The ABC-Type Multidrug Resistance Transporter LmrCD Is Responsible for an Extrusion-Based Mechanism of Bile Acid Resistance in Lactococcus lactis

Abstract

Upon prolonged exposure to cholate and other toxic compounds, Lactococcus lactis develops a multidrug resistance phenotype that has been attributed to an elevated expression of the heterodimeric ABC-type multidrug transporter LmrCD. To investigate the molecular basis of bile acid resistance in L. lactis and to evaluate the contribution of efflux-based mechanisms in this process, the drug-sensitive L. lactis NZ9000 ⌬lmrCD strain was challenged with cholate. A resistant strain was obtained that, compared to the parental strain, showed (i) significantly improved resistance toward several bile acids but not to drugs, (ii) morphological changes, and (iii) an altered susceptibility to antimicrobial peptides. Transcriptome and transport analyses suggest that the acquired resistance is unrelated to elevated transport activity but, instead, results from a multitude of stress responses, changes to the cell envelope, and metabolic changes. In contrast, wild-type cells induce the expression of lmrCD upon exposure to cholate, whereupon the cholate is actively extruded from the cells. Together, these data suggest a central role for an efflux-based mechanism in bile acid resistance and implicate LmrCD as the main system responsible in L. lactis.

Allelic Variation of Bile Salt Hydrolase Genes in Lactobacillus salivarius Does Not Determine Bile Resistance Levels

“…Another cholate-induced L. salivarius MDR transporter (see Fig. S3B, LSL_0032-3, in the supplemental material) is 52% and 53% identical to the L. lactis LmrCD cholate transporter, which also confers bile resistance on these bacteria (64). The Opp system (LSL_2026-7), located on the 44-kb plasmid pSF118-44, which is responsible for glycinebetaine uptake, was also induced by cholate.…”

Intestinal transport and metabolism of bile acids

“…The mechanisms for bacterial resistance to bile acids include expression of effl ux pumps and enzymes that biotransform bile acids. The bile acid effl ux systems include ATP-dependent (e.g., members of the ABC superfamily) (168)(169)(170)(171)(172) and proton motive force-dependent carriers (e.g., members of the resistance-nodulation-division family and major facilitator superfamily) ( 173, 174 ) and appear to be a common mechanism for bile acid resistance by gut bacteria. Indeed, expression of many of these effl ux systems is bile acid-inducible and their contribution to growth and bile resistance has been demonstrated directly by functional genetics ( 169, 172, 175 ).…”

Cholate-Stimulated Biofilm Formation by Lactococcus lactis Cells

“…The resultant strain was termed the L. lactis ⌬lmrCD r strain. Transcriptome and functional analysis suggests that the resistance by this strain is no longer transporter based but is due to cell envelope-and metabolism-related alterations (41). These cells show a decreased growth rate and an increased ability to flocculate, but it is unclear how they perform in biofilms.…”

“…The ABC-type multidrug resistance (MDR) transporter LmrCD is a major determinant of drug resistance in L. lactis, but it is also involved in bile acid resistance (41). Consequently, L. lactis cells that lack the lmrCD genes are sensitive to bile acids, such as cholate.…”

“…Consequently, L. lactis cells that lack the lmrCD genes are sensitive to bile acids, such as cholate. When these cells are exposed to stepwiseincreasing sublethal concentrations of cholate, they develop an improved resistance to cholate and several other bile acids (41). The resultant strain was termed the L. lactis ⌬lmrCD r strain.…”