Genetic dissection of the Type IV pilus biogenesis pathway in Neisseria gonorrhoeae.

Neisseria gonorrhoeae, the Gram negative etiologic agent of gonorrhea, is one of a large number of mucosal pathogens of man which express Type IV pili (Tfp). Tfp are filamentous surface appendages composed primarily of a single subunit termed pilin. In N. gonorrhoeae, these filaments facilitate colonization of the human host. In addition, their expression correlates with a number of phenotypes including: twitching motility, competence for natural transformation, autoagglutination and human epithelial cell adherence. The role of Tfp in these phenotypes has yet to be determined. Gonococcal PilT, a protein belonging to a large family of molecules with conserved nucleotide binding motifs, has recently been identified. This molecule is not required for pilus expression, but it is essential for expression of pilus-associated twitching motility and DNA uptake. In an attempt to define the function of PilT we have found that it influences the Tfp biogenesis pathway, as loss-of-function mutations in pilT suppress the biogenesis defect resulting from mutations in pilC, which encodes a putative pilus-associated adhesin. This finding indicates that PilT has an antagonistic effect on filament assembly. By constructing additional mutants, which lack PilT and carry lesions in other essential Tfp biogenesis genes, we have been able to define two discrete steps in the biogenesis pathway, filament assembly and extrusion across the outer membrane. This genetic approach has led to the discovery that PilT is involved in a pilin degradation pathway, which may account for its antagonistic role in filament assembly. Based on the findings presented in this thesis, we hypothesize that Tfp are dynamic structures, such that the biogenesis pathway is coupled to a PilT-mediated pilin degradation pathway. This model provides a molecular mechanism for pilus retraction and may have implications for the role of Tfp in twitching motility and DNA uptake. In addition, we have identified a prepilin-like molecule, termed ComP which is essential for DNA uptake, but dispensable for pilus expression and all other Tfp-associated phenotypes. Further examination of this molecule will help to define the role of Tfp and the Tfp biogenesis pathway in DNA uptake.Ph.D.Biological SciencesHealth and Environmental SciencesMicrobiologyMolecular biologyPathologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/132037/2/9938570.pd