Molecular analysis of the Enterococcus faecalis serotype 2 polysaccharide determinant

Area of Science:

• Microbiology
• Molecular Biology
• Bacterial Genetics

Background:

• Enterococcus faecalis produces a capsular polysaccharide (CPS) that is a key virulence factor.
• The 15-kb genetic cluster (cps2A-cps2K) in E. faecalis FA2-2 is known to be involved in serotype 2 CPS production.

Purpose of the Study:

• To elucidate the transcriptional regulation of the serotype 2 capsular polysaccharide gene cluster in Enterococcus faecalis.
• To confirm the functional role of the identified genetic pathway in serotype 2 CPS synthesis.

Main Methods:

• Transcriptional fusions using a promoterless lacZ gene were employed to identify regulatory elements.
• Primer extension and Reverse Transcription-PCR (RT-PCR) were used to map transcription initiation sites and confirm transcriptional linkage.
• Real-time RT-PCR quantified transcript levels during different growth phases.
• Heterologous expression in Escherichia coli was performed to assess pathway functionality.

Main Results:

• Two independent promoters were identified within the cps2A-cps2K genetic cluster.
• Transcription of the cluster is highest during the logarithmic growth phase.
• The heterologous expression of the pathway in E. coli resulted in reactivity with E. faecalis type 2 antiserum, confirming its role in serotype 2 CPS synthesis.

Conclusions:

• The identified promoters and transcriptional linkage are crucial for the regulated expression of the serotype 2 capsular polysaccharide pathway.
• The cps2A-cps2K gene cluster is essential and sufficient for the synthesis of the type-specific polysaccharide in Enterococcus faecalis.