Functional analysis of the proteins and multiprotein complexes involved in the biogenesis of type IV pili

Microbes are the most numerous living organism on the planet. They can cause diseases in other organisms, including humans, but they can also be helpful in everyday human activities, like digestion. Some are used to perform specific industrial or manufacturing processes such as the production of drugs or synthetic hormones, while others are used for the bioconversion of organic waste. A common trait for all these propertie is the necessity for microbes to interact with other organisms or their natural and man-made environments. For this purpose, they often use hair-like surface organelles known as pili, among which type IV pili (Tfp) are the most widespread. However, how Tfp are assembled and how they mediate the multiple functions they are linked with (adhesion to various surfaces, aggregation, motility and DNA import) remains poorly understood. We are therefore trying to answer these questions using as a model bacterium Neisseria meningitidis, which an important human pathogen responsible for 50% of the cases of bacterial meningitis worldwide. Besides helping develop more efficient therapies against human pathogens and leading to a better understanding of a fundamental biological problem, our work may lead to potential applications and benefits for other less tractable medically or commercially important species that express Tfp. In a first step, we have identified all the genes involved in Tfp assembly, which indicated that the corresponding machinery is complex since it numbers fifteen different protein components. This suggested that understanding how this machinery works would only be possibe through a reductionist approach, that is by first examining smaller pieces of it, which could explain the whole upon re-assembly. Therefore, we used bacterial genetics to determine that four different steps are necessary for the expression of functional Tfp and we could define at which one of these steps each of the above fifteen proteins is involved. The objectives of this research project are multiple and aim at understanding how the protein work together as subgroups in the above four steps to assemble functional Tfp. As a first thing, we will determine the basic biochemical properties of these fifteen proteins, such as teir abundance and localization in the cell. We will then identify the proteins that are interacting with each other in order to put the pieces of the puzzle back together. This will be done using two different and complementary techniques. Finally, we will move to a downstream stage which consists in determining the tri-dimensional structure and mode of function of the individual protein components. We will start by studying the PilW protein, which has particularly interesting properties.

Despite the fact that type IV pili (Tfp) are likely to be the most widespread pili in the bacterial world, their biogenesis and multiple functions remain poorly understood. Therefore, our research aims at contributing to a better understanding of Tfp biology. Using functional genomics and genetics approaches and Neisseria meningitidis as model organism, we previously demonstrated that Tfp biogenesis relies on 15 dedicated Pil proteins acting in four consecutive steps. The aim of this research project is to know more about these proteins by characterizing individually each of them and by identifying the multiprotein complexes they form. To achieve this we will: 1) systematically characterize the expression, localization and possible modifications of the above proteins. This invaluable information will be obtained by immunoblotting using an exhaustive collection of specific antibodies that we recently produced. 2) characterize multiprotein complexes formed by the above 15 proteins. Using our collection of antibodies, we will study by immunoblotting changes in stability or localization of one protein in the absence of the others, which is indicative of protein-protein interactions. In parallel, we will use a bacterial two-hybrid assay in Escherichia coli to identify functional interactions between pairs of proteins. 3) perform a detailed structure/function analysis of PilW. We will determine PilW's 3-D structure by X-ray crystallography, which will then be used to define its mode of function by site-directed mutagenesis and subsequent phenotypic analysis. This will indicate whether PilW, which affects the functionality of Tfp, does so by modulating pilus composition or modifying pilus components. It will also answer the question whether PilW-PilQ interactions are the molecular basis for PilW's role on the stability of PilQ multimers.