SpeB modulates fibronectin-dependent internalization of Streptococcus pyogenes by efficient proteolysis of cell-wall-anchored protein F1

SpeB is a cysteine proteinase and virulence determinant secreted by the important human pathogen Streptococcus pyogenes. Recent investigations have suggested a role for SpeB in streptococcal entry into human cells. However, conflicting data concerning the contribution of SpeB to internalization have been presented. Protein F1 is a cell-wall-attached fibronectin (Fn)-binding protein that is present in a majority of streptococcal isolates and is important for internalization. This study shows that protein F1 is efficiently degraded by SpeB, and that removal of protein F1 from the bacterial surface leads to reduced internalization. Whereas M1 protein and protein H, two additional surface proteins of S. pyogenes that bind human plasma proteins, are protected from proteolytic degradation by their ligands, protein F1 is readily cleaved by SpeB also when in complex with Fn. This finding, and the connection between the presence of Fn at the bacterial surface and entry into human cells, suggest that SpeB plays a role in the regulation of the internalization process.

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A Novel Sortase, SrtC2, from Streptococcus pyogenes Anchors a Surface Protein Containing a QVPTGV Motif to the Cell Wall

Journal of Bacteriology ◽

10.1128/jb.186.17.5865-5875.2004 ◽

2004 ◽

Vol 186 (17) ◽

pp. 5865-5875 ◽

Cited By ~ 38

Author(s):

Timothy C. Barnett ◽

Aman R. Patel ◽

June R. Scott

Keyword(s):

Cell Wall ◽

Streptococcus Pyogenes ◽

Group A Streptococcus ◽

Surface Protein ◽

Surface Proteins ◽

Human Pathogen ◽

Hydrophobic Region ◽

C Terminus ◽

Group A ◽

Covalently Linked

ABSTRACT The important human pathogen Streptococcus pyogenes (group A streptococcus GAS), requires several surface proteins to interact with its human host. Many of these are covalently linked by a sortase enzyme to the cell wall via a C-terminal LPXTG motif. This motif is followed by a hydrophobic region and charged C terminus, which are thought to retard the protein in the cell membrane to facilitate recognition by the membrane-localized sortase. Previously, we identified two sortase enzymes in GAS. SrtA is found in all GAS strains and anchors most proteins containing LPXTG, while SrtB is present only in some strains and anchors a subset of LPXTG-containing proteins. We now report the presence of a third sortase in most strains of GAS, SrtC. We show that SrtC mediates attachment of a protein with a QVPTGV motif preceding a hydrophobic region and charged tail. We also demonstrate that the QVPTGV sequence is a substrate for anchoring of this protein by SrtC. Furthermore, replacing this motif with LPSTGE, found in the SrtA-anchored M protein of GAS, leads to SrtA-dependent secretion of the protein but does not lead to its anchoring by SrtA. We conclude that srtC encodes a novel sortase that anchors a protein containing a QVPTGV motif to the surface of GAS.

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Extracellular Adherence Protein from Staphylococcus aureus Enhances Internalization into Eukaryotic Cells

Infection and Immunity ◽

10.1128/iai.71.5.2310-2317.2003 ◽

2003 ◽

Vol 71 (5) ◽

pp. 2310-2317 ◽

Cited By ~ 69

Author(s):

Axana Haggar ◽

Muzaffar Hussain ◽

Helena Lönnies ◽

Mathias Herrmann ◽

Anna Norrby-Teglund ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Mutant Strain ◽

Plasma Proteins ◽

Binding Capacity ◽

Clinical Isolates ◽

Eukaryotic Cells ◽

Isogenic Mutant ◽

Bacterial Surface ◽

Internalization Process ◽

Surface Localization

ABSTRACT In this study we have shown that Eap (extracellular adherence protein) plays a role in the internalization process of Staphylococcus aureus into eukaryotic cells. Eap is a protein that is mostly extracellularly and to a lesser extent is bound to the bacterial surface as a result of rebinding. Eap is able to bind to several plasma proteins, such as fibronectin, fibrinogen, and prothrombin. It has the capacity to form oligomers and is able to agglutinate S. aureus. A mutant strain, Newman mAH12 (eap:: Eryr), with a deficient eap gene was used in the present study. We have demonstrated that (i) strain Newman mAH12 could adhere to and become internalized to a higher extent by eukaryotic cells than the isogenic mutant, (ii) strain Newman mAH12 complemented with the eap gene displayed restoration of the internalization level, (iii) externally added Eap enhanced the internalization of laboratory and clinical S. aureus strains as well as of S. carnosus (a coagulase-negative species devoid of proteins important for internalization), and (iv) antibodies against Eap were able to block the internalization process in strain Newman mAH12 and clinical isolates. Eap, with its broad binding capacity and its surface localization, thus seems to contribute to the internalization of S. aureus into eukaryotic cells. We therefore propose a novel internalization pathway for S. aureus in which Eap plays an enhancing role.

