Contributions of σB and PrfA to Listeria monocytogenes salt stress under food relevant conditions

Abstract

Listeria monocytogenes is well known to survive and grow under several stress conditions, including salt stress, which is important for growth in certain foods as well as for host infection. To characterize the contributions, to salt stress response, of transcriptional regulators important for stress response and virulence (i.e., σ(B) and PrfA), we analyzed three L. monocytogenes parent strains and isogenic mutants (ΔsigB, ΔprfA, and ΔsigBΔprfA), representing different serotypes and lineages, for their ability to grow, at 25°C, in BHI with 1.9 M NaCl. With regard to growth rate, only the lineage IV strain presented a significant difference between the parent strain and both of its respective mutants lacking prfA (ΔprfA and ΔsigBΔprfA). Conversely, the lineage I and II parent strains showed significantly shorter lag phase in comparison to their respective ΔsigB mutant strains. Intestinal epithelial cell invasion assay and hemolytic activity assays showed a significant role for σ(B) in the former and for PrfA in the latter. To explore the mechanism that may contribute to the extended lag phase in the ΔsigB mutant strain and survival and growth of the parent strain upon salt shock, whole genome transcription profiling was performed to compare transcript levels between the lineage I, serotype 1/2b, parent strain and its isogenic ΔsigB mutant after 30 min of lag phase growth at 25°C in the presence of 1.9M NaCl (salt shock) without aeration. Microarray data showed significantly higher transcript levels for 173 genes in the parent strain as compared to the ΔsigB strain. Overall, 102 of the 173 σ(B) up-regulated genes had been identified in previous studies, indicating that 71 genes were newly identified as being up-regulated by σ(B) in this study. We hypothesize that, among these genes newly identified as σ(B) up-regulated, four genes (lmo2174, lmo0530, lmo0527 and lmo0529) may play a major role in response to salt stress. Lmo2174 contains domains that facilitate sensing and producing a transduction signal in the form of cyclic di-GMP, which may activate the enzymes Lmo0527, Lmo0529 and Lmo0530, which encode proteins similar to those responsible for synthesis of exopolysaccharides that may protect the cell by changing the cell wall structure during salt stress. Overall, our data showed that σ(B), but not PrfA, contributes to growth under salt stress. Moreover, we show that the σ(B) regulon of a L. monocytogenes lineage I strain challenged with salt shock includes salt stress-specific as well as previously unidentified σ(B) up-regulated genes.

Stochastic and Differential Activation of σB and PrfA in Listeria monocytogenes at the Single Cell Level under Different Environmental Stress Conditions

“…While previous studies have shown that a prfA null deletion had no effect on L. monocytogenes survival of 11% salt stress (Ribeiro et al, 2014), an increase in osmolarity may serve as a signal for the imminent encounter of the pathogen with host cells in the duodenum, where the osmolarity is higher than in previously encountered gastrointestinal compartments due to the breakdown of gastric contents into smaller molecules. Therefore, increased osmolarity may represent an environmental signal that is used by L. monocytogenes to sense its presence in a host environment where induction of PrfA activity is needed to facilitate invasion of host cells.…”

“…Specifically, we hypothesized that σ B and PrfA activities differ in individual bacteria in response to several environmental conditions. We chose two conditions shown previously to induce σ B activity: heat or salt stress (i.e., exposure to 45°C or 4.4% NaCl) (Sue et al, 2003; Hu et al, 2007; Abram et al, 2008; Somolinos et al, 2010; Ait-Ouazzou et al, 2012; Ringus et al, 2012; Ribeiro et al, 2014). Water phase salt concentrations in the range of 4% are typical in food preservation, and salt is used in the preservation of foods linked to human listeriosis cases and outbreaks (e.g., cheese or deli meat; Gottlieb et al, 2006; Currie et al, 2015; Heiman et al, 2016; McIntyre et al, 2015).…”

Genotypes Associated with Listeria monocytogenes Isolates Displaying Impaired or Enhanced Tolerances to Cold, Salt, Acid, or Desiccation Stress

“…LI strains have been shown to be more salt tolerant than LII strains (Bergholz et al, 2010) and serotype 4b strains to be more salt tolerant than serotype 1/2a and 1/2b strains (Van Der Veen et al, 2008; Bergholz et al, 2010; Ribeiro et al, 2014). In the present study, no significant differences were found between the growth rates of different serotypes in 6% NaCl, however, five times as many 4b isolates were classified as salt tolerant as were classified as salt sensitive.…”

“…To date, studies which evaluated the stress tolerances of L. monocytogenes isolates have focused on associating phenotypes with genetic lineages (Bergholz et al, 2010), serotypes (Junttila et al, 1988; Barbosa et al, 1994; Ribeiro et al, 2014), and isolation sources (Begot et al, 1997; Durack et al, 2013). However, few significant differences between these groups were observed, suggesting that the diversity among isolates within these means of classification is not definitive for predicting phenotypic behavior.…”

Transcriptomic Analysis of the Adaptation of Listeria monocytogenes to Growth on Vacuum-Packed Cold Smoked Salmon

“…A number of previous studies have analyzed the transcriptomes of foodborne pathogens under stress conditions commonly present on/in food matrices (including hyperosmotic stress, cold stress, hydrostatic pressure stress, antimicrobial stress, acid stress, and alkali stress), using laboratory media modified to simulate these conditions as a model (15)(16)(17)(18)(19)(20)(21)(22)(49)(50)(51)(52)(53)(54)(55)(56)(57)(58)(59)(60)(61)(62)(63). These laboratory media include BHI broth (18,20,21,59), BHI agar (19), Luria-Bertani broth (60,61), tryptic soy broth (62), M9-glucose (63), and Listeria minimal medium (22) as reference conditions.…”