Transcriptional and Phenotypic Responses of Listeria monocytogenes to Chlorine Dioxide

Abstract

Significant food-borne disease outbreaks have occurred from consumption of ready-to-eat foods, including produce, contaminated with Listeria monocytogenes. Challenging food matrices (e.g., cantaloupe, sprouts) with limited processing steps postharvest to reduce pathogen loads have underscored a need for new mitigation strategies. Chlorine dioxide (ClO 2 ) is increasingly being used in produce and other food systems to reduce food-borne pathogen levels. The goal of this study was to characterize the transcriptional response and survival of L. monocytogenes 10403S exposed to ClO 2 . The transcriptional profile of log-phase cells exposed to 300 mg/liter ClO 2 for 15 min was defined by whole-genome microarray. A total of 340 genes were significantly differentially expressed. Among the differentially expressed genes, 223 were upregulated (fold change > 1.5; adjusted P value < 0.05) in role categories responsible for protein fate, cellular processes, and energy metabolism. There were 113 and 16 genes differentially expressed belonging to regulatory networks of B and CtsR, respectively. We assessed L. monocytogenes 10403S survival after exposure to 100, 300, and 500 mg/liter aqueous ClO 2 in brain heart infusion (BHI) broth; there was a significant difference between cells exposed to 500 mg/liter ClO 2 and those exposed to all other conditions over time (P value < 0.05). Isogenic ⌬sigB and ⌬ctsR mutants exposed to 300 mg/liter ClO 2 were more sensitive to ClO 2 than the wild type under the same conditions. These results provide an initial insight into the mechanisms that L. monocytogenes employs to survive sublethal ClO 2 and further our understanding of the inactivation mechanisms of this increasingly used sanitizer.

Transcriptome Sequencing of Listeria monocytogenes Reveals Major Gene Expression Changes in Response to Lactic Acid Stress Exposure but a Less Pronounced Response to Oxidative Stress

“…Although other studies observed pronounced transcriptional shifts (Pleitner et al, 2014) and increased expression of a functionally characterized oxidative stress survival islet (Harter et al, 2017) following 15 and 10 min exposures to oxidative stress, respectively, our study observed relatively minor transcriptomic differences even after 30 min of oxidative stress exposure. Only nine DE genes were seen in the R479a hydrogen peroxide treatment ( Supplementary Table S3), although seven of these were also among the 50 DE genes of 6179 with the highest log2fold changes (Supplementary Table S4).…”

“…These studies also showed that lmo2230 is upregulated during early stationary phase (Raengpradub et al, 2008;Oliver et al, 2009), within the murine intestinal lumen (Toledo-Arana et al, 2009) and in response to osmotic and acid stress (Raengpradub et al, 2008;Utratna et al, 2011;Horlbog et al, 2019). Heat and cold stress have also been shown to induce lmo2230 expression (van der Veen et al, 2007;Hingston et al, 2017a), and lmo2230 is also upregulated after chlorine dioxide exposure (Pleitner et al, 2014).…”

“…Also unique to the 6719 hydrogen peroxide response was the upregulation of two oxidative stress protection genes: sodA (LM6179_2184), a superoxide dismutase upregulated by a log2fold change of 0.79, and LM6179_1300, annotated as a glutathione peroxidase and upregulated by a log2fold change of 0.56 (Chatterjee et al, 2006;Hingston et al, 2015). The importance of the superoxide dismutase in L. monocytogenes stress response has been documented before (Soni et al, 2011;Bucur et al, 2018), and upregulation of the EGD-e homolog of LM6179_1300, lmo0983, was observed following exposure to chlorine dioxide (Pleitner et al, 2014). However, stress survival islet 2 (SSI-2), an insert of two genes homologous to the Listeria innocua genes lin0464 and lin0465 (LM6179_0748 and LM6179_0749), was not differentially expressed in 6179 despite previous implication in oxidative stress (Harter et al, 2017).…”

“…In L. monocytogenes, transposon mutagenesis of ohrA resulted in decreased tolerance to hydrogen peroxide, diamide, and cumene hydroperoxide (Reniere et al, 2016). Additionally, exposure to chlorine dioxide resulted in increased levels of ohrA expression (Pleitner et al, 2014). Genes annotated as enzymes involved in certain steps of the citric acid cycle (citZ -citrate synthase, citB -aconitase, and citC -isocitrate dehydrogenase) were also upregulated in both 6179 and R479a following exposure to hydrogen peroxide.…”

“…In addition to differences in experimental conditions, the differences in upregulation of lmo2230 could be explained by different timepoints, different L. monocytogenes strains, and/or the usage of a microarray compared to RNA sequencing. Although upregulation of lmo2230 in response to stress has been observed in numerous studies (van der Veen et al, 2007;Raengpradub et al, 2008;Oliver et al, 2009;Toledo-Arana et al, 2009;Utratna et al, 2011;Pleitner et al, 2014;Hingston et al, 2017a;Horlbog et al, 2019), its function in L. monocytogenes remains uncharacterized. The weak amino acid identity of Lmo2230 to the arsenate reductase ArsC from B. subtilis might suggest a possible role in heavy metal detoxification.…”