Functional Characterization of the Streptococcal CpsA Protein in Streptococcus agalactiae
Carion, Thomas W.
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Streptococcal pathogens like Streptococcus agalactiae or Group B Streptococcus (GBS) capable of causing systemic disease remain a major health concern worldwide (Parsons et al., 2011). The streptococcal CpsA protein found in GBS is part of the LytR_cpsA_psr (LCP) family of proteins associated with regulatory control over cell surface physiology, including polysaccharide synthesis (Cieslewicz et al., 2001), cell wall processing (Chatfield et al., 2005) and response to antimicrobial stress (Utaida et al., 2003). The involvement of this protein family with these important virulence determinants highlights its potential as a possible target for virulence reduction, increased clearance by host immune function or antimicrobial therapy (Marques et al., 1992). Therefore, our aim was to characterize the functional properties of CpsA in GBS to better understand the role it may play during initiation and perpetuation of disease. In order to look at the importance of CpsA in virulence, a pLZ12-rofA-pro plasmid vector, and the purified CpsA truncations MBP-CpsA-full, MBP-CpsA-245- ΔLytR, MBP-CpsA-117-Δ1 were put in 515 WT and 515 ΔcpsA bacterial strains. The main difference between the 515 WT strain and the 515 ΔcpsA strain is the cpsA genes have been deleted in the ΔcpsA strain and therefore it cannot produce capsular polysaccharide. Percoll buoyant density assays showed the DNA-binding domain (MBPCpsA- 117-Δ1) of CpsA was sufficient for complementation of capsule levels in the ΔcpsA strain. These results support the hypothesis that CpsA is an activator of capsule production, and that it does this by binding to the capsule operon promoter. Time course Percoll buoyant density assays proved that capsule is regulated by the regulated growth phase pattern in both the 515 WT and ΔcpsA bacterial strains. The mutation or removal of different domains of CpsA does for example in the WT background, MBP-CpsA-117-Δ1 causes an overall higher overall capsular production trend at which capsule is produced at. In CpsA the modulation of capsule levels are not “on” or “off” but rather “higher” or “lower” to a certain degree to what the 515 WT would normally produce. In order to investigate the difference in capsule produced by the different CpsA domains in 515 WT and ΔcpsA strains, bacterial strains were grown in human whole blood to assess how differing levels of capsule affected the ability of the bacteria to survive. Strains with higher capsule levels did not always have higher survival rates. Despite the production of less capsule in the ΔcpsA strain, there was no major difference in ΔcpsA survival compared to WT. The constitutive expression of the different CpsA domains alters the regulatory scheme by enhancing or decreasing levels at different times, indicating that CpsA somehow exerts a regulatory effect that is growth phase dependent. Microscopy showed the presence of the different cpsA Domains in the 515 WT and ΔcpsA bacterial strains lead to changes in the chain length distribution. Both the 515 WT and ΔcpsA strains with the MBP-CpsA-245-ΔlytR target truncation exhibited longer chains than the only the plasmid vector alone in the 515 WT and ΔcpsA strains. The observed long chain phenotype could be restored to the short chain phenotype displayed by the WT and ΔcpsA parent strains in the presence of lysozyme. However, no difference was seen in antibiotic resistance when comparing the 515 WT and ΔcpsA parent strains containing the different truncated CpsA domains. Taken together, these results suggest that CpsA is a modular protein with different levels of regulatory capacity and that its regulation may include not only capsular synthesis, but also cell wall-associated factors.