Role ofBordetella pertussisRseA in the cell envelope stress response and adenylate cyclase toxin release

Abstract

Bordetella pertussis is the bacterial agent of the human disease, whooping cough. In many bacteria, the extracellular function sigma factor σE is central to the response to envelope stress, and its activity is negatively controlled by the RseA anti-sigma factor. In this study, the role of RseA in B. pertussis envelope stress responses was investigated. Compared with the wild-type strain, an rseA mutant showed elevated resistance to envelope stress and enhanced growth at 25°C. rpoH and other predicted σE target genes demonstrated increased transcription in the rseA mutant compared with the wild type parent. Transcription of those genes was also increased in wild type B. pertussis and Escherichia coli under envelope stress, whereas no stress-induced increase in transcription was observed in the rseA mutant. rseA inactivation was also associated with altered levels of certain proteins in culture supernatant fluids, which showed increased adenylate cyclase toxin (CyaA) levels. The increased CyaA in the mutant was correlated with an apparent increased stability of the extracellular toxin and increased production of CyaA-containing outer membrane vesicles. Consistent with this, compared with the wild type strain, rseA mutant cells produced increased numbers of large surface-associated vesicles.

Glutamate Limitation, BvgAS Activation, and (p)ppGpp Regulate the Expression of the Bordetella pertussis Type 3 Secretion System

“…Protein samples used for SDS-PAGE analysis were prepared as described previously, with some modifications (23). Briefly, proteins in the culture fluids were precipitated with 15% trichloroacetic acid after removing bacterial cells by centrifugation at 7,500 rpm at 4°C for 10 min and passing through a 0.45-m-pore-size membrane filter.…”

“…Proteins separated by SDS-PAGE were transferred to a polyvinylidene difluoride (PVDF) membrane and reacted with diluted antibodies at room temperature for 1 h. Unless described elsewhere, dilution ratios were 1,500-fold for antipertactin (anti-Prn) (24), anti-pertussis toxin (anti-PTX) (Santa Cruz Biotechnology, Inc.), and anti-adenylate cyclase toxin (anti-ACT) (Santa Cruz Biotechnology, Inc.) antibodies from mouse and anti-Bsp22 (kind gift from Akio Abe, Kitasato University) and anti-filamentous hemagglutinin (anti-FHA) antibodies from rabbit (23). Rabbit anti-BspR antibody (kind gift from Akio Abe, Kitasato University) was diluted 800-fold.…”

Modulation of Pertussis and Adenylate Cyclase Toxins by Sigma Factor RpoE in Bordetella pertussis

“…In Bordetella bronchiseptica, Sig E has been shown to facilitate survival of membrane perturbations and is required for virulence in immunocompromised mice (12). It has also been shown that in B. pertussis RpoE influences alleviation of membrane stress (13). RpoE is controlled by an anti-sigma factor known as RseA.…”

“…RseA sequesters RpoE to the inner membrane, which renders it transcriptionally inactive; however, proteolytic cleavage of RseA or genetic mutation to rseA results in transcriptional activation of RpoE. Deletion of rseA from B. pertussis strain UT25 resulted in an increased amount and diversity of periplasmic proteins (13). Surprisingly, it was shown that the rseA gene deletion mutant (PM18) of the B. pertussis strain UT25 also released a large amount of adenylate cyclase toxin (ACT) into the culture media (13).…”

“…Deletion of rseA from B. pertussis strain UT25 resulted in an increased amount and diversity of periplasmic proteins (13). Surprisingly, it was shown that the rseA gene deletion mutant (PM18) of the B. pertussis strain UT25 also released a large amount of adenylate cyclase toxin (ACT) into the culture media (13). Not only was more ACT released but it also was of greater stability (13).…”