Identification of a CO2 Responsive Regulon in Bordetella Sara E. Hester1,2, Minghsun Lui3, Tracy Nicholson 4 , Daryl Nowacki1,2, Eric T. Harvill1* 1 Department of Veterinary and Biomedical Sciences, The Pennsylvania State University, University Park, Pennsylvania, United States of America, 2 Graduate Program in Biochemistry, Microbiology, and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania, United States of America, 3 Department of Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine at UCLA, University of California Los Angeles, Los Angeles, California, United States of America, 4 National Animal Disease Center, Agricultural Research Service, United States Department of Agriculture, Ames, Iowa, United State of America Abstract Sensing the environment allows pathogenic bacteria to coordinately regulate gene expression to maximize survival within or outside of a host. Here we show that Bordetella species regulate virulence factor expression in response to carbon dioxide levels that mimic in vivo conditions within the respiratory tract. We found strains of Bordetella bronchiseptica that did not produce adenylate cyclase toxin (ACT) when grown in liquid or solid media with ambient air aeration, but produced ACT and additional antigens when grown in air supplemented to 5% CO2. Transcriptome analysis and quantitative real time-PCR analysis revealed that strain 761, as well as strain RB50, increased transcription of genes encoding ACT, filamentous hemagglutinin (FHA), pertactin, fimbriae and the type III secretion system in 5% CO2 conditions, relative to ambient air. Furthermore, transcription of cyaA and fhaB in response to 5% CO2 was increased even in the absence of BvgS. In vitro analysis also revealed increases in cytotoxicity and adherence when strains were grown in 5% CO2. The human pathogens B. pertussis and B. parapertussis also increased transcription of several virulence factors when grown in 5% CO2, indicating that this response is conserved among the classical bordetellae. Together, our data indicate that Bordetella species can sense and respond to physiologically relevant changes in CO2 concentrations by regulating virulence factors important for colonization, persistence and evasion of the host immune response. Citation: Hester SE, Lui M, Nicholson T, Nowacki D, Harvill ET (2012) Identification of a CO2 Responsive Regulon in Bordetella. PLoS ONE 7(10): e47635. doi:10.1371/journal.pone.0047635 Editor: Nancy E. Freitag, University of Illinois at Chicago College of Medicine, United States of America Received February 13, 2012; Accepted September 19, 2012; Published October 24, 2012 This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Funding: This work was supported by National Institutes of Health grant GM083113 (to ETH) and by the Agriculture and Food Research Initiative Competitive Grants Program Grant no. 2010-65110-20488 from the USDA National Institute of Food and Agriculture. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: harvill@psu.edu Introduction Many cues, such as temperature, oxygen (O2), iron, pH, osmolarity and bicarbonate, allow bacteria to distinguish between environments within a host and outside of a host, as well as various microenvironments within a host [1]. In sensing multiple cues, bacteria are able to synchronize gene expression to adapt and ultimately thrive [2]. One cue, carbon dioxide (CO2), has been shown to affect regulation of virulence factor expression in many bacterial pathogens. Bacillus anthracis responds to elevated levels of CO2 by increasing expression of the genes encoding edema toxin, lethal factor and protective antigen [3–5]. In response to 10% CO2, Streptococcus pyogenes increases transcription of M protein, an important virulence factor that prevents the deposition of complement onto the bacterial surface [6]. In increased CO2, M protein has been shown to be regulated by a trans-acting positive regulatory protein that binds to the promoter of the emm gene [6,7]. CO2 regulation in B. anthracis appears to be more complicated since the transcriptional regulator of the toxins is not increased transcriptionally in response to growth in CO2 [3]. Additionally, Staphylococcus aureus, Salmonella enterocolitica and Borrelia burgdorferri are responsive to increased CO2 concentrations, suggesting this ability is useful to a variety of pathogens [8–11]. Bordetella bronchiseptica is a Gram-negative bacterium that infects a wide range of hosts causing respiratory disease varying from asymptomatic persistence in the nasal cavity for the life of the host to lethal pneumonia [12–14]. B. bronchiseptica is very closely related to the other two classical bordetellae, Bordetella pertussis and Bordetella parapertussis, the causative agents of whooping cough in humans [13,15,16]. Several virulence factors are produced by B. bronchiseptica such as, pertactin (PRN), filamentious hemaglutinin (FHA), two serotypes of fimbriae, and the two cytotoxic mechanisms, adenylate cyclase toxin (ACT) and the Type III Secretion System (TTSS) [13,16]. ACT, a member of the repeats- in-toxin (RTX) family, is a bi-functional adenylate cyclase/ hemolysin that converts ATP to cAMP, disrupting oxidative burst, phagocytosis, chemotaxis and eventually leads to apoptosis in macrophages and neutrophils [17–19]. ACT has also been shown to contribute to pathology, efficient colonization and persistence of B. bronchiseptica and B. pertussis species [20–22]. Regulation of virulence factors in bordetellae occurs via the BvgAS two-component system [23]. BvgS, the sensor in the cytoplasmic membrane, is thought to directly sense changes in the environment and, through a phosphorylation-transfer mechanism, activates BvgA, the response regulator [24–26]. Once BvgA is activated (Bvg+ phase), it binds to high and low affinity motifs in the genome, resulting in increased expression of the genes encoding toxins and adhesins, while expression of Bvg2 phase genes involved in motility and uptake of certain nutrients are repressed; the opposite occurs in the Bvg2 phase [27–32]. An intermediate phase has been described in which a subset of virulence factors are expressed, along with a unique set of factors PLOS ONE | www.plosone.org 1 October 2012 | Volume 7 | Issue 10 | e47635