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

Abstract

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 (<0.1% identification rate). Through the use of various genetic techniques, 28 genes have been identified that lead to the lack-of-secretion phenotype. These genes encode various proteins including transcription regulators, sporulation factors, two-component regulatory systems, and potentially a protein which may play a role in secretion during cell division. Through genetic complementation/knockout trials the last major unknown physiological trait of Gram+ bacteria, protein secretion, has been studied here. C. perfringens is the causative agent of gastrointestinal disease and gas gangrene. Gastrointestinal infection is typically self-limiting but may be treated with antibiotics which can lead to unintended clearing of normal healthy microflora. As for gangrenous infection, if left untreated it is 100% fatal. Even with swift treatment, amputation may still be required due to the rapid spreading of bacteria in tissue (cm/hour). Antibiotic treatment is nearly ineffective due to induced blood clotting. This leads to an anaerobic environment where most antibiotics will either not be able to function or even reach the cells. For both routes of infection, bacteriophages could supplement or entirely replace antibiotics. This could help greatly reduce the amount of collateral damage to the microbiome in gastrointestinal infection due to high phage specificity. A more speculatory approach is administering phages to tissue surrounding gangrene infection to diminish bacterial spread and density. Phages used as an alternative treatment could also help slow the observance of antibiotic resistance seen throughout the medical industry. In order to develop an effective phage therapy, phage docking sites need to be identified. Docking sites would allow for implementation of phage engineering and aid in the determination of untested phages that would be predicted to bind. Phage therapy may be a promising alternative treatment method to better treat both types of C. perfringens infections.