protein or lipid). Expression and purification of Sortase takes ∼3 d, and Sortase-mediated reactions take only a few minutes, but reaction times can be extended to increase yields.
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m13 bacteriophage display framework that allows Sortase mediated modification of surface accessible phage proteins
Bioconjugate Chemistry, 2012Co-Authors: Gaelen T. Hess, Juan J. Cragnolini, Maximilian Weilin Popp, Stephanie K Dougan, Eric Spooner, Arianna M Belcher, Hidde L Ploegh, Mark Allen, Carla P. GuimaraesAbstract:We exploit bacterial Sortases to attach a variety of moieties to the capsid proteins of M13 bacteriophage. We show that pIII, pIX, and pVIII can be functionalized with entities ranging from small molecules (e.g., fluorophores, biotin) to correctly folded proteins (e.g., GFP, antibodies, streptavidin) in a site-specific manner, and with yields that surpass those of any reported using phage display technology. A case in point is modification of pVIII. While a phage vector limits the size of the insert into pVIII to a few amino acids, a phagemid system limits the number of copies actually displayed at the surface of M13. Using Sortase-based reactions, a 100-fold increase in the efficiency of display of GFP onto pVIII is achieved. Taking advantage of orthogonal Sortases, we can simultaneously target two distinct capsid proteins in the same phage particle and maintain excellent specificity of labeling. As demonstrated in this work, this is a simple and effective method for creating a variety of structures, thu...
David R Liu - One of the best experts on this subject based on the ideXlab platform.
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laboratory evolution of a Sortase enzyme that modifies amyloid β protein
Nature Chemical Biology, 2021Co-Authors: Brent M. Dorr, Christopher J Podracky, Alexandra Desousa, Dominic M Walsh, David R LiuAbstract:Epitope-specific enzymes are powerful tools for site-specific protein modification but generally require genetic manipulation of the target protein. Here, we describe the laboratory evolution of the bacterial transpeptidase Sortase A to recognize the LMVGG sequence in endogenous amyloid-β (Aβ) protein. Using a yeast display selection for covalent bond formation, we evolved a Sortase variant that prefers LMVGG substrates from a starting enzyme that prefers LPESG substrates, resulting in a >1,400-fold change in substrate preference. We used this evolved Sortase to label endogenous Aβ in human cerebrospinal fluid, enabling the detection of Aβ with sensitivities rivaling those of commercial assays. The evolved Sortase can conjugate a hydrophilic peptide to Aβ42, greatly impeding the ability of the resulting protein to aggregate into higher-order structures. These results demonstrate laboratory evolution of epitope-specific enzymes toward endogenous targets as a strategy for site-specific protein modification without target gene manipulation and enable potential future applications of Sortase-mediated labeling of Aβ peptides. Laboratory evolution of the bacterial transpeptidase Sortase A coupled with yeast display selection enables a change of the enzyme’s substrate preference to recognize and covalently label endogenous amyloid-β protein, impeding the protein’s ability to aggregate.
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Laboratory evolution of a Sortase enzyme that modifies amyloid-β protein.
Nature chemical biology, 2021Co-Authors: Christopher J Podracky, Brent M. Dorr, Alexandra Desousa, Dominic M Walsh, David R LiuAbstract:Epitope-specific enzymes are powerful tools for site-specific protein modification but generally require genetic manipulation of the target protein. Here, we describe the laboratory evolution of the bacterial transpeptidase Sortase A to recognize the LMVGG sequence in endogenous amyloid-β (Aβ) protein. Using a yeast display selection for covalent bond formation, we evolved a Sortase variant that prefers LMVGG substrates from a starting enzyme that prefers LPESG substrates, resulting in a >1,400-fold change in substrate preference. We used this evolved Sortase to label endogenous Aβ in human cerebrospinal fluid, enabling the detection of Aβ with sensitivities rivaling those of commercial assays. The evolved Sortase can conjugate a hydrophilic peptide to Aβ42, greatly impeding the ability of the resulting protein to aggregate into higher-order structures. These results demonstrate laboratory evolution of epitope-specific enzymes toward endogenous targets as a strategy for site-specific protein modification without target gene manipulation and enable potential future applications of Sortase-mediated labeling of Aβ peptides.
Robert T. Clubb - One of the best experts on this subject based on the ideXlab platform.
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Sortase Transpeptidases: Structural Biology and Catalytic Mechanism.
