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. Johnson and Cotter are experts in Bordetella pathogenesis and molecular biology, and Dr. Rosenthal is an expert in using bacterial single-cell RNA sequencing to study physiologically relevant bacterial subpopulations. Together, we have established a collaboration to leverage our combined expertise to identify the PlrSR operon. Our preliminary data indicate that the expression of plrR is heterogenous in the Bordetella population, which may explain the failure of our prior attempts to identify the regulon using bulk RNA-sequencing. We are also proposing the first single-cell RNA seq characterization of the heterogeneity of Bordetella recovered from the mammalian respiratory tract. Identification of the PlrSR regulon is low risk/high reward and will lay the foundation for a R01 project. This proposal builds on the established expertise of the Cotter-Johnson lab and includes an innovative approach using the expertise of the Rosenthal lab. Our preliminary data suggest that the single-cell RNAseq approach is successful using in vitro grown cultures of Bordetella and that we can recover sufficient bacterial cells from the murine LRT for the proposed analyses. 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.