Impact of a Dual Function Type VI Secretion System (T6SS) Immunity Protein on Airway Microbiota

About This Grant

SUMMARY/ABSTRACT Dense microbial communities known as the microbiota are intimately associated with human health. Invading pathogens must overcome the microbiota to establish and successfully cause infections. In settings like the gastrointestinal tract, antagonistic interactions between microbes can strongly influence colonization outcomes. One highly prevalent pathway known to mediate these interactions the type VI secretion system (T6SS) which mediates contact-dependent killing via the translocation of toxic effector proteins into recipient cells. How the T6SS affects pathogen fitness and microbiota composition in other body sites, like the respiratory tract, is not well understood. A model disease for studying microbial ecology and interspecies interactions is Cystic Fibrosis, where polymicrobial communities of microorganisms colonize the respiratory tract due to defects in ion secretion and pathological mucus accumulation. Pseudomonas aeruginosa (Pa) causes chronic infections, dominates the airways of people with cystic fibrosis (pwCF) and is a major cause of morbidity and mortality. Pa harbors three T6SS with a variety of effectors delivered to host and bacterial competitors. We have recently reported that a T6SS effector, TseT, is delivered by the H2-T6SS and regulates microbial diversity in the upper respiratory tract of pwCF. In addition to TseT, the tseT operon encodes an immunity protein, TsiT, which our preliminary studies show also regulates Pa biofilm, in addition to its role as a bona fide immunity protein. TsiT has sequential and structural homology to general LysR-type transcriptional regulators (LTTR), which are known to regulate diverse genes including those involved in biofilm, metabolism, and quorum sensing. We have also identified diverse homologs for tsiT in a variety of Gram-negative organisms, including Burkholderiales. These results have led us to the overarching hypothesis that TsiT is a dual-function immunity protein that both regulates biofilm and neutralizes the TseT effector. In this proposal, we will define the mechanism by which TsiT regulates biofilm, investigate the evolution of tsiT homologs, and determine their impact on microbial ecology in polymicrobial communities in the respiratory tract. To this end, we propose three aims: 1) Test the hypothesis that TsiT is a transcriptional regulator that mediates Pa biofilm; 2) Test the hypothesis that tsiT diversity outside of P. aeruginosa confers protection against Pa TseT intoxication; 3) Test the hypothesis that tsiT genes modulate P. aeruginosa fitness during biofilm growth in a model CF respiratory microbiota.