Identification of a CO2 responsive regulon in Bordetella.

Sara E Hester,Eric T Harvill,Tracy Nicholson,Minghsun Lui,Daryl Nowacki,Nancy E Freitag

doi:10.1371/journal.pone.0047635

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.

Highlights

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]
Through screening of 73 isolates based on hemolysis on blood agar plates and PCR amplification of the genes encoding adenylate cyclase toxin (ACT), 4 B. bronchiseptica isolates were found to be non-hemolytic, but still retained the genes for production of ACT (Fig. 1, data not shown)
RB50 was hemolytic even in ambient air (,0.03% CO2) (Fig. 1, compare panels A and C), but there appeared to be more hemolysis when it was grown in 5% CO2 conditions, suggesting both strains produce more ACT in response to growth in 5% CO2 conditions

Summary

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]. 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 [3,4,5]. 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]. Staphylococcus aureus, Salmonella enterocolitica and Borrelia burgdorferri are responsive to increased CO2 concentrations, suggesting this ability is useful to a variety of pathogens [8,9,10,11]

Methods

Results

Conclusion