Differential regulation of type III secretion and virulence genes inBordetella pertussisandBordetella bronchisepticaby a secreted anti-σ factor

Abstract

The BvgAS phosphorelay regulates ∼10% of the annotated genomes of Bordetella pertussis and Bordetella bronchiseptica and controls their infectious cycles. The hierarchical organization of the regulatory network allows the integration of contextual signals to control all or specific subsets of BvgAS-regulated genes. Here, we characterize a regulatory node involving a type III secretion system (T3SS)-exported protein, BtrA, and demonstrate its role in determining fundamental differences in T3SS phenotypes among Bordetella species. We show that BtrA binds and antagonizes BtrS, a BvgAS-regulated extracytoplasmic function (ECF) sigma factor, to couple the secretory activity of the T3SS apparatus to gene expression. In B. bronchiseptica, a remarkable spectrum of expression states can be resolved by manipulating btrA, encompassing over 80 BtrA-activated loci that include genes encoding toxins, adhesins, and other cell surface proteins, and over 200 BtrA-repressed genes that encode T3SS apparatus components, secretion substrates, the BteA effector, and numerous additional factors. In B. pertussis, BtrA retains activity as a BtrS antagonist and exerts tight negative control over T3SS genes. Most importantly, deletion of btrA in B. pertussis revealed T3SS-mediated, BteA-dependent cytotoxicity, which had previously eluded detection. This effect was observed in laboratory strains and in clinical isolates from a recent California pertussis epidemic. We propose that the BtrA-BtrS regulatory node determines subspecies-specific differences in T3SS expression among Bordetella species and that B. pertussis is capable of expressing a full range of T3SS-dependent phenotypes in the presence of appropriate contextual cues.virulence gene regulation | ECF sigma factor | T3SS | Bordetella | host adaptation

The BvgAS Regulon of Bordetella pertussis

“…Our analyses confirm and extend results from previous microarray analyses (9,31,32). As expected, we observed the activation of multiple well-known vags, such as genes encoding adhesins and toxins (Table S2), T3SS (Table S2), and transcriptional regulators (Table 1).…”

“…Among these regulators is brpL, the gene for the ECF sigma factor BrpL, which is responsible for expression of the T3SS operon. Its expression is increased Ͼ100-fold by the presence of BvgA~P (Table 1) (32,46). Our in vitro transcription analyses with B. pertussis RNAP and BvgA~P demonstrate that BvgA~P directly activates P brpL .…”

“…Known vags, including those encoding adhesins and toxins, were expressed consistently in both species in the Bvg(ϩ) mode, while more species-specific differences were observed in the Bvg(Ϫ) mode. Recently, Ahuja et al reported the first transcriptome sequencing (RNA-seq) analysis in a Bordetella species, but it was limited to B. bronchiseptica (32). Here, we report the first RNA-seq examination of the BvgAS regulon in B. pertussis.…”

“…(A) Denaturing gels showing products of FeBABE cleavage using complexes formed with P brpL (left) or P fhaB (right), 32 P end labeled on either the template or the nontemplate strand, B. pertussis RNAP, and (as indicated) either phosphorylated BvgA T194C -FeBABE or phosphorylated BvgA V148C -FeBABE. The GϩA lanes correspond to DNA ladders generated by Maxam-Gilbert sequencing with the positions relative to the transcription start sites indicated (36).…”

“…Although some previous work had failed to detect T3SS proteins in B. pertussis and concluded that the T3SS may be inactive (46), other work has challenged this idea by demonstrating that clinical isolates and laboratory-adapted B. pertussis strains under certain nutrition-limited cultures produce T3SS proteins (50). In addition, Ahuja et al (32) recently reported that in both B. pertussis and B. bronchiseptica, BtrA (BP2233) functions as an anti-sigma factor for BrpL, the ECF sigma factor responsible for T3SS expression. This observation suggests that T3SS gene expression can be activated by the downregulation of BtrA.…”