Abstract
Listeria monocytogenes continues to be a food safety challenge owing to its stress tolerance and virulence traits. Several listeriosis outbreaks have been linked to the consumption of contaminated ready-to-eat food products. Numerous interventions, including nisin application, are presently employed to mitigate against L. monocytogenes risk in food products. In response, L. monocytogenes deploys several defense mechanisms, reducing nisin efficacy, that are not yet fully understood. Cold shock proteins (Csps) are small, highly conserved nucleic acid-binding proteins involved in several gene regulatory processes to mediate various stress responses in bacteria. L. monocytogenes possesses three csp gene paralogs; cspA, cspB, and cspD. Using a panel of single, double, and triple csp gene deletion mutants, the role of Csps in L. monocytogenes nisin tolerance was examined, demonstrating their importance in nisin stress responses of this bacterium. Without csp genes, a L. monocytogenes ΔcspABD mutant displayed severely compromised growth under nisin stress. Characterizing single (ΔcspA, ΔcspB, and ΔcspD) and double (ΔcspBD, ΔcspAD, and ΔcspAB) csp gene deletion mutants revealed a hierarchy (cspD > cspB > cspA) of importance in csp gene contributions toward the L. monocytogenes nisin tolerance phenotype. Individual eliminations of either cspA or cspB improved the nisin stress tolerance phenotype, suggesting that their expression has a curbing effect on the expression of nisin resistance functions through CspD. Gene expression analysis revealed that Csp deficiency altered the expression of DltA, MprF, and penicillin-binding protein-encoding genes. Furthermore, the ΔcspABD mutation induced an overall more electronegative cell surface, enhancing sensitivity to nisin and other cationic antimicrobials as well as the quaternary ammonium compound disinfectant benzalkonium chloride. These observations demonstrate that the molecular functions of Csps regulate systems important for enabling the constitution and maintenance of an optimal composed cell envelope that protects against cell-envelope-targeting stressors, including nisin. Overall, our data show an important contribution of Csps for L. monocytogenes stress protection in food environments where antimicrobial peptides are used. Such knowledge can be harnessed in the development of better L. monocytogenes control strategies. Furthermore, the potential that Csps have in inducing cross-protection must be considered when combining hurdle techniques or using them in a series.
Highlights
Listeria monocytogenes is a serious public health and food safety challenge and a major economic burden worldwide
We initially examined the functional relevance of csp genes in L. monocytogenes nisin tolerance by comparing nisin stress growth phenotypes between the wild type (WT) strain and a cspABD mutant of L. monocytogenes EGDe
The growth parameters total area under the curve (AUC) capturing overall growth dynamics, lag phase duration (LPD), maximum growth rate (MGR), and final maximum cell density (MD), determined for L. monocytogenes EGDe WT and cspABD strains in nisin-supplemented brain heart infusion (BHI) and normalized for growth of each strain in normal BHI, were compared
Summary
Listeria monocytogenes is a serious public health and food safety challenge and a major economic burden worldwide. Some of the nisin stress mitigation responses documented in this bacterium to date include cell envelope composition and net cell surface charge changes that are mediated through Dalanylation and lysinylation of cell wall teichoic acids and membrane phospholipids, respectively (Abachin et al, 2002; Thedieck et al, 2006). These responses involving the Dlt and MprF protein systems, respectively, and are in part regulated through the VirABRS four-component regulatory protein system (Collins et al, 2010a; Grubaugh et al, 2018; Jiang et al, 2019). Other nisin-protective response molecular mechanisms documented in this bacterium are orchestrated through elaborate regulatory cascade loops that involve CesRK, LisRK, and LiaFSR regulatory protein systems, which, upon sensing nisin stress, implement protection responses through expression regulation of various genes in their regulons (Cotter et al, 2002; Kallipolitis et al, 2003; Fritsch et al, 2011; Nielsen et al, 2012; Bergholz et al, 2013: Draper et al, 2015)
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