High-pressure processing-induced transcriptome response during recovery of Listeria monocytogenes.

BACKGROUND: High-pressure processing (HPP) is a commonly used technique in the food industry to inactivate pathogens, including L. monocytogenes. It has been shown that L. monocytogenes is able to recover from HPP injuries and can start to grow again during long-term cold storage. To date, the gene expression profiling of L. monocytogenes during HPP damage recovery at cooling temperature has not been studied. In order identify key genes that play a role in recovery of the damage caused by HPP treatment, we performed RNA-sequencing (RNA-seq) for two L. monocytogenes strains (barotolerant RO15 and barosensitive ScottA) at nine selected time points (up to 48 h) after treatment with two pressure levels (200 and 400 MPa). RESULTS: The results showed that a general stress response was activated by SigB after HPP treatment. In addition, the phosphotransferase system (PTS; mostly fructose-, mannose-, galactitol-, cellobiose-, and ascorbate-specific PTS systems), protein folding, and cobalamin biosynthesis were the most upregulated genes during HPP damage recovery. We observed that cell-division-related genes (divIC, dicIVA, ftsE, and ftsX) were downregulated. By contrast, peptidoglycan-synthesis genes (murG, murC, and pbp2A) were upregulated. This indicates that cell-wall repair occurs as a part of HPP damage recovery. We also observed that prophage genes, including anti-CRISPR genes, were induced by HPP. Interestingly, a large amount of RNA-seq data (up to 85%) was mapped to Rli47, which is a non-coding RNA that is upregulated after HPP. Thus, we predicted that Rli47 plays a role in HPP damage recovery in L. monocytogenes. Moreover, gene-deletion experiments showed that amongst peptidoglycan biosynthesis genes, pbp2A mutants are more sensitive to HPP. CONCLUSIONS: We identified several genes and mechanisms that may play a role in recovery from HPP damage of L. monocytogenes. Our study contributes to new information on pathogen inactivation by HPP.

The σB-dependent regulatory sRNA Rli47 represses isoleucine biosynthesis in Listeria monocytogenes through a direct interaction with the ilvA transcript.

The facultative intracellular pathogen Listeria monocytogenes can persist and grow in a diverse range of environmental conditions, both outside and within its mammalian host. The alternative sigma factor Sigma B (σB) plays an important role in this adaptability and is critical for the transition into the host. While some of the functions of the σB regulon in facilitating this transition are understood the role of σB-dependent small regulatory RNAs (sRNAs) remain poorly characterized. In this study, we focused on elucidating the function of Rli47, a σB-dependent sRNA that is highly induced in the intestine and in macrophages. Using a combination of in silico and in vivo approaches, a binding interaction was predicted with the Shine-Dalgarno region of the ilvA mRNA, which encodes threonine deaminase, an enzyme required for branched-chain amino acid biosynthesis. Both ilvA transcript levels and threonine deaminase activity were increased in a deletion mutant lacking the rli47 gene. The Δrli47 mutant displayed a shorter growth lag in isoleucine-depleted growth media relative to the wild-type, and a similar phenotype was also observed in a mutant lacking σB. The impact of the Δrli47 on the global transcription profile of the cell was investigated using RNA-seq, and a significant role for Rli47 in modulating amino acid metabolism was uncovered. Taken together, the data point to a model where Rli47 is responsible for specifically repressing isoleucine biosynthesis as a way to restrict growth under harsh conditions, potentially contributing to the survival of L. monocytogenes in niches both outside and within the mammalian host.

The Cobalamin-Dependent Gene Cluster of Listeria monocytogenes: Implications for Virulence, Stress Response, and Food Safety.

Several genes of the eut, pdu, and cob/cbi operons are responsible for the metabolism of ethanolamine (EA) and 1,2-propanediol (PD) and are essential during the pathogenic lifecycles of various enteric pathogens. Studies concerning EA and PD metabolism have primarily focused on bacterial genera from the family Enterobacteriaceae, especially the genus Salmonella. Listeria monocytogenes is a member of the Firmicutes phylum and is the causative agent of the rare but highly fatal foodborne disease listeriosis. The eut, pdu, and cob/cbi operons are organized as a single large locus collectively referred to as the cobalamin-dependent gene cluster (CDGC). The CDGC is well conserved in L. monocytogenes; however, functional characterization of the genes in this cluster and how they may contribute to Listeria virulence and stress tolerance in food production environments is highly limited. Previous work suggests that the degradation pathway of PD is essential for L. monocytogenes establishment in the gastrointestinal tract. In contrast, EA metabolism may be more important during intracellular replication. Other studies indicate that the CDGC is utilized when L. monocytogenes is exposed to food and food production relevant stress conditions. Perhaps most noteworthy, L. monocytogenes exhibits attenuated growth at cold temperatures when a key EA utilization pathway gene was deleted. This review aims to summarize the current knowledge of these pathways in L. monocytogenes and their significance in virulence and stress tolerance, especially considering recent developments.

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.

Listeria monocytogenes is a well-characterized pathogen that represents a major threat to food safety. In this study, we examine the chromosomal and plasmid transcriptomes of two different L. monocytogenes strains, 6179 [belonging to sequence type (ST) 121] and R479a (ST8), in response to 30 min exposure to oxidative (0.01% hydrogen peroxide) and acid (1% lactic acid, pH 3.4) stress. The exposure to oxidative stress resulted in 102 and 9 differentially expressed (DE) genes in the chromosomal transcriptomes of 6179 and R479a, respectively. In contrast, 2280 and 2151 DE genes were observed in the respective chromosomal transcriptomes of 6179 and R479a in response to lactic acid stress. During lactic acid stress, we observed upregulation of numerous genes known to be involved in the L. monocytogenes stress response, including multiple members of the σB regulon, many of which have not been functionally characterized. Among these genes, homologs of lmo2230 were highly upregulated in both strains. Most notably, the σB-dependent non-coding RNA Rli47 was by far the most highly expressed gene in both 6179 and R479a, accounting for an average of 28 and 38% of all mapped reads in the respective chromosomal transcriptomes. In response to oxidative stress, one DE gene was identified in the 6179 plasmid transcriptome, and no DE genes were observed in the transcriptome of the R479a plasmid. However, lactic acid exposure resulted in upregulation of the stress response gene clpL, among others, on the 6179 plasmid. In R479a, a number of uncharacterized plasmid genes were upregulated, indicating a potential role in stress response. Furthermore, an average of 65% of all mapped transcriptome reads for the R479a plasmid following acid stress were mapped to an intergenic region bearing similarity to riboswitches involved in transition metal resistance. The results of this study support the conclusion that members of the σB regulon, particularly lmo2230 and the non-coding RNA Rli47, play an integral role in the response of L. monocytogenes to acid stress. Furthermore, we report the first global transcriptome sequencing analysis of L. monocytogenes plasmid gene expression and identify a putative, plasmid-encoded riboswitch with potential involvement in response to acid exposure.