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Cysteine proteinase SpeB from Streptococcus pyogenes – a potent modifier of immunologically important host and bacterial proteins

Biological Chemistry ◽

10.1515/bc.2011.208 ◽

2011 ◽

Vol 392 (12) ◽

pp. 1077-1088 ◽

Cited By ~ 83

Author(s):

Daniel C. Nelson ◽

Julia Garbe ◽

Mattias Collin

Keyword(s):

Streptococcus Pyogenes ◽

Group A Streptococcus ◽

Wide Spectrum ◽

Cysteine Proteinase ◽

Complement Components ◽

Bacterial Surface ◽

Bacterial Proteins ◽

Disease States ◽

Group A ◽

Important Host

AbstractGroup A streptococcus (Streptococcus pyogenes) is an exclusively human pathogen that causes a wide spectrum of diseases ranging from pharyngitis, to impetigo, to toxic shock, to necrotizing fasciitis. The diversity of these disease states necessitates thatS. pyogenespossess the ability to modulate both the innate and adaptive immune responses. SpeB, a cysteine proteinase, is the predominant secreted protein fromS. pyogenes. Because of its relatively indiscriminant specificity, this enzyme has been shown to degrade the extracellular matrix, cytokines, chemokines, complement components, immunoglobulins, and serum protease inhibitors, to name but a few of the known substrates. Additionally, SpeB regulates other streptococcal proteins by degrading them or releasing them from the bacterial surface. Despite the wealth of literature on putative SpeB functions, there remains much controversy about this enzyme because many of reported activities would produce contradictory physiological results. Here we review all known host and bacterial protein substrates for SpeB, their cleavage sites, and discuss the role of this enzyme in streptococcal pathogenesis based on the current literature.

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A Proline-Rich Region with a Highly Periodic Sequence in Streptococcal β Protein Adopts the Polyproline II Structure and Is Exposed on the Bacterial Surface

Journal of Bacteriology ◽

10.1128/jb.184.22.6376-6393.2002 ◽

2002 ◽

Vol 184 (22) ◽

pp. 6376-6383 ◽

Cited By ~ 16

Author(s):

Thomas Areschoug ◽

Sara Linse ◽

Margaretha Stålhammar-Carlemalm ◽

Lars-Olof Hedén ◽

Gunnar Lindahl

Keyword(s):

Cell Wall ◽

Surface Expression ◽

Surface Proteins ◽

Rich Region ◽

Bacterial Surface ◽

Polyproline Ii ◽

Β Protein ◽

Typical Sequence ◽

Sequence Periodicity ◽

Proline Rich Region

ABSTRACT Proline-rich regions have been identified in many surface proteins of pathogenic streptococci and staphylococci. These regions have been suggested to be located in cell wall-spanning domains and/or to be required for surface expression of the protein. Because little is known about these regions, which are found in extensively studied and biologically important surface proteins, we characterized the proline-rich region in one such protein, the β protein of group B streptococci. The proline-rich region in β, designated the XPZ region, has a proline at every third position, and the sequence is highly periodic in other respects. Immunochemical analysis showed that the XPZ region was not associated with the cell wall but was exposed on the bacterial surface. Moreover, characterization of a β mutant lacking the XPZ region demonstrated that this region was not required for surface expression of the β protein. Comparison of the XPZ region in different β proteins showed that it varied in size but always retained the typical sequence periodicity. Circular dichroism spectroscopy indicated that the XPZ region had the structure of a polyproline II helix, an extended and solvent-exposed structure with exactly three residues per turn. Because of the three-residue sequence periodicity in the XPZ region, it is expected to be amphipathic and to have distinct nonpolar and polar surfaces. This study identified a proline-rich structure with unique properties that is exposed on the surface of an important human pathogen.

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Characterization of a virulence-associated and cell-wall-located DNase of Streptococcus pyogenes

Microbiology ◽

10.1099/mic.0.031955-0 ◽

2010 ◽

Vol 156 (1) ◽

pp. 184-190 ◽

Cited By ~ 38

Author(s):

Tadao Hasegawa ◽

Masaaki Minami ◽

Akira Okamoto ◽

Ichiro Tatsuno ◽

Masanori Isaka ◽

...

Keyword(s):

Cell Wall ◽

Streptococcus Pyogenes ◽

Protein A ◽

Insoluble Fraction ◽

Infection Model ◽

Two Dimensional Gel Electrophoresis ◽

Mouse Infection Model ◽

Peptide Mass ◽

A Cell

We investigated culture supernatant proteins from the M1 serotype of Streptococcus pyogenes by two-dimensional gel electrophoresis and peptide mass mapping analysis, and characterized the single protein spots. Among them, we analysed the Spy0747 protein. This protein is homologous to the SsnA protein, a cell-wall-located DNase expressed in Streptococcus suis serotype 2. We designated the Spy0747 protein as SpnA. SpnA protein was also detected in the insoluble fraction of whole-cell lysates using shotgun proteomic analysis, suggesting that SpnA is also located in the cell wall. SpnA was expressed as a glutathione S-transferase-fusion protein in Escherichia coli. We confirmed that the recombinant protein had DNase activity that was dependent on Ca2+ and Mg2+, like SsnA. Blood bactericidal assays and mouse infection model experiments showed that the spnA knockout strain was less virulent than the parental strain, thus suggesting that SpnA could play an important role in virulence. Using PCR, we found that the spnA gene was present in all clinical S. pyogenes strains we examined. Our results, together with a previous report identifying Spy0747 as a surface-associated protein, suggest that SpnA is an important cell-wall-located DNase that is generally produced in S. pyogenes and is involved in virulence.

