The LysR-type regulator LeuO regulates the acid tolerance response in Vibrio cholerae

Abstract

Vibrio cholerae is a neutrophilic enteric pathogen that is extremely sensitive to acid. As V. cholerae passages through the host gastrointestinal tract it is exposed to a variety of environmental stresses including low pH and volatile fatty acids. Exposure to acidic environments induces expression of the V. cholerae acid tolerance response. A key component of the acid tolerance response is the cad system, which is encoded by cadC and the cadBA operon. CadB is a lysine/cadaverine antiporter and CadA is a lysine decarboxylase and these function together to counter low intracellular and extracellular pH. CadC is a membrane-associated transcription factor that activates cadBA expression in response to acidic conditions. Herein we investigated the role of the LysR-type transcriptional regulator LeuO in the V. cholerae acid tolerance response. Transcriptional reporter assays revealed that leuO expression repressed cadC transcription, indicating that LeuO was a cadC repressor. Consistent with this, leuO expression was inversely linked to lysine decarboxylase production and leuO overexpression resulted in increased sensitivity to organic acids. Overexpression of leuO in a cadA mutant potentiated killing by organic acids, suggesting that the function of leuO in the acid tolerance response extended beyond its regulation of the cad system. Collectively, these studies have identified a new physiological role for LeuO in V. cholerae acid tolerance.

Vibrio cholerae LeuO Links the ToxR Regulon to Expression of Lipid A Remodeling Genes

“…ToxR also activates leuO transcription in response to bile salts and cyclic dipeptides (10, 11). LeuO has been linked to multiple phenotypes in V. cholerae, including biofilm production, virulence repression, acid tolerance, and bile resistance (10,11,19,20). Taken together, these results suggest that in addition to reg- …”

“…The above-described results suggested that LeuO acts directly at the carRS promoter; however, they did not exclude the possibility that LeuO affected carRS expression via an intermediate protein that was conserved in E. coli and V. cholerae. To determine if LeuO was capable of acting directly on the carRS promoter, we performed an EMSA with MBP-LeuO and the carRS promoter as previously described (19). The results showed that incubation of MBP-LeuO with a DNA probe spanning the carRS promoter resulted in a mobility shift in the carRS probe starting at 2.5 M MBP-LeuO (Fig.…”

“…Its entry into the human host represents a dramatic environmental shift where the cells are confronted with a number of barriers to colonization, including exposure to bile salts and products of the innate immune system. ToxR plays a critical role in the transition to the human host and overcoming these barriers (10,11,19,(45)(46)(47). In addition to regulating the production of essential virulence factors, ToxR also functions to modulate the composition of the outer membrane to fine-tune its barrier properties.…”

“…Protein purification for the electrophoretic mobility shift assays (EMSAs) was done as previously described (19). Briefly, overnight cultures of E. coli ER2566 carrying pMAL-c2 or pMAL-c2::leuO (pVA175) were diluted 100-fold into fresh LB broth and incubated at 37°C with shaking until they reached an OD 600 of ϳ0.5 when isopropyl ␤-D-1-thiogalactopyranoside (IPTG) was added at 0.3 mM.…”

“…EMSAs were performed as previously described (19). The DNA fragment containing the carRS promoter (the nucleotide sequence between Ϫ400 and ϩ20 relative to the carR translational start site) was PCR amplified from the N16961 genome using carRS-F-EMSA and …”