PlyC, a novel bacteriophage lysin for compartment‐ dependent proteomics of group A streptococci

Köller, Thomas; Nelson, Daniel; Nakata, Masao; Kreutzer, Michael; Fischetti, Vincent A.; Glocker, Michael O.; Podbielski, Andreas; Kreikemeyer, Bernd

doi:10.1002/pmic.200700001

Cited by 27 publications

(27 citation statements)

References 34 publications

(37 reference statements)

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“…As a second set of experiments, we aimed to detect surface-localized FctA in immunoblot experiments. The cell wall fraction of the WT and mutant strains was extracted with PlyC treatment (25). Expressed FctA in the protein extracts was detected using a mouse polyclonal antiserum against recombinant FctA (Fig.…”

Section: Resultsmentioning

confidence: 99%

Mode of Expression and Functional Characterization of FCT-3 Pilus Region-Encoded Proteins in Streptococcus pyogenes Serotype M49

et al. 2009

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The human pathogen Streptococcus pyogenes (group A streptococcus [GAS]) pilus components, suggested to play a role in pathogenesis, are encoded in the variable FCT (fibronectin-and collagen-binding T-antigen) region. We investigated the functions of sortase A (SrtA), sortase C2 (SrtC2), and the FctA protein of the most prevalent type 3 FCT region from a serotype M49 strain. Although it is considered a housekeeping sortase, SrtA's activity is involved in pilus formation in addition to its essentiality for GAS extracellular matrix protein binding, host cell adherence/internalization, survival in human blood, and biofilm formation. SrtC2 activity is crucial for pilus formation but dispensable for the other phenotypes tested in vitro. FctA is the major pilus backbone protein, simultaneously acting as the M49 T antigen, and requires SrtC2 and LepA, a signal peptidase I homologue, for monomeric surface expression and polymerization, respectively. Collagen-binding protein Cpa expression supports pilus formation at the pilus base. Immunofluorescence microscopy and fluorescence-activated cell sorting analysis revealed several unexpected expression patterns, as follows: (i) the monomeric pilus protein FctA was found exclusively at the old poles of GAS cells, (ii) FctA protein expression increased with lower temperatures, and (iii) FctA protein expression was restricted to 20 to 50% of a given GAS M49 population, suggesting regulation by a bistability mode. Notably, disruption of pilus assembly by sortase deletion rendered GAS serotype M49 significantly more aggressive in a dermonecrotic mouse infection model, indicating that sortase activity and, consequently, pilus expression allow a subpopulation of this GAS serotype to be less aggressive. Thus, pilus expression may not be a virulence attribute of GAS per se.Streptococcus pyogenes (group A streptococcus [GAS]) is a bacterial pathogen that is perfectly adapted to colonization, infection, and persistence in its human host (8,10,14,22). Many of the associated virulence factors expressed by this bacterium are encoded in discrete regions of the GAS genome (28). Two of these pathogenicity regions (Mga and FCT [see below]) each comprise genes for secreted and surface-exposed virulence factors and at least one stand-alone transcriptional regulator. These genes have been shown to act together in a growth phase-dependent regulatory network to coordinate GAS host cell adherence, internalization, and intracellular persistence (summarized in references 28 and 29).The FCT region (fibronectin-and collagen-binding T-antigen region) (6) is present in all GAS genomes specifically tested (26). The FCT region always contains a RALP transcriptional regulator whose type correlates with the type of emm pathogenicity region and the preferred infection site of the GAS strain, i.e., the throat or the skin. Since a similar association was also shown for the fibronectin-and collagenbinding proteins encoded by this region, the FCT region gene products could contribute to tissue-specific GAS infecti...