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Further Evidence that a Cell Wall Precursor [C55-MurNAc-(Peptide)-GlcNAc] Serves as an Acceptor in a Sorting Reaction

Journal of Bacteriology ◽

10.1128/jb.184.8.2141-2147.2002 ◽

2002 ◽

Vol 184 (8) ◽

pp. 2141-2147 ◽

Cited By ~ 60

Author(s):

Alexey Ruzin ◽

Anatoly Severin ◽

Frank Ritacco ◽

Keiko Tabei ◽

Guy Singh ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Cell Wall ◽

Thin Layer ◽

Surface Proteins ◽

Natural Substrate ◽

Lipid Ii ◽

A Cell ◽

In Vitro Biosynthesis ◽

Layer Chromatography

ABSTRACT Previous studies suggested that a Gly-containing branch of cell wall precursor [C55-MurNAc-(peptide)-GlcNAc], which is often referred to as lipid II, might serve as a nucleophilic acceptor in sortase-catalyzed anchoring of surface proteins in Staphylococcus aureus. To test this hypothesis, we first simplified the procedure for in vitro biosynthesis of Gly-containing lipid II by using branched UDP-MurNAc-hexapeptide isolated from the cytoplasm of Streptomyces spp. Second, we designed a thin-layer chromatography-based assay in which the mobility of branched but not linear lipid II is shifted in the presence of both sortase and LPSTG-containing peptide. These results and those of additional experiments presented in this study further suggest that lipid II indeed serves as a natural substrate in a sorting reaction.

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Human pathogen Candida dubliniensis: A cell wall mannan with a high content of β-1,2-linked mannose residues

Carbohydrate Polymers ◽

10.1016/j.carbpol.2007.03.007 ◽

2007 ◽

Vol 70 (1) ◽

pp. 89-100 ◽

Cited By ~ 12

Author(s):

Izabela Ližičárová ◽

Mária Matulová ◽

Peter Capek ◽

Eva Machová

Keyword(s):

Cell Wall ◽

Candida Dubliniensis ◽

Human Pathogen ◽

A Cell ◽

Cell Wall Mannan

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Differential Recognition of Surface Proteins in Streptococcus pyogenes by Two Sortase Gene Homologs

Journal of Bacteriology ◽

10.1128/jb.184.8.2181-2191.2002 ◽

2002 ◽

Vol 184 (8) ◽

pp. 2181-2191 ◽

Cited By ~ 85

Author(s):

Timothy C. Barnett ◽

June R. Scott

Keyword(s):

Cell Wall ◽

Streptococcus Pyogenes ◽

Group A Streptococcus ◽

Surface Proteins ◽

Reading Frame ◽

Gram Positive Bacteria ◽

Differential Recognition ◽

Surface Localization ◽

Group A ◽

Protein F

ABSTRACT 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.

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Purification and Characterization of LPXTGase from Staphylococcus aureus: the Amino Acid Composition Mirrors That Found in the Peptidoglycan

Journal of Bacteriology ◽

10.1128/jb.188.2.389-398.2006 ◽

2006 ◽

Vol 188 (2) ◽

pp. 389-398 ◽

Cited By ~ 5

Author(s):

Sung G. Lee ◽

Vincent A. Fischetti

Keyword(s):

Amino Acids ◽

Staphylococcus Aureus ◽

Streptococcus Pyogenes ◽

Surface Proteins ◽

Purification And Characterization ◽

Bacterial Surface ◽

Gram Positive Bacteria ◽

Wall Assembly ◽

Attachment Process

ABSTRACT Bacterial surface proteins are important molecules in the infectivity and survival of pathogens. Surface proteins on gram-positive bacteria have been shown to attach via a transpeptidase, termed sortase, that cleaves an LPXTG sequence found close to the C termini of nearly all surface proteins on these bacteria. We previously identified a unique enzyme (LPXTGase) from Streptococcus pyogenes that also cleaves the LPXTG motif with a catalytic activity higher than that of sortase, suggesting that it plays an important role in the attachment process. We have now purified and characterized an LPXTGase from Staphylococcus aureus and found that it has both similar and unique features compared to the S. pyogenes enzyme. The S. aureus enzyme is glycosylated and contains unusual amino acids, like its streptococcal counterpart. Like the streptococcal enzyme, staphylococcal LPXTGase has an overrepresentation of amino acids found in the peptidoglycan, i.e., glutamine/glutamic acid, glycine, alanine, and lysine, and furthermore, we find that these amino acids are present in the enzyme at precisely the same ratio at which they are found in the peptidoglycan for the respective organism. This suggests that enzymes responsible for wall assembly may also play a role in the construction of LPXTGase.

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