Rga is a regulator of adherence and pilus formation in Streptococcus agalactiae

Abstract

Streptococcus agalactiae is the leading cause of bacterial sepsis and meningitis in neonates and is also the causative agent of several serious infections in immunocompromised adults. S. agalactiae encounters multiple niches during an infection, suggesting that regulatory mechanisms control the expression of specific virulence factors in this bacterium. The present study describes the functional characterization of a gene from S. agalactiae, designated rga, which encodes a protein with significant similarity to members of the RofA-like protein (RALP) family of transcriptional regulators. After deletion of the rga gene in the genome of S. agalactiae, the mutant strain exhibited significantly reduced expression of the genes srr-1 and pilA, which encode a serine-rich repeat surface glycoprotein and a pilus protein, respectively, and moderately increased expression of the fbsA gene, which encodes a fibrinogen-binding protein. Electrophoretic mobility shift assays demonstrated specific DNA binding of purified Rga to the promoter regions of pilA and fbsA, suggesting that Rga directly controls pilA and fbsA. Adherence assays revealed significantly reduced binding of the Δrga mutant to epithelial HEp-2 cells and to immobilized human keratin 4, respectively. In contrast, the adherence of the Δrga mutant to A549 cells and its binding to human fibrinogen was significantly increased. Immunoblot and immunoelectron microscopy revealed that the quantity of pilus structures was significantly reduced in the Δrga mutant compared with the parental strain. The wild-type phenotype could be restored by plasmid-mediated expression of rga, demonstrating that the mutant phenotypes resulted from a loss of Rga function.

Regulation and Function of Pilus Island 1 in Group B Streptococcus

“…S2 in the supplemental material). Poor expression of PI-2a in strain 2603 is likely due, at least in part, to one or both of the frameshift mutations that abrogate expression of the two regulatory proteins, RogB and Rga, that normally activate PI-2a gene transcription (3,4,29). These results indicated that pilus 2a is expressed poorly or not at all in strain 2603, so pilus 1 is the only potentially functional pilus in this strain (see below).…”

“…We found a modest increase (about 2-fold) in PI-2a gene expression in 515 ⌬csrR, a csrR deletion mutant in the type Ia GBS strain 515 (unpublished data). Two RofA family proteins, RogB and Rga, were reported to be activators of PI-2a pilus expression in NEM316 (3,4,29). However, frameshift mutations are found in both rogB and rga in GBS 2603, consistent with the poor expression of the PI-2a pilus.…”

“…Samen et al found that inactivation of CsrRS did not change PI-2a expression in GBS strain NEM316 (29). We found a modest increase (about 2-fold) in PI-2a gene expression in 515 ⌬csrR, a csrR deletion mutant in the type Ia GBS strain 515 (unpublished data).…”

“…We chose to work with type V GBS strain 2603V/R (referred to henceforth as strain 2603) because it harbors inactivating mutations in RogB, the transcriptional activator associated with PI-2a, and in Rga, another activator of PI-2a gene expression (3,4,29). Presumably as a consequence of one or both mutations, strain 2603 produces undetectable amounts of PI-2a pili.…”

CsrRS and Environmental pH Regulate Group B Streptococcus Adherence to Human Epithelial Cells and Extracellular Matrix

“…Transcriptional regulators such as RovS, a member of the Rgg protein family (34); Rga, a RofA-like protein (33); and the RgfA/C two-component system (1,42) have been found to modulate GBS adherence to host cells or ECM, but the molecular basis of the coordinated expression of GBS adherence factors remains incompletely understood.…”