Cold Shock Proteins Promote Nisin Tolerance in Listeria monocytogenes Through Modulation of Cell Envelope Modification Responses

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.

Deciphering the global roles of Cold shock proteins in Listeria monocytogenes nutrient metabolism and stress tolerance

“…On the other hand, csp mutants were more tolerant than the WT on some substances, e.g., EGDe_∆cspABD was more tolerant than its WT on five substances. We also previously found that cspD expressed alone in an EGDe ∆cspAB mutant increased tolerance to antimicrobials such as nisin and daptomycin (Muchaamba et al, 2021b). The mechanisms underlying these phenotypes are unknown, but we can only currently speculate that Csp loss might cause an alteration in expression of genes that increase resistance.…”

“…At the mechanistic level, csp mutants were more sensitive to the cell wall and membrane-targeting substances such as cloxacillin and BC, suggesting a role for Csps in the proper function of these cellular systems. Recently, we demonstrated that, among other functions, Csps also affect the expression of Penicillin-binding protein genes and cell wall modification systems contributing to such stress responses ( Muchaamba et al, 2021b ). An explanation is that csp mutants could have altered cell wall structures that increase permeability to some chemicals and antibiotics thus increasing sensitivity.…”

“…An explanation is that csp mutants could have altered cell wall structures that increase permeability to some chemicals and antibiotics thus increasing sensitivity. Furthermore, the cell envelope in a Δ cspABD mutant was more negatively charged than the WT, enhancing interaction and sensitivity to positively charged stressors such as nisin and BC ( Muchaamba et al, 2021b ). In the present study, the csp mutants were also more sensitive to 30S subunit targeting inhibitors suggesting that this subunit might be the Csps site of action on the ribosomes and that Csps might be involved in the initiation steps of protein synthesis ( Weber et al, 2001 ).…”

“…Lm wildtype (WT) strain representatives of LI, LII, and LIII genetic backgrounds were used to create marker-less in-frame csp gene deletion mutants. The csp gene deletion mutant series and complemented strains in Lm EGDe have been previously described (Schmid et al, 2009;Muchaamba et al, 2021b). Deletion of csp genes in the other Lm strains was performed through a double homologous recombination and allelic exchange process (Smith and Youngman, 1992;Camilli et al, 1993), mediated by the pKSV7-ΔcspA and pKSV7-ΔcspB plasmids as previously described (Schmid et al, 2009).…”

“…In agreement with this, Csps have been shown to be important in different bacterial processes including normal growth, metabolism, virulence, and tolerance of various stress conditions such as osmotic stress, antibiotics, and nutrient starvation ( Phadtare, 2004 ; Michaux et al, 2012 ; Keto-Timonen et al, 2016 ; Zhang et al, 2018 ). Similarly, characterization of csp gene deletion mutants in Lm has so far revealed that in addition to promoting cold stress, their functions are also essential for virulence, motility, biofilm formation, and resisting food-relevant stress conditions including, elevated NaCl concentrations, nisin, desiccation, and oxidative environments ( Schmid et al, 2009 ; Loepfe et al, 2010 ; Schärer et al, 2013 ; Eshwar et al, 2017 ; Kragh et al, 2020 ; Muchaamba et al, 2021b ). Our understanding of the exact molecular mechanisms through which Csps influence such a wide range of cellular processes and phenotypes is, however, currently limited.…”