Contributions of a LysR Transcriptional Regulator to Listeria monocytogenes Virulence and Identification of Its Regulons

Abstract

The capacity of Listeria monocytogenes to adapt to environmental changes is facilitated by a large number of regulatory proteins encoded by its genome. Among these proteins are the uncharacterized LysR-type transcriptional regulators (LTTRs). LTTRs can work as positive and/or negative transcription regulators at both local and global genetic levels. Previously, our group determined by comparative genome analysis that one member of the LTTRs (NCBI accession no. WP_003734782) was present in pathogenic strains but absent from nonpathogenic strains. The goal of the present study was to assess the importance of this transcription factor in the virulence of L. monocytogenes strain F2365 and to identify its regulons. An L. monocytogenes strain lacking lysR (the F2365ΔlysR strain) displayed significant reductions in cell invasion of and adhesion to Caco-2 cells. In plaque assays, the deletion of lysR resulted in a 42.86% decrease in plaque number and a 13.48% decrease in average plaque size. Furthermore, the deletion of lysR also attenuated the virulence of L. monocytogenes in mice following oral and intraperitoneal inoculation. The analysis of transcriptomics revealed that the transcript levels of 139 genes were upregulated, while 113 genes were downregulated in the F2365ΔlysR strain compared to levels in the wild-type bacteria. lysR-repressed genes included ABC transporters, important for starch and sucrose metabolism as well as glycerolipid metabolism, flagellar assembly, quorum sensing, and glycolysis/gluconeogenesis. Conversely, lysR activated the expression of genes related to fructose and mannose metabolism, cationic antimicrobial peptide (CAMP) resistance, and beta-lactam resistance. These data suggested that lysR contributed to L. monocytogenes virulence by broad impact on multiple pathways of gene expression.IMPORTANCEListeria monocytogenes is the causative agent of listeriosis, an infectious and fatal disease of animals and humans. In this study, we have shown that lysR contributes to Listeria pathogenesis and replication in cell lines. We also highlight the importance of lysR in regulating the transcription of genes involved in different pathways that might be essential for the growth and persistence of L. monocytogenes in the host or under nutrient limitation. Better understanding L. monocytogenes pathogenesis and the role of various virulence factors is necessary for further development of prevention and control strategies.

Keywords: Listeria; Listeria monocytogenes; RNA sequence; transcription regulator; virulence.

FIG 1

F2365ΔlysR strain exhibits normal growth patterns in BHI broth, as enriched medium (A), and MM (B). Bacterial growth was determined by optical density measurements at 600 nm. All growth data are the results from three independent experiments. The growth assay was conducted with wild-type F2365, F2365ΔlysR, and complement strains.

FIG 2

F2365ΔlysR strain showed reduction in adhesion (A), invasion (B), plaque number (C), and plaque size (D) with respect to the wild type. Adherence and invasion were performed with Caco-2 cells at an MOI of 10 bacteria to 1 Caco-2 cell. The experiment was performed three independent times with four replicates. Data represent mean numbers of CFU from four replicates. Error bars reflect standard errors from each means. Asterisks indicate significant differences (P < 0.05) compared to the wild type. Plaque assays were performed with mouse L2 fibroblasts at an MOI of 10 bacteria to 1 fibroblast cell. The plaque number and plaque size were determined 96 h postinfection. The experiment was repeated 3 independent times. At least 10 plaques were measured each time. Error bars represent the standard errors of the means. The wild-type strain was set at 100%.

FIG 3

Strain lacking lysR is attenuated for virulence in a mouse model following oral (A and B) and intraperitoneal (C and D) infections. Mice (5 mice per group) were infected orally or intraperitoneally with 5.9 × 10⁷ or 1 × 10⁵ CFU/ml. The number of CFU in liver and spleen was determined by serial dilution and plating on BHI plates. Each dot represents the bacterial concentration in one mouse. Median numbers for each strain are indicated by horizontal lines. Data were analyzed using a nonparametric Mann-Whitney test. Data from two independent experiments (n = 5) are shown. Letter a, significant difference (P < 0.05) from the wild type.

FIG 4

Volcano plot highlighting the differential expression genes modulated by J774A.1 macrophage cells at 4 h of infection by F2365ΔlysR and wild-type strains at an MOI range from 1 to 10. The fold change in expression of each gene is plotted against mean gene expression. Upregulated genes are plotted in red, and downregulated genes are in green. Numbers 1 to 5 represent the top upregulated genes, and 6 to 10 represent the top downregulated genes. Number 1 is type I 3-dehydroquinate dehydratase 1 (aroD). Number 2 is the DUF5067 domain-containing protein. Number 3 is a hypothetical protein. Number 4 is a hypothetical protein. Number 5 is an ABC transporter protein (extracellular solute-binding protein/arabinogalactan oligomer/maltooligosaccharide transport). Number 6 is an activator of the mannose operon, transcriptional antiterminator. Number 7 is mannosylglycerate hydrolase. Number 8 is PTS fructose transporter subunit IIC. Number 9 is an ABC transporter permease. Number 10 is a PspC domain-containing protein.

FIG 5

GO functional enrichment analysis for all differentially expressed genes. The colors represent different GO types.

FIG 6

GO categories for upregulated and downregulated genes based on differentially expressed genes in the RNA-seq.

FIG 7

RT-PCR analyses of selected differentially expressed genes. (A) Upregulated genes; (B) downregulated genes. The data represent means ± standard errors from four biological replicates.