The Type III Secreted Protein BspR Regulates the Virulence Genes in Bordetella bronchiseptica

Bordetella bronchiseptica is closely related with B. pertussis and B. parapertussis, the causative agents of whooping cough. These pathogenic species share a number of virulence genes, including the gene locus for the type III secretion system (T3SS) that delivers effector proteins. To identify unknown type III effectors in Bordetella, secreted proteins in the bacterial culture supernatants of wild-type B. bronchiseptica and an isogenic T3SS-deficient mutant were compared with iTRAQ-based, quantitative proteomic analysis method. BB1639, annotated as a hypothetical protein, was identified as a novel type III secreted protein and was designated BspR (Bordetella secreted protein regulator). The virulence of a BspR mutant (ΔbspR) in B. bronchiseptica was significantly attenuated in a mouse infection model. BspR was also highly conserved in B. pertussis and B. parapertussis, suggesting that BspR is an essential virulence factor in these three Bordetella species. Interestingly, the BspR-deficient strain showed hyper-secretion of T3SS-related proteins. Furthermore, T3SS-dependent host cell cytotoxicity and hemolytic activity were also enhanced in the absence of BspR. By contrast, the expression of filamentous hemagglutinin, pertactin, and adenylate cyclase toxin was completely abolished in the BspR-deficient strain. Finally, we demonstrated that BspR is involved in the iron-responsive regulation of T3SS. Thus, Bordetella virulence factors are coordinately but inversely controlled by BspR, which functions as a regulator in response to iron starvation

Progress Overview of Bacterial Two-Component Regulatory Systems as Potential Targets for Antimicrobial Chemotherapy

Bacteria adapt to changes in their environment using a mechanism known as the two-component regulatory system (TCS) (also called “two-component signal transduction system” or “two-component system”). It comprises a pair of at least two proteins, namely the sensor kinase and the response regulator. The former senses external stimuli while the latter alters the expression profile of bacterial genes for survival and adaptation. Although the first TCS was discovered and characterized in a non-pathogenic laboratory strain of Escherichia coli, it has been recognized that all bacteria, including pathogens, use this mechanism. Some TCSs are essential for cell growth and fitness, while others are associated with the induction of virulence and drug resistance/tolerance. Therefore, the TCS is proposed as a potential target for antimicrobial chemotherapy. This concept is based on the inhibition of bacterial growth with the substances acting like conventional antibiotics in some cases. Alternatively, TCS targeting may reduce the burden of bacterial virulence and drug resistance/tolerance, without causing cell death. Therefore, this approach may aid in the development of antimicrobial therapeutic strategies for refractory infections caused by multi-drug resistant (MDR) pathogens. Herein, we review the progress of TCS inhibitors based on natural and synthetic compounds

BspR negatively regulates the expression of T3SS-associated genes.

<p><i>B. bronchiseptica</i> strains were grown in SS medium under vigorous shaking at 37°C. The wild-type strain cultured in the presence of MgSO₄ (40 mM) was used as a negative control. (<b>A</b>) The secreted proteins isolated from bacterial culture supernatants were separated by SDS-PAGE and stained with CBB. Arrows indicate the bands corresponding to type III secreted proteins: A, BteA (multimerized); B, BopB; N, BopN; D, BopD; and 22, Bsp22. Arrowheads indicate the bands that disappeared in the absence of BspR. (<b>B</b>) Whole bacterial lysates were prepared from bacterial cultures and analyzed by immunoblotting using the following antibodies: anti-BspR, anti-BteA, anti-BopB, anti-BopN, anti-BopD, and anti-Bsp22. (<b>C</b>) Total RNA isolated from <i>B. bronchiseptica</i> strains was subjected to quantitative RT-PCR analysis. The graphs show the relative amounts of <i>bsp22</i>, <i>bopN</i>, <i>bteA</i>, and <i>btrS</i> mRNA normalized to the mRNA for the housekeeping gene <i>recA</i>. The wild-type strain cultured in the presence of MgSO₄ (40 mM) was used as a negative control. The values represent the means ± SE from three independent experiments. ***, P<0.001.</p

Increased proteins by the <i>bspR</i> deletion.

1<p>Ratio of the amount of whole cell lysate proteins of Δ<i>bspR</i> against wild-type <i>B</i>. <i>bronchiseptica</i>. P-value <0.05.</p

BspR is a type III secreted protein conserved in pathogenic <i>Bordetella</i> species.

<p>(<b>A</b>) Genomic organization of the <i>bsc</i> and <i>btr</i> loci in <i>B. bronchiseptica</i>. Sequence data were obtained from the <i>B. bronchiseptica</i> sequencing group at the Sanger Institute (<a href="ftp://ftp.sanger.ac.uk/pub/pathology/bb/" target="_blank">ftp://ftp.sanger.ac.uk/pub/pathology/bb/</a>). (<b>B</b>) BLAST alignment of BspR: BB1639 in <i>B</i>. <i>bronchiseptica</i> (Bbr), BPP2242 in <i>B</i>. <i>parapertussis</i> (Bpp), and BP2233 in <i>B</i>. <i>pertussis</i> (Bpe). The dots represent identical amino acid sequence in their respective <i>Bordetella</i> spp. The asterisk indicates the putative start methionine codon predicted by MetaGeneAnnotator (<a href="http://metagene.cb.k.u-tokyo.ac.jp/" target="_blank">http://metagene.cb.k.u-tokyo.ac.jp/</a>). (<b>C</b>) Immunoblot analysis using anti-BspR antibodies. Wild-type and ΔT3SS <i>B. bronchiseptica</i> were grown on SS medium in the absence or presence of MgSO₄ under vigorous shaking at 37°C for 18 h. Whole-cell lysates (WCL) and secreted protein fraction in the supernatant (Sup) were prepared from the bacterial cultures and analyzed by immunoblot analysis using anti-BspR antibodies. The arrowhead indicates the specific signal for BspR.</p

Signaling under iron starvation conditions is dysregulated in the absence of BspR.

<p>Wild-type, Δ<i>bspR</i>, and Δ<i>bspR</i>/p<i>bspR B. bronchiseptica</i> strains were grown in SS medium supplemented with iron at a final concentration of 0, 36, or 360 µM. The standard SS medium commonly used elsewhere contains iron at a concentration of 36 µM. The secreted proteins isolated from bacterial culture supernatants were separated by SDS-PAGE and stained with CBB. Arrows indicate the bands corresponding to the type III secreted proteins BteA (multimerized), BopB, BopN, BopD, and Bsp22 (A, B, N, D, and 22, respectively).</p

Primers used in this study.

*<p>The underlined portions (GGATCC) indicate the BamHI site for the self-ligation of the inverse PCR products in the construction of mutants.</p

T3SS -dependent and -independent secreted proteins in <i>B. bronchiseptica.</i>

1<p>Ratio of the amount of secreted protein of wild-type <i>B</i>. <i>bronchiseptica</i> against ΔT3SS strain.</p>2<p>Significance in the differences between wild type and ΔT3SS (t-test).</p