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Section: Resultsmentioning

confidence: 99%

Mode of Expression and Functional Characterization of FCT-3 Pilus Region-Encoded Proteins in Streptococcus pyogenes Serotype M49

et al. 2009

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“…Subsequently, bacterial suspensions were digested under ambient conditions with phage lysin C (100 U/ml) (23,36). This enzymatic treatment proved to be superior to the mutanolysin digestion described in a recent study (23). The digestion was terminated after 20 min, and phage lysin C-released surface proteins found in the supernatant of the digest were subsequently dialyzed (Serva dialysis tubes; molecular mass cutoff, 3,000 Da).…”

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The Streptococcus pyogenes Serotype M49 Nra-Ralp3 Transcriptional Regulatory Network and Its Control of Virulence Factor Expression from the Novel eno ralp3 epf sagA Pathogenicity Region

et al. 2007

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Many Streptococcus pyogenes (group A streptococcus [GAS]) virulence factor-and transcriptional regulatorencoding genes cluster together in discrete genomic regions. Nra is a central regulator of the FCT region. Previous studies exclusively described Nra as a transcriptional repressor of adhesin and toxin genes. Here transcriptome and proteome analysis of a serotype M49 GAS strain and an isogenic Nra mutant of this strain revealed the complete Nra regulon profile. Nra is active in all growth phases tested, with the largest regulon in the transition phase. Almost exclusively, virulence factor-encoding genes are repressed by Nra; these genes include the GAS pilus operon, the capsule synthesis operon, the cytolysin-mediated translocation system genes, all Mga region core virulence genes, and genes encoding other regulators, like the Ihk/Irr system, Rgg, and two additional RofA-like protein family regulators. Surprisingly, our experiments revealed that Nra additionally acts as a positive regulator, mostly for genes encoding proteins and enzymes with metabolic functions. Epidemiological investigations revealed strong genetic linkage of one particular Nra-repressed regulator, Ralp3 (SPy0735), with a gene encoding Epf (extracellular protein factor from Streptococcus suis). In a serotypespecific fashion, this ralp3 epf gene block is integrated, most likely via transposition, into the eno sagA virulence gene block, which is present in all GAS serotypes. In GAS serotypes M1, M4, M12, M28, and M49 this novel discrete genetic region is therefore designated the eno ralp3 epf sagA (ERES) pathogenicity region. Functional experiments showed that Epf is a novel GAS plasminogen-binding protein and revealed that Ralp3 activity counteracts Nra and MsmR regulatory activity. In addition to the Mga and FCT regions, the ERES region is the third discrete chromosomal pathogenicity region. All of these regions are transcriptionally linked, adding another level of complexity to the known GAS growth phase-dependent regulatory network.

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“…Thus, in contrast to many bacteriophages that recognize the same epitope by their tail fibers and by the lysins (3)(4)(5), the bacteriophage C1 tail spike has evolved to become highly specific to only one group of streptococci. Although bacteriophage C1 lysin protein has been extensively studied (6)(7)(8)(9), the structure of the phage has remained unknown.…”

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Structural investigations of a Podoviridae streptococcus phage C1, implications for the mechanism of viral entry

Aksyuk

Bowman

Kaufmann

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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The Podoviridae phage C1 was one of the earliest isolated bacteriophages and the first virus documented to be active against streptococci. The icosahedral and asymmetric reconstructions of the virus were calculated using cryo-electron microscopy. The capsid protein has an HK97 fold arranged into a T ¼ 4 icosahedral lattice. The C1 tail is terminated with a φ29-like knob, surrounded by a skirt of twelve long appendages with novel morphology. Several C1 structural proteins have been identified, including a candidate for an appendage. The crystal structure of the knob has an N-terminal domain with a fold observed previously in tube forming proteins of Siphoviridae and Myoviridae phages. The structure of C1 suggests the mechanisms by which the virus digests the cell wall and ejects its genome. Although there is little sequence similarity to other phages, conservation of the structural proteins demonstrates a common origin of the head and tail, but more recent evolution of the appendages.bacteriophage C1 tail | tail knob | X-ray crystallography

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PlyC, a novel bacteriophage lysin for compartment‐ dependent proteomics of group A streptococci

Cited by 27 publications

References 34 publications

Mode of Expression and Functional Characterization of FCT-3 Pilus Region-Encoded Proteins in Streptococcus pyogenes Serotype M49

Mode of Expression and Functional Characterization of FCT-3 Pilus Region-Encoded Proteins in Streptococcus pyogenes Serotype M49

The Streptococcus pyogenes Serotype M49 Nra-Ralp3 Transcriptional Regulatory Network and Its Control of Virulence Factor Expression from the Novel eno ralp3 epf sagA Pathogenicity Region

Structural investigations of a Podoviridae streptococcus phage C1, implications for the mechanism of viral entry

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