Functional analysis of the CpsA protein of Streptococcus agalactiae

Abstract

Streptococcal 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 ΔcpsA 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 ΔcpsA strain, was attenuated in human whole blood. However, the ΔcpsA 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.

Fig 1

GBS 515 CpsA protein. (A) Genes in the capsule operon of GBS 515. Putative promoter sequences within the capsule operon are indicated by bent arrows. (B) Membrane topology of CpsA. (C) Arrangement of the CpsA protein where domains are shown as TM (transmembrane domains), DNA_PPF (DNA polymerase processivity factor domain), and LytR (LytR_cpsA_psr family domain). Below are truncations made to MBP fusions of CpsA representing the full protein, a truncation at amino acid 245, a truncation at amino acid 117, and a truncation at amino acid 39.

Fig 2

EMSA demonstrating binding of GBS MBP-CpsA-full (10 pmol) to either the labeled GBS cpsA-pro probe (A) or the labeled GBS cpsE-pro probe (B).

Fig 3

EMSA results showing binding of MBP-CpsA-full (10 pmol) (A), MBP-CpsA-117 (52 pmol) (B), or MBP-CpsA-39 (7 pmol) (C) to either the labeled GBS cpsA-pro or labeled GBS cpsE-pro probes in the absence or presence of competitor DNA representing unlabeled GBS cpsA-pro, GBS cpsE-pro, or S. iniae cpsA-pro (unlabeled nonspecific). Unbound labeled probe (“U”) and bound labeled probe (“B”) are indicated to the left.

Fig 4

Percoll buoyant density assay reflecting capsule levels of the GBS WT or ΔcpsA strains harboring the vector plasmid or a plasmid containing MBP-CpsA-full, MBP-CpsA-245, or MBP-CpsA-117. Error bars represent the standard errors of the mean. The significance between the GBS WT/vector and the ΔcpsA/vector is indicated (*, P < 0.01).

Fig 5

Whole-blood assay measuring the log₁₀ level of CFU killed for bacterial strains incubated in human whole blood for 3 h. Error bars represent the standard deviations.

Fig 6

Zebrafish infection study tracking survival over time of zebrafish infected intramuscularly with either the GBS 515 WT or the ΔcpsA strain compared to a medium-only mock infection.

Fig 7

Visualization of chain length at ×1,000 magnification for GBS 515 WT and ΔcpsA strains, carrying the plasmid vector, MBP-CpsA-full, MBP-CpsA-245, or MBP-CpsA-117 as indicated on the top of the panel.

Fig 8

Quantification of the chain length for parent strains of GBS 515, (A) WT and (B) ΔcpsA, carrying the plasmid vector, MBP-CpsA-full, MBP-CpsA-245, or MBP-CpsA-117 as indicated on the bottom of the panel. Error bars represent the standard deviations.

Fig 9

Visualization of the chain length at ×1,000 magnification for GBS 515 WT and ΔcpsA strains, carrying the plasmid vector, MBP-CpsA-full, MBP-CpsA-245, or MBP-CpsA-117 as indicated on the top of the panel when grown in the presence of a subinhibitory concentration of lysozyme.