Inactivation of the group A Streptococcus regulator srv results in chromosome wide reduction of transcript levels, and changes in extracellular levels of Sic and SpeB

Department of Microbiology and Immunology, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA.
FEMS Immunology & Medical Microbiology (Impact Factor: 3.08). 12/2006; 48(2):283-92. DOI: 10.1111/j.1574-695X.2006.00150.x
Source: PubMed


Group A Streptococcus is characterized by the ability to cause a diverse number of human infections including pharyngitis, necrotizing fasciitis, toxic shock syndrome, and acute rheumatic fever, yet the regulation of streptococcal genes involved in disease processes and survival in the host is not completely understood. Genome scale analysis has revealed a complex regulatory network including 13 two-component regulatory systems and more than 100 additional putative regulators, the majority of which remain uncharacterized. Among these is the streptococcal regulator of virulence, Srv, the first Group A Streptococcus member of the Crp/Fnr family of transcriptional regulators. Previous work demonstrated that the loss of srv resulted in a significant decrease in Group A Streptococcus virulence. To begin to define the gene products influenced by Srv, we combined microarray and two-dimensional gel electrophoresis analysis. Loss of srv results in a chromosome wide reduction of gene transcription and changes in the production of the extracellular virulence factors Sic (streptococcal inhibitor of complement) and SpeB (cysteine proteinase). Sic levels are reduced in the srv mutant, whereas the extracellular concentration and activity of SpeB is increased. These data link Srv to the increasingly complex GAS regulatory network.

