Genetic studies <italic>Neisseria gonorrhoeae</italic> multicellular behaviour and human cell adherence mediated by Type IV pili.

Bacterial adherence is an important early step in infection and results from the specific interaction between a bacterial protein, called an adhesin, and a host cell receptor. This specific interaction is often mediated by bacterial cell surface supramolecular fibers known as pili, or fimbriae that are composed of protein subunits. Although the major structural subunit can confer the adhesive characteristics of pili, more often the adhesin is a component of the pilus shaft interpersed along the fiber or localized to a tip fibrillar structure. This study focuses on the Type IV pili (Tfp) of Neisseria gonorrhoeae , a unique class of adhesive pili defined by their shared biochemical and structural features which are expressed by a wide variety of important human mucosal pathogens. Gonococcal Tfp not only play a critical role in binding to receptors on human epithelial cells by adhesin presentation, but also correlates with diverse phenotypes which may contribute to the host-microbe interaction including bacterial autoagglutination and twitching motility. The primary objective of these studies was to use both genetic and functional analyses to define relationships between pilus and pilus-associated phenotypes. The gonococcal pilU gene whose product is a member of TrbB-like family proteins was identified and characterized. Mutant construction and analyses showed that the PilU protein is dispensable for pilus biogenesis but modifies the expression of Tfp-associated multicellular behavior and epithelial cell adherence. Comparative alignment of the PilU primary structure with other members of the TrbB-like proteins revealed a conserved carboxy terminal domain unique to members of the family which specifically modify Tfp-associated phenotypes. Furthermore, although precise functions served by PilU remain obscure, unique features of findings in gonococcus lead us to propose a model in which PilU exerts its effects by antagonizing the activities of the related PilT protein required for twitching motility. As a complementary approach to addressing these questions, spontaneous mutants which were defective in Tfp-associated aggregative were isolated and shown to carry missense mutations in pilE encoding the pilus subunit protein. These aggregation mutants were quantitatively unaltered in fiber expression, making it possible to assess in a more direct manner the relative contributions of cell-cell aggregation to host cell interaction. The findings define the critical role of residues within the amino-terminus of gonococcal pilin subunit to cell-cell aggregation, and established a strong correlation between bacterial self-aggregation and the net increase of adherence to host tissues, which directly demonstrates the potential significance of bacterial multicellular interactions for host colonization, the first critical step in pathogenesis.Ph.D.Biological SciencesGeneticsMicrobiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/132217/2/9959835.pd