Harris, T. N. | bioRxiv Science

bioRxiv2025

Deletion of major shell protein of ethanolamine utilization microcompartment reduces intrinsic antibiotic resistance, biofilm and intracellular survival of Salmonella Typhimurium

With the high rise in Salmonella infection and emergence of antibiotic-resistant variants, developing a novel strategy to control the pathogen is imperative. Earlier studies revealed that Salmonella deploys ethanolamine (EA) metabolic machinery to disseminate in the intestine. Salmonella with a defect in EA metabolism manifests with lower intestinal colonization efficiency. Remarkably, the potential of EA metabolism as a therapeutic target is yet to explore. Our study revealed that supplementation of EA and vitamin B12 in both rich and minimal media enhanced biofilm formation, increased motility, and increased tolerance of Salmonella to some antibiotics. Conversely, mutants deficient in EA metabolic enzymes exhibited no physiological fitness. In Salmonella, EA metabolic enzymes are localized within a proteinaceous microcompartment (MCP) shell composed of thousands of copies of shell proteins encoded by five genes from the eut operon. Fascinatingly, bacterial cells with defective MCP shell due to mutation in the major shell proteins showed enhanced susceptibility towards a number of antibiotics in minimal media. The mutants were unable to form biofilm, produced lower curli expression and were defective in flagellar motility. Also, mutation in one of the major shell proteins reduced intramacrophagic viability of Salmonella. Notably, phenotypes were restored upon ectopic expression of corresponding genes. It was evident that mutation in the MCP shell proteins downregulated the expression of genes related to pathogenicity. Overall, this study sheds new light on understanding the relationship between EA metabolism and bacterial physiology that would pave the way for developing novel therapeutic interventions against Salmonella.

microbiology↗

bioRxiv2023

Mutation of mltG increases peptidoglycan fragment release, cell size, and antibiotic susceptibility in Neisseria gonorrhoeae

Infection with the Gram-negative species Neisseria gonorrhoeae leads to inflammation that is responsible for the disease symptoms of gonococcal urethritis, cervicitis, and pelvic inflammatory disease. During growth these bacteria release significant amounts of peptidoglycan (PG) fragments which elicit inflammatory responses in the human host. To better understand the mechanisms involved in PG synthesis and breakdown in N. gonorrhoeae, we characterized the effects of mutation of mltG. MltG has been identified in other bacterial species as a terminase that stops PG strand growth by cleaving the growing glycan. Mutation of mltG in N. gonorrhoeae did not affect bacterial growth rate but resulted in increased PG turnover, more cells of large size, decreased autolysis under non-growth conditions, and increased sensitivity to antibiotics that affect PG crosslinking. An mltG mutant released greatly increased amounts of PG monomers, PG dimers, and larger oligomers. In the mltG background, mutation of either ltgA or ltgD, encoding the lytic transglycosylases responsible for PG monomer liberation, resulted in wild-type levels of PG monomer release. Bacterial two-hybrid assays identified positive interactions of MltG with synthetic penicillin-binding proteins PBP1 and PBP2 and the PG-degrading endopeptidase PBP4 (PbpG). These data are consistent with MltG acting as a terminase in N. gonorrhoeae and suggest that absence of MltG activity results in excessive PG growth and extra PG in the sacculus that must be degraded by lytic transglycosylases including LtgA and LtgD. Furthermore, absence of MltG causes a cell wall defect that is manifested as large cell size and antibiotic sensitivity. ImportanceNeisseria gonorrhoeae is unusual in that the bacteria release larger amounts of cell wall material as they grow as compared to related bacteria, and the released cell wall fragments induce inflammation that leads to tissue damage in infected people. The study of MltG revealed the importance of this enzyme for controlling cell wall growth, cell wall fragment production, and bacterial cell size and suggest a role for MltG in a cell wall synthesis and degradation complex. The increased antibiotic sensitivities of an mltG mutant suggest that an antimicrobial drug inhibiting MltG would be useful in combination therapy to restore the sensitivity of the bacteria to cell wall targeting antibiotics to which the bacteria are currently resistant.

microbiology↗