Systematic analysis in Neisseria meningitidis of proteins that fine-­tune functions mediated by type IV pili

Abstract

Type IV pili (Tfp) are widespread virulence factors whose multifunctional ability sets them apart from other pili. Tfp mediate adhesion as well as aggregation, competence for DNA transformation and twitching motility (a non-­flagellar form of locomotion) in many bacterial pathogens. The molecular mechanisms of these functions and the biogenesis of these pili are not yet fully understood. To better understand Tfp biology, our group started a systematic characterization of all of the proteins involved in Tfp biology in a well-­defined genetic background, Neisseria meningitidis strain 8013. Screening of a large library of mutants in this strain followed by an in-­depth mining of the genome resulted in the discovery of 23 genes that may be involved in Tfp biology. Seven of these genes encode proteins deemed “accessory” for Tfp biogenesis as the corresponding mutants are piliated. Since previous analysis of one these genes (pilX) led to the finding that it modulates one Tfp-­linked function (aggregation), the first aim of my project was to phenotypically characterize mutants in the remaining genes along with double mutants in which filament retraction is abolished by a concurrent mutation in the pilT gene, and strains overexpressing the corresponding proteins with regards to levels of piliation, adhesion, aggregation and DNA competence. This was achieved using a battery of quantitative and qualitative methods well-­established in our research group. Results from this study showed that each of the seven proteins plays a role in fine-­tuning of Tfp-­linked function(s). This completed our systematic study of mutants in N. meningitidis Tfp biology, and gave us a complete picture of the roles of these proteins in Tfp function. This provides us with a platform for further in-­depth study of these proteins. In parallel, we attempted to further characterize one of these proteins, PilZ, by determining its localization and protein structure, however, we have been unsuccessful in both efforts so far. Finally we attempted to improve upon the current pilus purification method using a His-­tagged version of the major pilus subunit PilE to purify fibres by affinity chromatography. This could be a very useful tool for future studies.