Identifying the Bordetella PlrSR regulon

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PROJECT SUMMARY Pertussis (aka whooping cough) is a serious, re-emerging public health concern despite available vaccines. The acellular vaccine against Bordetella pertussis, used in the U.S. since the 1990s, prevents serious disease, but not colonization or transmission, resulting in a larger reservoir from which infants, who are most vulnerable, can be infected. New vaccines that protect against both colonization and disease are needed. The BvgAS and PlrSR two-component systems control Bordetella virulence. The BvgAS two-component regulatory system (TCS) has long been considered the master regulator of Bordetella virulence. It controls production of all know protein virulence factors, including those in the acellular vaccine. We discovered another TCS, called PlrSR, that is essential for Bordetella viability and for BvgAS activity in the lower respiratory tract (LRT). We found that PlrSR is required for LRT infection even when BvgAS is constitutively active, indicating that PlrSR controls expression of unidentified but critical virulence functions independently of BvgAS. These currently unknown virulence factors could serve as therapeutic targets or new vaccine components, and hence their identification is critical for controlling pertussis in the future. Drs. Cotter and Julio are experts in Bordetella pathogenesis and molecular biology, and Dr. Bourret is an expert in TCS biochemistry. Together, we have characterized PlrS and PlrR proteins biochemically and have discovered a way to bypass the apparent essentiality of plrS in vitro so that we can construct strains to collect gene expression data without knowing the stimuli sensed by PlrS. In Aim 1, we will identify genes regulated by PlrSR using RNA-Seq to reveal positive or negative regulation, and ChIP-Seq to reveal direct or indirect regulation. In Aim 2, we will investigate PlrSR signaling by making reporter fusions to key PlrSR-regulated genes and assessing responses to physiologically relevant stimuli, as well as the consequences of using PlrS lacking PDC or PAS sensory domains. Identification of the PlrSR regulon is low risk/high reward and will lay the foundation for a R01 project. The proposed methods are well-established, suggesting a high probability of achieving our Aims. Identifying the PlrSR regulon will be transformative in understanding Bordetella pathogenesis and enable a future R01 project in which we can determine (i) why PlrR is essential for viability, (ii) the roles of PlrSR regulated gene products in LRT infection by Bordetella, (iii) how PlrSR regulates gene expression, (iv) connections between the PlrSR and BvgAS TCSs, and (v) perhaps gain insight into the stimuli sensed by PlrS.