The role of the Type IV pili ATPase PilT as a surface sensor in regulating cell division

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Type IV pili (TFP) play a major role in bacterial pathogenesis. Although they were originally discovered and described in Gram-negative bacteria, we discovered that nearly all Clostridia species and some other Gram- positive bacteria have TFP. We demonstrated that C. perfringens TFP are needed for bacterial adherence to mouse muscle cells (myoblasts), providing a model for their role in gas gangrene infections. For full virulence in animal hosts, bacteria need to sense the presence of a surface. Gram-negative bacteria can use TFP as surface sensing organelles, since TFP are extended from the bacterium and contact nearby cell surfaces. Surface sensing using TFP has not been studied in Gram-positive bacteria but we are using C. perfringens as a model system to determine how they do this. C. perfringens increases its cell length when grown on surfaces and we discovered that a mutation in the gene encoding the retraction ATPase, PilT, in C. perfringens lost this ability. pilT is the first gene in an operon with ftsA and ftsZ, suggesting a direct link between TFP and divisome functions. Our main hypothesis is that PilT functions as a signal transducer that communicates the presence of a surface to the components of the divisome (FtsA and FtsZ) to regulate the rate of cell division and, as consequence, cell length. PilT does this by partner switching between the PilM ring located at the base of the TFP assembly apparatus in liquid grown cells to the FtsA protein on surfaces. FtsA is important for formation of the Z-ring that is used to constrict the cell envelope during the division process and PilT binding may inactivate FtsA and inhibit cell division. We have designed two aims to test this hypothesis: Aim 1. Determine the relative affinities of the protein interactome of the retraction ATPase PilT in vitro. Purified PilT, PilM, FtsA and PilB2 will be used in co-immunoprecipitation, pull down, surface plasmon resonance, isothermal titration calorimetry and protein cross-linking assays to determine their relative binding affinity to PilT and each other. To identify other proteins that may interact with PilT, co-immunoprecipitation and pull down assays will be done in whole cell extracts. Aim 2: Define the in vivo partner switching kinetics for the PilT protein when grown in liquid media and then placed on a surface. Using a reversible split fluorescence reporter and our novel pulse-chase methods for tracking fluorescent proteins in C. perfringens, the location and protein-protein interactions of PilT will be determined when cells are grown in liquid and transferred to a solid surface. These studies will provide insights into how Gram-positive pathogens sense surfaces, which can be used to design strategies to block these sensing mechanisms and lower their virulence.