Functional Analysis of the CpsA Protein of Streptococcus agalactiae

Abstract

bStreptococcal pathogens, such as the group B streptococcus (GBS) Streptococcus agalactiae, are an important cause of systemic disease, which is facilitated in part by the presence of a polysaccharide capsule. The CpsA protein is a putative transcriptional regulator of the capsule locus, but its exact contribution to regulation is unknown. To address the role of CpsA in regulation, full-length GBS CpsA and two truncated forms of the protein were purified and analyzed for DNA-binding ability. Assays demonstrated that CpsA is able to bind specifically to two putative promoters within the capsule operon with similar affinity, and full-length protein is required for specificity. Functional characterization of CpsA confirmed that the ⌬cpsA strain produced less capsule than did the wild type and demonstrated that the production of full-length CpsA or the DNA-binding region of CpsA resulted in increased capsule levels. In contrast, the production of a truncated form of CpsA lacking the extracellular LytR domain (CpsA-245) in the wild-type background resulted in a dominant-negative decrease in capsule production. GBS expressing CpsA-245, but not the ⌬cpsA strain, was attenuated in human whole blood. However, the ⌬cpsA strain showed significant attenuation in a zebrafish infection model. Furthermore, chain length was observed to be variable in a CpsA-dependent manner, but could be restored to wild-type levels when grown with lysozyme. Taken together, these results suggest that CpsA is a modular protein influencing multiple regulatory functions that may include not only capsule synthesis but also cell wall associated factors.

Streptococcus agalactiae Capsule Polymer Length and Attachment Is Determined by the Proteins CpsABCD

“…Thus, a possible redundancy between LCP proteins remains a possibility. It is noteworthy that we find no evidence for CpsA involvement in the transcriptional regulation of the cps operon, in contrast to previous reports in the literature (14,15). We speculate that the CpsH polymerase and CpsA compete for the same substrate (the nascent CPS).…”

“…Strains-CpsA has previously been reported to be involved in cps operon transcription (14,15). We used real-time quantitative RT-PCR to analyze the transcription of the operon in all of the mutant strains.…”

“…Their function in GBS has been previously investigated through the construction and characterization of knock-out mutants, suggesting that CpsA is required for transcription of the cps operon, whereas CpsC and CpsD have a more undefined role in polymerization/export of CPS (14). Moreover, recombinant CpsA was shown to bind the cps operon promoter in vitro (15). CpsA is a 485-aa membrane protein with a major extracellular portion.…”

On the origin of Group B Streptococcus from disease outbreaks in wild marine fish in Australia

“…In GBS cpsA is considered to be a transcriptional activator (Hanson et al, 2012). The function of cpsB is still uncertain but its inactivation have profound effect on capsule production (Cieslewicz et al, 2001).…”

“…In GBS, mutations in the capsular polysaccharide have been associated in reduced complement deposition and phagocytosis (Martins et al, 2010). GBS cpsA deletion mutant produced less capsule and was significantly less virulent than wild type strain in a zebrafish infection model (Hanson et al, 2012).…”