Date of Award

2022

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Microbiology and Immunology

Abstract

Antimicrobial-resistant (AMR) microorganisms are an increasingly seriousproblem worldwide, accounting for thousands of fatalities annually. The current literature on combating AMR pathogens often centers on commensal microbes such as Lactobacillus species, which are most frequently found in asymptomatic ‘healthy’ patients and are believed to elicit protective effects. In this dissertation, I sought to investigate the protective effect of Lactobacillus crispatus, utilizing our vast collection of clinical isolates. Previous work from our lab had found that cell-free-supernatant (CFS) of L. crispatus is robustly bactericidal, and that this killing activity does not depend on hydrogen peroxide or lactic acid. We identified phenyl-lactic acid (PLA) as an antimicrobial compound produced by L. crispatus clinical isolate LC9873. We determined that it exhibits robust killing activity against E. coli and other microbes. Like CFS, PLA’s bactericidal activity is sensitive to pH in a concentration-dependent manner, but it is durable over time and under extreme temperatures. We hypothesized that PLA effected cell death in E. coli by triggering PMF collapse, primarily by dampening the Cpx stress response necessary for its maintenance. We found that deletion of proteins that hamper PMF maintenance upon dysregulation improved cell survival in response to PLA, which corresponded to a decrease in Thioflavin T fluorescence, a fluorescent voltage sensor that diffuses across the cell membrane and is retained intracellularly upon PMF damage. We found that deletion of Cpx system components and proteins that respond to membrane damage and safeguard the PMF increased susceptibility to PLA, which corresponded to an increase in ThT fluorescence. These findings support our overall mechanistic model whereby PLA effects cell death by triggering PMF collapse. After investigating the mode of action of PLA, we characterized the range of susceptibility to both CFS and PLA across Gram-positive, Gram-negative, AMR, and fungal uropathogens. We found these pathogens highly susceptible to both CFS and PLA, though Gram-positive pathogen Enterococcus faecalis was less susceptible to PLA than CFS. We found that both CFS and PLA affected several L. gasseri and L. crispatus clinical isolates in a bacteriostatic, rather than bactericidal fashion, suggesting a means of protection against the effects of PLA.

Creative Commons License

Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.

Download

Included in

Biology Commons

Share

COinS