The LisRK signal transduction system determines the sensitivity of Listeria monocytogenes to nisin and cephalosporins

Abstract

The Listeria monocytogenes two-component signal transduction system, LisRK, initially identified in strain LO28, plays a significant role in the virulence potential of this important food-borne pathogen. Here, it is shown that, in addition to its major contribution in responding to ethanol, pH, and hydrogen peroxide stresses, LisRK is involved in the ability of the cell to tolerate important antimicrobials used in food and in medicine, e.g., the lantibiotic nisin and the cephalosporin family of antibiotics. A (Delta)lisK mutant (lacking the LisK histidine kinase sensor component) displays significantly enhanced resistance to the lantibiotic nisin, a greatly enhanced sensitivity to the cephalosporins, and a large reduction in the expression of three genes thought to encode a penicillin-binding protein, another histidine kinase (other than LisK), and a protein of unknown function. Confirmation of the role of LisRK was obtained when the response regulator, LisR, was overexpressed using both constitutive and inducible (nisin-controlled expression) systems. Under these conditions we observed a reversion of the (Delta)lisK mutant to wild-type growth kinetics in the presence of nisin. It was also found that overexpression of LisR complemented the reduced expression of two of the aforementioned genes. These results demonstrate the important role of LisRK in the response of L. monocytogenes to a number of antimicrobial agents.

FIG. 1.

Growth of LO28ΔlisK in the presence of antimicrobial agents. Shown is growth of LO28 (circles) and LO28ΔlisK (squares) in TSB-YE with 0 (A), 50 (B), 100 (C), 200 (D), and 300 (E) μg of nisin powder/ml. Growth was determined by using a Spectra Max 340 spectrophotometer (Molecular Devices) over a 20-h period. Error bars, standard deviations from the means of quadruplicate experiments.

FIG. 2.

(A and B) RT-PCRs to compare levels of transcripts for genes whose products show homology to histidine kinase (HK), a protein of unknown function (gene C), and a PBP in strains LO28 (L) and LO28ΔlisK (ΔK) (A) and LO28ΔlisK(pNZ44lisR) (+R) and LO28ΔlisK(pNZ44) (−R) (B). (C) Confirmation of the overexpression of lisR by RT-PCR using lisR-specific PCR primers to amplify cDNA templates of equal concentrations generated from LO28ΔlisKNICE(pNZ8048) or LO28ΔlisKNICE(pNZ8048-lisR) RNA following nisin induction. In all cases control PCR primers were used to ensure the complete removal of DNA from RNA preparations prior to reverse transcription and to ensure that levels of cDNA for samples that were to be compared were equal.

FIG. 3.

Complementation of the nisin resistance phenotype of LO28ΔlisK by using the NICE system. Growth of LO28ΔlisKNICE(pNZ8048) (squares) and LO28ΔlisKNICE(pNZ8048-lisR) (circles) in TSB-YE with 0 (A), 50 (B), 100 (C), 200 (D), and 300 (E) μg of nisin powder/ml. Growth was determined by using a Spectra Max 340 spectrophotometer (Molecular Devices) over a 20-h period. Error bars, standard deviations from the means of quadruplicate experiments.