Characterization of a Type IV Pilus Biogenesis Operon in Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic bacterial pathogen and frequent cause of nosocomial infections. Its intrinsic antibiotic resistance makes it challenging to treat. Additionally, P. aeruginosa is the dominant pathogen responsible for chronic pulmonary infection in individuals with cystic fibrosis (CF). Type IV pili (Tfp) are retractile surface appendages that promote P. aeruginosa virulence by mediating i) bacterial adherence to host tissue, ii) twitching motility (TM), a form of surface-associated bacterial translocation that aids in bacterial dissemination, and iii) formation of biofilm communities. In P. aeruginosa, Tfp fibers are primarily composed of a single repeating subunit termed pilin, which is encoded by the pilA gene. In addition, there are several less abundant proteins associated with the fiber that play key structural and functional roles. The five `pilin-like' proteins (FimU, PilV, PilW, PilX, PilE), which share the highly conserved N-terminal -helical region of pilin, are encoded by an operon (fimU-pilVWXY1Y2E (fimU)) that also encodes the structurally distinct protein, PilY1. The pilin-like proteins and PilY1 are incorporated into the mature Tfp fiber and are required for Tfp biogenesis; however, PilY1 has additional roles in antagonizing pilus retraction and mediating attachment to host epithelial cells. Here we describe the regulatory mechanisms that control expression of the fimU Tfp biogenesis operon. Specifically, we identified two linked, but independent networks involving the transcriptional regulators AlgR and Vfr that converge to control fimU promoter activity. Additionally, we revealed a positive feedback mechanism that results in fimU activation when either the pilin-like proteins or PilY1 is not expressed. We determined that enhanced fimU expression involves upregulation of the TCS AlgZ/R. Based on the recently solved PilY1 crystal structure, which revealed an EF-hand-like calcium-binding site within the C-terminus of the protein; we investigated the role of calcium binding in PilY1 function. Both in vitro and in vivo studies demonstrated the importance of calcium binding and release in the control of pilus extension/retraction. Together, these studies detail the regulatory mechanisms involved in controlling both fimU transcription and PilY1 function and provide an enhanced understanding of Tfp biogenesis and regulation.Doctor of Philosoph