Advances in Protein Chemistry, 2017Co-Authors: Alex William Jacobitz, Kattke, Jeff Wereszczynski, Robert T. ClubbAbstract:Abstract Gram-positive bacteria use Sortase cysteine transpeptidase enzymes to covalently attach proteins to their cell wall and to assemble pili. In pathogenic bacteria Sortases are potential drug targets, as many of the proteins that they display on the microbial surface play key roles in the infection process. Moreover, the Staphylococcus aureus Sortase A (SaSrtA) enzyme has been developed into a valuable biochemical reagent because of its ability to ligate biomolecules together in vitro via a covalent peptide bond. Here we review what is known about the structures and catalytic mechanism of Sortase enzymes. Based on their primary sequences, most Sortase homologs can be classified into six distinct subfamilies, called class A–F enzymes. Atomic structures reveal unique, class-specific variations that support alternate substrate specificities, while structures of Sortase enzymes bound to sorting signal mimics shed light onto the molecular basis of substrate recognition. The results of computational studies are reviewed that provide insight into how key reaction intermediates are stabilized during catalysis, as well as the mechanism and dynamics of substrate recognition. Lastly, the reported in vitro activities of Sortases are compared, revealing that the transpeptidation activity of SaSrtA is at least 20-fold faster than other Sortases that have thus far been characterized. Together, the results of the structural, computational, and biochemical studies discussed in this review begin to reveal how Sortases decorate the microbial surface with proteins and pili, and may facilitate ongoing efforts to discover therapeutically useful small molecule inhibitors.
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crystal structure of the streptomyces coelicolor Sortase e1 transpeptidase provides insight into the binding mode of the novel class e sorting signal
PLOS ONE, 2016Co-Authors: Michele D. Kattke, Michael R. Sawaya, Albert H Chan, Andrew Duong, Danielle L Sexton, Duilio Cascio, Marie A Elliot, Robert T. ClubbAbstract:Many species of Gram-positive bacteria use Sortase transpeptidases to covalently affix proteins to their cell wall or to assemble pili. Sortase-displayed proteins perform critical and diverse functions for cell survival, including cell adhesion, nutrient acquisition, and morphological development, among others. Based on their amino acid sequences, there are at least six types of Sortases (class A to F enzymes); however, class E enzymes have not been extensively studied. Class E Sortases are used by soil and freshwater-dwelling Actinobacteria to display proteins that contain a non-canonical LAXTG sorting signal, which differs from 90% of known sorting signals by substitution of alanine for proline. Here we report the first crystal structure of a class E Sortase, the 1.93 A resolution structure of the SrtE1 enzyme from Streptomyces coelicolor. The active site is bound to a tripeptide, providing insight into the mechanism of substrate binding. SrtE1 possesses β3/β4 and β6/β7 active site loops that contact the LAXTG substrate and are structurally distinct from other classes. We propose that SrtE1 and other class E Sortases employ a conserved tyrosine residue within their β3/β4 loop to recognize the amide nitrogen of alanine at position P3 of the sorting signal through a hydrogen bond, as seen here. Incapability of hydrogen-bonding with canonical proline-containing sorting signals likely contributes to class E substrate specificity. Furthermore, we demonstrate that surface anchoring of proteins involved in aerial hyphae formation requires an N-terminal segment in SrtE1 that is presumably positioned within the cytoplasm. Combined, our results reveal unique features within class E enzymes that enable them to recognize distinct sorting signals, and could facilitate the development of substrate-based inhibitors of this important enzyme family.
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structural and computational studies of the staphylococcus aureus Sortase b substrate complex reveal a substrate stabilized oxyanion hole
Journal of Biological Chemistry, 2014Co-Authors: Alex William Jacobitz, Jeff Wereszczynski, Sung Wook Yi, Brendan R Amer, Grace L Huang, Angelyn V Nguyen, Andrew J Mccammon, Michael E Jung, Michael R. Sawaya, Robert T. ClubbAbstract:Sortase cysteine transpeptidases covalently attach proteins to the bacterial cell wall or assemble fiber-like pili that promote bacterial adhesion. Members of this enzyme superfamily are widely distributed in Gram-positive bacteria that frequently utilize multiple Sortases to elaborate their peptidoglycan. Sortases catalyze transpeptidation using a conserved active site His-Cys-Arg triad that joins a sorting signal located at the C terminus of their protein substrate to an amino nucleophile located on the cell surface. However, despite extensive study, the catalytic mechanism and molecular basis of substrate recognition remains poorly understood. Here we report the crystal structure of the Staphylococcus aureus Sortase B enzyme in a covalent complex with an analog of its NPQTN sorting signal substrate, revealing the structural basis through which it displays the IsdC protein involved in heme-iron scavenging from human hemoglobin. The results of computational modeling, molecular dynamics simulations, and targeted amino acid mutagenesis indicate that the backbone amide of Glu224 and the side chain of Arg233 form an oxyanion hole in Sortase B that stabilizes high energy tetrahedral catalytic intermediates. Surprisingly, a highly conserved threonine residue within the bound sorting signal substrate facilitates construction of the oxyanion hole by stabilizing the position of the active site arginine residue via hydrogen bonding. Molecular dynamics simulations and primary sequence conservation suggest that the sorting signal-stabilized oxyanion hole is a universal feature of enzymes within the Sortase superfamily.