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    • "We have previously shown that SpeB was constitutively produced following allelic replacement of the streptococcal regulator of virulence (Srv) in MGAS5005, a M1T1 GAS clinical isolate that produces low levels of SpeB during late exponential and early stationary phases of planktonic growth [34], [35], [36]. We have also demonstrated that constitutive SpeB production by MGAS5005Δsrv results in decreased in vitro biofilm formation, and biofilm formation can be restored following chemical or genetic inactivation of speB/SpeB [37], [38]. "
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    ABSTRACT: Group A Streptococcus (GAS) is a human specific pathogen capable of causing both mild infections and severe invasive disease. We and others have shown that GAS is able to form biofilms during infection. That is to say, they form a three-dimensional, surface attached structure consisting of bacteria and a multi-component extracellular matrix. The mechanisms involved in regulation and dispersal of these GAS structures are still unclear. Recently we have reported that in the absence of the transcriptional regulator Srv in the MGAS5005 background, the cysteine protease SpeB is constitutively produced, leading to increased tissue damage and decreased biofilm formation during a subcutaneous infection in a mouse model. This was interesting because MGAS5005 has a naturally occurring mutation that inactivates the sensor kinase domain of the two component regulatory system CovRS. Others have previously shown that strains lacking covS are associated with decreased SpeB production due to CovR repression of speB expression. Thus, our results suggest the inactivation of srv can bypass CovR repression and lead to constitutive SpeB production. We hypothesized that Srv control of SpeB production may be a mechanism to regulate biofilm dispersal and provide a mechanism by which mild infection can transition to severe disease through biofilm dispersal. The question remained however, is this mechanism conserved among GAS strains or restricted to the unique genetic makeup of MGAS5005. Here we show that Srv mediated control of SpeB and biofilm dispersal is conserved in the invasive clinical isolates RGAS053 (serotype M1) and MGAS315 (serotype M3), both of which have covS intact. This work provides additional evidence that Srv regulated control of SpeB may mediate biofilm formation and dispersal in diverse strain backgrounds.
    PLoS ONE 12/2011; 6(12):e28640. DOI:10.1371/journal.pone.0028640 · 3.23 Impact Factor
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    • "While speB is highly conserved and present in almost all strains of GAS, speB expression is variable between strains [30]. Production of SpeB in MGAS5005 planktonic culture is detected during late exponential and early stationary phases of growth, however, high levels of SpeB are present in MGAS5005Δsrv culture after only two hours of growth [29], [31]. "
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    ABSTRACT: Group A Streptococcus (GAS) is a Gram-positive human pathogen best known for causing pharyngeal and mild skin infections. However, in the 1980's there was an increase in severe GAS infections including cellulitis and deeper tissue infections like necrotizing fasciitis. Particularly striking about this elevation in the incidence of severe disease was that those most often affected were previously healthy individuals. Several groups have shown that changes in gene content or regulation, as with proteases, may contribute to severe disease; yet strains harboring these proteases continue to cause mild disease as well. We and others have shown that group A streptococci (MGAS5005) reside within biofilms both in vitro and in vivo. That is to say that the organism colonizes a host surface and forms a 3-dimensional community encased in a protective matrix of extracellular protein, DNA and polysaccharide(s). However, the mechanism of assembly or dispersal of these structures is unclear, as is the relationship of these structures to disease outcome. Recently we reported that allelic replacement of the streptococcal regulator srv resulted in constitutive production of the streptococcal cysteine protease SpeB. We further showed that the constitutive production of SpeB significantly decreased MGAS5005Δsrv biofilm formation in vitro. Here we show that mice infected with MGAS5005Δsrv had significantly larger lesion development than wild-type infected animals. Histopathology, Gram-staining and immunofluorescence link the increased lesion development with lack of disease containment, lack of biofilm formation, and readily detectable levels of SpeB in the tissue. Treatment of MGAS5005Δsrv infected lesions with a chemical inhibitor of SpeB significantly reduced lesion formation and disease spread to wild-type levels. Furthermore, inactivation of speB in the MGAS5005Δsrv background reduced lesion formation to wild-type levels. Taken together, these data suggest a mechanism by which GAS disease may transition from mild to severe through the Srv mediated dispersal of GAS biofilms.
    PLoS ONE 04/2011; 6(4):e18984. DOI:10.1371/journal.pone.0018984 · 3.23 Impact Factor
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    • "We have recently shown that inactivation of the streptococcal regulator of virulence (Srv), a proposed transcriptional regulator with homology to the Listeria monocytogenes regulator PrfA, results in a mutant strain exhibiting a significant reduction in biofilm formation [13,22]. Unlike in the wild-type parental strain, the streptococcal cysteine protease (SpeB) is constitutively produced by the srv mutant suggesting Srv contributes to the control of SpeB production [23]. SpeB is capable of cleaving both host (vitronectin, fibronectin, C3b) and self (M-protein, C5a peptidase, Fba, Sda1) extracellular proteins [21,24-30]. "
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    ABSTRACT: Group A Streptococcus (GAS) is a Gram-positive human pathogen that is capable of causing a wide spectrum of human disease. Thus, the organism has evolved to colonize a number of physiologically distinct host sites. One such mechanism to aid colonization is the formation of a biofilm. We have recently shown that inactivation of the streptococcal regulator of virulence (Srv), results in a mutant strain exhibiting a significant reduction in biofilm formation. Unlike the parental strain (MGAS5005), the streptococcal cysteine protease (SpeB) is constitutively produced by the srv mutant (MGAS5005Δsrv) suggesting Srv contributes to the control of SpeB production. Given that SpeB is a potent protease, we hypothesized that the biofilm deficient phenotype of the srv mutant was due to the constitutive production of SpeB. In support of this hypothesis, we have previously demonstrated that treating cultures with E64, a commercially available chemical inhibitor of cysteine proteases, restored the ability of MGAS5005Δsrv to form biofilms. Still, it was unclear if the loss of biofilm formation by MGAS5005Δsrv was due only to the constitutive production of SpeB or to other changes inherent in the srv mutant strain. To address this question, we constructed a ΔsrvΔspeB double mutant through allelic replacement (MGAS5005ΔsrvΔspeB) and tested its ability to form biofilms in vitro. Allelic replacement of speB in the srv mutant background restored the ability of this strain to form biofilms under static and continuous flow conditions. Furthermore, addition of purified SpeB to actively growing wild-type cultures significantly inhibited biofilm formation. The constitutive production of SpeB by the srv mutant strain is responsible for the significant reduction of biofilm formation previously observed. The double mutant supports a model by which Srv contributes to biofilm formation and/or dispersal through regulation of speB/SpeB.
    BMC Research Notes 11/2010; 3(1):281. DOI:10.1186/1756-0500-3-281
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