Comparative analysis of the sigma B-dependent stress responses in Listeria monocytogenes and Listeria innocua strains exposed to selected stress conditions.
ABSTRACT: The alternative sigma factor sigma(B) contributes to transcription of stress response and virulence genes in diverse gram-positive bacterial species. The composition and functions of the Listeria monocytogenes and Listeria innocua sigma(B) regulons were hypothesized to differ due to virulence differences between these closely related species. Transcript levels in stationary-phase cells and in cells exposed to salt stress were characterized by microarray analyses for both species. In L. monocytogenes, 168 genes were positively regulated by sigma(B); 145 of these genes were preceded by a putative sigma(B) consensus promoter. In L. innocua, 64 genes were positively regulated by sigma(B). sigma(B) contributed to acid stress survival in log-phase cells for both species but to survival in stationary-phase cells only for L. monocytogenes. In summary, (i) the L. monocytogenes sigma(B) regulon includes >140 genes that are both directly and positively regulated by sigma(B), including genes encoding proteins with importance in stress response, virulence, transcriptional regulation, carbohydrate metabolism, and transport; (ii) a number of L. monocytogenes genes encoding flagellar proteins show higher transcript levels in the Delta sigB mutant, and both L. monocytogenes and L. innocua Delta sigB null mutants have increased motility compared to the respective isogenic parent strains, suggesting that sigma(B) affects motility and chemotaxis; and (iii) although L. monocytogenes and L. innocua differ in sigma(B)-dependent acid stress resistance and have species-specific sigma(B)-dependent genes, the L. monocytogenes and L. innocua sigma(B) regulons show considerable conservation, with a common set of at least 49 genes that are sigma(B) dependent in both species.
Project description:In several gram-positive bacterial genera including Bacillus, Staphylococcus, and Listeria, sigma B (σB) has been identified as a stress-responsive alternative sigma factor responsible for initiating transcription of genes (the σB regulon) involved in response to stress-inducing environmental conditions. In L. monocytogenes, a foodborne pathogen of considerable threat to public health and the food industry, σB is involved in regulation of stress response and virulence gene expression. We have defined the σB regulon in L. monocytogenes during early stationary phase and under salt stress (0.3M NaCl) conditions using whole-genome microarrays, identifying 168 genes that generated ≥2.0-fold higher signals in the parental strain 10403S than in an isogenic sigB null mutant (ΔsigB), categorized into nine functional groups including stress-response genes (12), virulence genes (5), and genes related to transport (26) and metabolism (45). To gain a broader biological perspective of the σB stress response system, we applied these microarrays to Listeria innocua under the same environmental conditions. Our studies revealed 64 candidates in the L. innocua σB regulon with ≥2.0-fold higher signals in the parent than in a ΔsigB mutant; 49 of the 64 genes overlap with the L. monocytogenes σB regulon, indicating extensive overlap in σB-controlled genes between the two species. Further transcriptional analysis using TaqMan quantitative real time RT-PCR was performed for selected genes that displayed contrasting fold changes among the four microarray data sets (two stress conditions per species). We report novel members of the L. monocytogenes σB regulon, as well as the initial definition of the L. innocua σB regulon. Our comparative studies of the σB stress response systems in L. monocytogenes and L. innocua revealed features of the σB regulon that are conserved and unique to the two species. Keywords: Listeria monocytogenes, Listeria innocua, SigB regulon, salt stress, stationary phase Overall design: Independent RNA isolations were performed for each growth experiment (log phase cells exposed to BHI+0.3M NaCl and early stationary phase cells). Three biological replicates were used in competitive whole-genome microarray experiments. For each set of hybridizations (salt-stressed and early stationary phase), RNA from the parent strain L. monocytogenes 10403S were hybridized to RNA from the isogenic sigB null mutant.
