Determining the mechanism of protein transit through the peptidoglycan layer in Gram-positive bacteria and identifying phage-docking sites on Clostridium perfringens

Clostridium perfringens is a Gram-positive (Gram+) anaerobic spore-forming bacterium that can act as a lethal human pathogen. Depending on the pathway of entry and strain, infection can lead to gastrointestinal disease or gas gangrene. Disease, as with nearly all bacterial pathogens, is primarily the result of secreted proteins, better known as exotoxins. These toxins can aid in the breakdown of host tissue leading to increased nutrient availability for the pathogen. Toxins of particular interest include collagenase, perfringolysin O, and phospholipase C. Most toxins, assuming they are not directly injected into host cells, need to be secreted. This is no trivial task, as the Gram+ cell wall (the outermost structure) is a very thick, dense, mesh-like screen comprised primarily of peptidoglycan (PG) and teichoic acid side chains. Due to this barrier, which ordinarily benefits the bacterium in both defense and resisting osmotic stress, secretion can be an issue. Globular proteins smaller than 25 kDa have been found to passively move through the PG, but most toxins and larger macromolecules exceed this size threshold. A solution to this issue could be either a temporary opening in the PG layer or a designated protein-lined channel for this event to occur. In order to determine this mechanism, the aforementioned toxins come into play as they are all between 45-120 kilodaltons (kDa) in size. Each is passed through the cytoplasmic membrane via the general secretion system (Sec) in a linear fashion. From there it is highly supported that proper toxin folding occurs in the periplasmic-like space. Following this step, the cell wall secretion mechanism has yet to be identified. Such a mechanism could be a target for antimicrobials rendering the cell avirulent, but viable. The strain used in this project, HN13, is both non-sporulating and only has two characterized secretion pathways (Sec and pilus-dependent). Random transposon mutagenesis and several negative selection rounds were used to construct multiple mariner mutant libraries. These libraries were screened on specific agar to visualize respective toxin secretion with approximately 200,000 mutant colonies screened. From the initial secretion screen mutants undergo an additional secondary screen through which only 186 mutants of interest were found (