Cell envelope stress responses and the mechanism of antibiotic tolerance in Gram-negative pathogens

Project SummaryBacteria often resist killing by normally bactericidal antibiotics, resulting in clinical treatment failure and thedevelopment of antibiotic resistance. The ability to survive damage elicited by exposure to antibiotics is termedtolerance. Tolerance is likely responsible for the recurrence of infections after discontinuation of antimicrobialtherapy, and provides a reservoir of a bacterial population that can develop full scale resistance. An extremecase of tolerance is the formation of persister cells, which do not experience antibiotic-induced damage due todormancy. However, we and others have found that many Gram-negative pathogens (Vibrio cholerae,Pseudomonas aeruginosa, Enterobacter cloacae, Haemophilus influenzae and Acinetobacter baumannii) arefully susceptible to damage induced by cell wall acting antibiotics (beta lactams), but yet survive at very highlevels. Survival is enabled through the formation of viable spheres that are devoid of detectable cell wallmaterial and that recover to normal shape upon withdrawal of the antibiotic. In our model organism, the cholerapathogen V. cholerae, tolerance is promoted by cell envelope stress responses, especially the two-componentsystem WigKR. WigKR is induced by cell wall acting antibiotics and mounts a complex response that ultimatelyenables recovery from the spherical state. This response includes upregulation of cell wall synthesis functions,outer membrane synthesis, phospholipid synthesis and downregulation of motility and iron acquisition genes.How this response promotes tolerance is poorly understood, and so are the mechanisms of tolerance in otherGram-negative bacteria. Here, we aim to interrogate V. cholerae's cell envelope stress responses and theirrelationship with beta lactam tolerance and post-antibiotic recovery. Using genetic and biochemicalapproaches, we will find the elusive induction signal sensed by the histidine kinase WigK. Leveragingextensive datasets comprehensively describing the WigKR regulon, we will measure each individual regulonmember's contribution to beta lactam tolerance. Lastly, we will apply what we have learned in the V. choleraemodel to other Gram-negative pathogens exhibiting high beta lactam tolerance, specifically E. cloacae and P.aeruginosa. Our experiments will yield novel insight into the mechanisms of antibiotic tolerance and result inthe identification of candidate drug targets for anti-tolerance adjuvants of beta lactams.