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Sortase enzymes in Gram-positive bacteria.
Molecular Microbiology, 2011Co-Authors: Thomas Spirig, Ethan M. Weiner, Robert T. ClubbAbstract:Summary In Gram-positive bacteria proteins are displayed on the cell surface using Sortase enzymes. These cysteine transpeptidases join proteins bearing an appropriate sorting signal to strategically positioned amino groups on the cell surface. Working alone, or in concert with other enzymes, Sortases either attach proteins to the cross-bridge peptide of the cell wall or they link proteins together to form pili. Because surface proteins play a fundamental role in microbial physiology and are frequently virulence factors, Sortase enzymes have been intensely studied since their discovery a little more than a decade ago. Based on their primary sequences and functions Sortases can be partitioned into distinct families called class A to F enzymes. Most bacteria elaborate their surfaces using more than one type of Sortase that function non-redundantly by recognizing unique sorting signals within their protein substrates. Here we review what is known about the functions of these enzymes and the molecular basis of catalysis. Particular emphasis is placed on ‘pilin’ specific class C Sortases that construct structurally complex pili. Exciting new data have revealed that these enzymes are amazingly promiscuous in the substrates that they can employ and that there is a startling degree of diversity in their mechanism of action. We also review recent data that suggest that Sortases are targeted to specific sites on the cell surface where they work with other Sortases and accessory factors to properly function.
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Sortase enzymes in Gram‐positive bacteria
Molecular microbiology, 2011Co-Authors: Thomas Spirig, Ethan M. Weiner, Robert T. ClubbAbstract:In Gram-positive bacteria proteins are displayed on the cell surface using Sortase enzymes. These cysteine transpeptidases join proteins bearing an appropriate sorting signal to strategically positioned amino groups on the cell surface. Working alone, or in concert with other enzymes, Sortases either attach proteins to the cross-bridge peptide of the cell wall or they link proteins together to form pili. Because surface proteins play a fundamental role in microbial physiology and are frequently virulence factors, Sortase enzymes have been intensely studied since their discovery a little more than a decade ago. Based on their primary sequences and functions Sortases can be partitioned into distinct families called class A to F enzymes. Most bacteria elaborate their surfaces using more than one type of Sortase that function non-redundantly by recognizing unique sorting signals within their protein substrates. Here we review what is known about the functions of these enzymes and the molecular basis of catalysis. Particular emphasis is placed on 'pilin' specific class C Sortases that construct structurally complex pili. Exciting new data have revealed that these enzymes are amazingly promiscuous in the substrates that they can employ and that there is a startling degree of diversity in their mechanism of action. We also review recent data that suggest that Sortases are targeted to specific sites on the cell surface where they work with other Sortases and accessory factors to properly function.
Vicente Monedero - One of the best experts on this subject based on the ideXlab platform.
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Contribution of Sortase SrtA2 to Lactobacillus casei BL23 inhibition of Staphylococcus aureus internalization into bovine mammary epithelial cells
PLoS ONE, 2017Co-Authors: Renata Faria Silva Souza, Damien Bouchard, Lucie Rault, Chantal Cauty, Luis G. Bermúdez-humarán, Nubia Seyffert, Julien Jardin, Vicente Monedero, Philippe Langella, Vasco AzevedoAbstract:Probiotics have been considered as a promising strategy to prevent various diseases in both humans and animals. This approach has gained interest in recent years as a potential means to control bovine mastitis. In a previous study, we found that several L. casei strains, including BL23, were able to inhibit the internalization of S. aureus, a major etiologic agent of mastitis, into bovine mammary epithelial cells (bMEC). This antagonism required a direct contact between L. casei and bMEC or S. aureus, suggesting the inhibition relied on interactions between L. casei cell surface components and bMEC. In this study, we have investigated the impact of some candidates which likely influence bacteria host cell interactions. We have shown that L. casei BL23 fbpA retained its inhibitory potential, indicating that L. casei BL23 antagonism did not rely (solely) on competition between S. aureus and L. casei fibronectin-binding proteins for adhesion to bMEC. We have then investigated the impact of four Sortase mutants, srtA1, srtA2, srtC1 and srtC2, and a double mutant (srtA1-srtA2) on L. casei BL23 inhibitory potential. Sortases are responsible for the anchoring on the bacterial cell wall of LPXTG-proteins, which reportedly play an important role in bacteria-host cell interaction. All the srt mutants tested presented a reduced inhibition capacity, the most pronounced effect being observed with the srtA2 mutant. A lower internalization capacity of L. casei srtA2 into bMEC was also observed. This was associated with several changes at the surface of L. casei BL23 srtA2 compared to the wild type (wt) strain, including altered abundance of some LPXTG- and moonlighting proteins, and modifications of cell wall structure. These results strongly support the role of Sortase A2 in L. casei BL23 inhibition against S. aureus internalization. Deciphering the contribution of the cell surface components altered in srtA2 strain in the inhibition will require further investigation.