Project description:While the stress-responsive alternative sigma factor sigma(B) has been identified in different species of Bacillus, Listeria, and Staphylococcus, the sigma(B) regulon has been extensively characterized only in B. subtilis. We combined biocomputing and microarray-based strategies to identify sigma(B)-dependent genes in the facultative intracellular pathogen Listeria monocytogenes. Hidden Markov model (HMM)-based searches identified 170 candidate sigma(B)-dependent promoter sequences in the strain EGD-e genome sequence. These data were used to develop a specialized, 208-gene microarray, which included 166 genes downstream of HMM-predicted sigma(B)-dependent promoters as well as selected virulence and stress response genes. RNA for the microarray experiments was isolated from both wild-type and Delta sigB null mutant L. monocytogenes cells grown to stationary phase or exposed to osmotic stress (0.5 M KCl). Microarray analyses identified a total of 55 genes with statistically significant sigma(B)-dependent expression under the conditions used in these experiments, with at least 1.5-fold-higher expression in the wild type over the sigB mutant under either stress condition (51 genes showed at least 2.0-fold-higher expression in the wild type). Of the 55 genes exhibiting sigma(B)-dependent expression, 54 were preceded by a sequence resembling the sigma(B) promoter consensus sequence. Rapid amplification of cDNA ends-PCR was used to confirm the sigma(B)-dependent nature of a subset of eight selected promoter regions. Notably, the sigma(B)-dependent L. monocytogenes genes identified through this HMM/microarray strategy included both stress response genes (e.g., gadB, ctc, and the glutathione reductase gene lmo1433) and virulence genes (e.g., inlA, inlB, and bsh). Our data demonstrate that, in addition to regulating expression of genes important for survival under environmental stress conditions, sigma(B) also contributes to regulation of virulence gene expression in L. monocytogenes. These findings strongly suggest that sigma(B) contributes to L. monocytogenes gene expression during infection.
Project description:BACKGROUND:Ecological, biochemical and genetic resemblance as well as clear differences of virulence between L. monocytogenes and L. innocua make this bacterial clade attractive as a model to examine evolution of pathogenicity. This study was attempted to examine the population structure of L. innocua and the microevolution in the L. innocua-L. monocytogenes clade via profiling of 37 internalin genes and multilocus sequence typing based on the sequences of 9 unlinked genes gyrB, sigB, dapE, hisJ, ribC, purM, gap, tuf and betL. RESULTS:L. innocua was genetically monophyletic compared to L. monocytogenes, and comprised four subgroups. Subgroups A and B correlated with internalin types 1 and 3 (except the strain 0063 belonging to subgroup C) and internalin types 2 and 4 respectively. The majority of L. innocua strains belonged to these two subgroups. Subgroup A harbored a whole set of L. monocytogenes-L. innocua common and L. innocua-specific internalin genes, and displayed higher recombination rates than those of subgroup B, including the relative frequency of occurrence of recombination versus mutation (rho/theta) and the relative effect of recombination versus point mutation (r/m). Subgroup A also exhibited a significantly smaller exterior/interior branch length ratio than expected under the coalescent model, suggesting a recent expansion of its population size. The phylogram based on the analysis with correction for recombination revealed that the time to the most recent common ancestor (TMRCA) of L. innocua subgroups A and B were similar. Additionally, subgroup D, which correlated with internalin type 5, branched off from the other three subgroups. All L. innocua strains lacked seventeen virulence genes found in L. monocytogenes (except for the subgroup D strain L43 harboring inlJ and two subgroup B strains bearing bsh) and were nonpathogenic to mice. CONCLUSIONS:L. innocua represents a young species descending from L. monocytogenes and comprises four subgroups: two major subgroups A and B, and one atypical subgroup D serving as a link between L. monocytogenes and L. innocua in the evolutionary chain. Although subgroups A and B appeared at approximately the same time, subgroup A seems to have experienced a recent expansion of the population size with higher recombination frequency and effect than those of subgroup B, and might represent the possible evolutionary direction towards adaptation to environments. The evolutionary history in the L. monocytogenes-L. innocua clade represents a rare example of evolution towards reduced virulence of pathogens.
Project description:Some Listeria monocytogenes internalins are recognized as contributing to invasion of mammalian tissue culture cells. While PrfA is well established as a positive regulator of L. monocytogenes virulence gene expression, the stress-responsive sigma(B) has been recognized only recently as contributing to expression of virulence genes, including some that encode internalins. To measure the relative contributions of PrfA and sigma(B) to internalin gene transcription, we used reverse transcription-PCR to quantify transcript levels for eight internalin genes (inlA, inlB, inlC, inlC2, inlD, inlE, inlF, and inlG) in L. monocytogenes 10403S and in isogenic Delta prfA, Delta sigB, and Delta sigB Delta prfA strains. Strains were grown under defined conditions to produce (i) active PrfA, (ii) active sigma(B) and active PrfA, (iii) inactive PrfA, and (iv) active sigma(B) and inactive PrfA. Under the conditions tested, sigma(B) and PrfA contributed differentially to the expression of the various internalins such that (i) both sigma(B) and PrfA contributed to inlA and inlB transcription, (ii) only PrfA contributed to inlC transcription, (iii) only sigma(B) contributed to inlC2 and inlD transcription, and (iv) neither sigma(B) nor PrfA contributed to inlF and inlG transcription. inlE transcript levels were undetectable. The important role for sigma(B) in regulating expression of L. monocytogenes internalins suggests that exposure of this organism to environmental stress conditions, such as those encountered in the gastrointestinal tract, may activate internalin transcription. Interplay between sigma(B) and PrfA also appears to be critical for regulating transcription of some virulence genes, including inlA, inlB, and prfA.
