| Description |
Uropathogenic Escherichia coli (UPEC) is the leading cause of urinary tract infections (UTIs), gram-negative bacteremia, and urosepsis. More than half of UPEC isolates secrete a pore-forming toxin called alpha-hemolysin (HlyA). At the molecular level, HlyA intoxication of cells activates proteolytic and signaling cascades such as MAP kinase, caspases, and more recently discovered serine proteases. Previous work by our lab demonstrated that serine proteases, in particular Trypsinogen 4 (Try4), were activated in response to HlyA intoxication and trafficked into the nucleus. Here I present work that begins to determine how HlyA promotes the activation of the host serine protease Try4 and also how Try4 traffics into the nucleus. Using confocal microscopy and Try4 truncations mutants, I discovered that the mesotrypsin core domain was necessary and sufficient for Try4 translocation into the nucleus. We identified that Try4 trafficked into the nucleolus, a subnuclear organelle. Inhibitor and drug studies in bladder epithelial cells showed that Try4 trafficking into the nucleolus required potassium efflux. Using mass spectrometry, we discovered potential Try4 interacting partners during HlyA intoxication. One interacting partner was identified as caspase-14, a relatively unknown caspase. Using caspase-14 over-expression constructs and confocal microscopy, I observed that Try4 trafficking into the nucleolus was significantly increased by caspase-14 over-expression. To iv ascertain the relevance of Try4 activation in vivo, we used two models of infection a UTI and sepsis. We found that inhibiting activation of Try4 was beneficial to the host, resulting in increased survival during a sepsis challenge and decreased inflammation during a UTI. At the cellular level, these studies begin to elucidate the intracellular mechanism of PFT-mediated activation and recruitment of Try4 into the nucleolus. On the organismal level, my work reveals that HlyA-mediated activation of Try4 is harmful to the host. |
