|dc.description.abstract||The bacterial cell wall is essential for viability, and thus represents an attractive target for antibiotic therapy. Although beta-lactams and certain other antibiotics target enzymes involved in cell wall synthesis, pathogens are rapidly becoming resistant. One poorly understood class of enzymes that may represent promising new therapeutic targets are endopeptidases, which cleave the cell wall. They are thought to be essential for insertion of new peptidoglycan into the wall and growth. In Escherichia coli, the three peptidoglycan endopeptidases, MepS, MepM, and MepH, are functionally redundant, with at least one of them being required for growth and viability. Although peptidoglycan hydrolysis is important for cell growth, uncontrolled hydrolysis can compromise the integrity of the cell wall and lead to lysis. MepS activity is controlled through the protease Prc, underscoring the importance of regulating these potentially lethal enzymes.
Pseudomonas aeruginosa is a common opportunistic pathogen that infects over 51,000 people in the United States annually. Over 6,000 cases per year are due to multidrug-resistant strains, leading the WHO to categorize it as one of the top three critical pathogens for which antibiotics are urgently needed. A better understanding of essential cellular processes, such as cell wall biogenesis, could help address the urgent need for new antibiotics to target this pathogen. The goals of this project were to identify cell wall endopeptidases in P. aeruginosa and investigate their regulation, which could have implications for future antibiotic development.
Bioinformatic analysis was employed to identify potential endopeptidases in P. aeruginosa. Based on this analysis, three genes were identified. In-frame deletion mutants of each gene were made either singly or in combination. A strain with deletion of two genes was inviable for growth as assessed through spot dilutions, suggesting that they together compromise an essential set of endopeptidases in P. aeruginosa.
Suppressor mutants that grow despite lacking these essential endopeptidases were sequenced. Loss-of-function mutations in one gene, encoding a protease, suppressed the growth defect and allowed the cells to grow. This was verified through inducing its expression, through a plasmid, in the loss-of-function suppressor mutants. This restricted growth in these suppressor mutants. This suggests that there may be additional redundant endopeptidase(s) proteolyzed by this protease. When the protease was deleted, these additional redundant endopeptidases can accumulate and compensate for deletion of the two other endopeptidases. In summary, P. aeruginosa was confirmed to have an essential set of endopeptidases analogous to E. coli. In addition, endopeptidases may be regulated by a protease.||