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Surface protein-CAT reporter fusions demonstrate differential gene expression in the vir regulon of Streptococcus
A Podbielski1, J A Peterson, P Cleary
1Institut fur Medische Mikrobiologie, Klinikum RWTH, Aachen, Germany.
Abstract:
Streptococcus pyogenes expresses at least two virulence factors, the anti-phagocytic M protein and an inhibitor of chemotaxis, the C5a peptidase (ScpA), under control of the virR locus. To facilitate studies of this regulatory unit, we constructed a new shuttle vector with a staphylococcal chloramphenicol acetyl transferase (CAT) reporter box which replicates in S. pyogenes. We cloned polymerase chain reaction (PCR)-derived potential promoter regions of the virR, M protein (emm12), and ScpA (scpA) genes from an M type 12 S. pyogenes, strain CS24. Promoter activity was assessed by measurements of specific mRNAs, transacetylase activity, and minimum inhibitory concentrations (MICs) for chloramphenicol resistance. We demonstrated that VirR is a necessary but not always sufficient positive trans-acting regulator of emm12 and scpA expression; however, virR is not autoregulated. A potential virR-binding consensus sequence is postulated for emm12, scpA and other M-like protein genes. Promoter activity of the structural genes was found to be dramatically influenced by growth conditions such as anaerobiosis. Levels of control, over and above the requirement for virR, are realized. The virR and scpA promoters were mapped for the first time using primer extension analysis. The observed mRNA start sites did not completely agree within the sequence predicted start sites. Data suggest that scpA could be subject to transcription attenuation.
Insights
Streptococcus pyogenes virulence factors, M protein and C5a peptidase (ScpA), are regulated by VirR. This study characterized the virR locus and its control over virulence gene expression in S. pyogenes.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Streptococcus pyogenes possesses virulence factors like M protein and C5a peptidase (ScpA).
- These factors are crucial for S. pyogenes pathogenesis and are regulated by the virR locus.
- Understanding this regulation is key to developing strategies against S. pyogenes infections.
Purpose of the Study:
- To construct a novel shuttle vector for studying the virR regulatory unit in S. pyogenes.
- To investigate the role of VirR in the transcriptional regulation of emm12 and scpA genes.
- To identify potential regulatory sequences and map promoter regions.
Main Methods:
- Construction of a staphylococcal chloramphenicol acetyl transferase (CAT) reporter shuttle vector.
- Cloning of promoter regions from virR, emm12, and scpA genes using PCR.
- Assessment of promoter activity via mRNA levels, transacetylase activity, and chloramphenicol resistance.
- Primer extension analysis for promoter mapping.
Main Results:
- VirR acts as a necessary positive trans-acting regulator for emm12 and scpA expression.
- VirR is not autoregulated, and a potential binding consensus sequence was identified.
- Gene expression is significantly influenced by growth conditions, such as anaerobiosis.
- Primer extension analysis revealed mRNA start sites and suggested potential transcription attenuation for scpA.
Conclusions:
- VirR is a key regulator of S. pyogenes virulence factors M protein and ScpA.
- Environmental factors significantly impact virulence gene expression.
- Further research into transcription attenuation mechanisms is warranted.