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Functional Analysis of the Lactobacillus casei BL23 Sortases
Applied and Environmental Microbiology, 2012Co-Authors: Diego Muñoz-provencio, Luis G. Bermúdez-humarán, Maria Carmen Collado, Jesús Rodríguez-díaz, Philippe Langella, Vicente MonederoAbstract:Sortases are a class of enzymes that anchor surface proteins to the cell wall of Gram-positive bacteria. Lactobacillus casei BL23 harbors four Sortase genes, two belonging to class A (srtA1 and srtA2) and two belonging to class C (srtC1 and srtC2). Class C Sortases were clustered with genes encoding their putative substrates that were homologous to the SpaEFG and SpaCBA proteins that encode mucus adhesive pili in Lactobacillus rhamnosus GG. Twenty-three genes encoding putative Sortase substrates were identified in the L. casei BL23 genome with unknown (35%), enzymatic (30%), or adhesion-related (35%) functions. Strains disrupted in srtA1, srtA2, srtC1, and srtC2 and an srtA1 srtA2 double mutant were constructed. The transcription of all four Sortase encoding genes was detected, but only the mutation of srtA1 resulted in a decrease in bacterial surface hydrophobicity. The β-N-acetyl-glucosaminidase and cell wall proteinase activities of whole cells diminished in the srtA1 mutant and, to a greater extent, in the srtA1 srtA2 double mutant. Cell wall anchoring of the staphylococcal NucA reporter protein fused to a cell wall sorting sequence was also affected in the srtA mutants, and the percentages of adhesion to Caco-2 and HT-29 intestinal epithelial cells were reduced for the srtA1 srtA2 strain. Mutations in srtC1 or srtC2 result in an undetectable phenotype. Together, these results suggest that SrtA1 is the housekeeping Sortase in L. casei BL23 and SrtA2 would carry out redundant or complementary functions that become evident when SrtA1 activity is absent.
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Functional Analysis of the Lactobacillus casei BL23 Sortases
Applied and Environmental Microbiology, 2012Co-Authors: Diego Muñoz-provencio, Maria Carmen Collado, Luis G. Bermúdez-humarán, Jesús Rodríguez-díaz, Philippe Langella, Vicente MonederoAbstract:Sortases are a class of enzymes that anchor surface proteins to the cell wall of Gram-positive bacteria. Lactobacillus casei BL23 harbors four Sortase genes, two belonging to class A (srtA1 and srtA2) and two belonging to class C (srtC1 and srtC2). Class C Sortases were clustered with genes encoding their putative substrates that were homologous to the SpaEFG and SpaCBA proteins that encode mucus adhesive pili in Lactobacillus rhamnosus GG. Twenty-three genes encoding putative Sortase substrates were identified in the L. casei BL23 genome with unknown (35%), enzymatic (30%), or adhesion-related (35%) functions. Strains disrupted in srtA1, srtA2, srtC1, and srtC2 and an srtA1 srtA2 double mutant were constructed. The transcription of all four Sortase encoding genes was detected, but only the mutation of srtA1 resulted in a decrease in bacterial surface hydrophobicity. The beta-N-acetyl-glucosaminidase and cell wall proteinase activities of whole cells diminished in the srtA1 mutant and, to a greater extent, in the srtA1 srtA2 double mutant. Cell wall anchoring of the staphylococcal NucA reporter protein fused to a cell wall sorting sequence was also affected in the srtA mutants, and the percentages of adhesion to Caco-2 and HT-29 intestinal epithelial cells were reduced for the srtA1 srtA2 strain. Mutations in srtC1 or srtC2 result in an undetectable phenotype. Together, these results suggest that SrtA1 is the housekeeping Sortase in L. casei BL23 and SrtA2 would carry out redundant or complementary functions that become evident when SrtA1 activity is absent.