Project description:Transcription of the Listeria monocytogenes positive regulatory factor A protein (PrfA) is initiated from either of two promoters immediately upstream of prfA (prfAp(1) and prfAp(2)) or from the upstream plcA promoter. We demonstrate that prfAp(2) is a functional sigma(B)-dependent promoter and that a sigB deletion mutation affects the virulence phenotype of L. monocytogenes. Thus, the alternative sigma factor sigma(B) contributes to virulence in L. monocytogenes.
Project description:In the last decade, atypical Listeria monocytogenes and L. innocua strains have been detected in food and the environment. Because of mutations in the major virulence genes, these strains have different virulence intensities in eukaryotic cells. In this study, we performed phenotypic and genotypic characterization of atypical L. monocytogenes and L. innocua isolates obtained from swine slaughterhouses and meat markets. Forty strains were studied, including isolates of L. monocytogenes and L. innocua with low-hemolytic activity. The isolates were characterized using conventional phenotypic Listeria identification tests and by the detection and analysis of L. monocytogenes-specific genes. Analysis of 16S?rRNA was used for the molecular identification of the Listeria species. The L. monocytogenes isolates were positive for all of the virulence genes studied. The atypical L. innocua strains were positive for hly, plcA, and inlC. Mutations in the InlC, InlB, InlA, PI-PLC, PC-PLC, and PrfA proteins were detected in the atypical isolates. Further in vitro and transcriptomic studies are being developed to confirm the role of these mutations in Listeria virulence.
Project description:Listeria innocua is considered a nonpathogenic Listeria species. Natural atypical hemolytic L. innocua isolates have been reported but have not been characterized in detail. Here, we report the genomic and functional characterization of representative isolates from the two known natural hemolytic L. innocua clades. Whole-genome sequencing confirmed the presence of Listeria pathogenicity islands (LIPI) characteristic of Listeria monocytogenes species. Functional assays showed that LIPI-1 and inlA genes are transcribed, and the corresponding gene products are expressed and functional. Using in vitro and in vivo assays, we show that atypical hemolytic L. innocua is virulent, can actively cross the intestinal epithelium, and spreads systemically to the liver and spleen, albeit to a lesser degree than the reference L. monocytogenes EGDe strain. Although human exposure to hemolytic L. innocua is likely rare, these findings are important for food safety and public health. The presence of virulence traits in some L. innocua clades supports the existence of a common virulent ancestor of L. monocytogenes and L. innocua.
Project description:Listeria monocytogenes sigma(B) positively regulates the transcription of class II stress response genes; CtsR negatively regulates class III stress response genes. To identify interactions between these two stress response systems, we constructed L. monocytogenes DeltactsR and DeltactsR DeltasigB strains, as well as a DeltactsR strain expressing ctsR in trans under the control of an IPTG (isopropyl-beta-d-thiogalactopyranoside)-inducible promoter. These strains, along with a parent and a DeltasigB strain, were assayed for motility, heat resistance, and invasion of human intestinal epithelial cells, as well as by whole-genome transcriptomic and quantitative real-time PCR analyses. Both DeltactsR and DeltactsR DeltasigB strains had significantly higher thermotolerances than the parent strain; however, full heat sensitivity was restored to the DeltactsR strain when ctsR was expressed in trans. Although log-phase DeltactsR was not reduced in its ability to infect human intestinal cells, the DeltactsR DeltasigB strain showed significantly lower invasion efficiency than either the parent strain or the DeltasigB strain, indicating that interactions between CtsR and sigma(B) contribute to invasiveness. Statistical analyses also confirmed interactions between the ctsR and the sigB null mutations in both heat resistance and invasion phenotypes. Microarray transcriptomic analyses and promoter searches identified (i) 42 CtsR-repressed genes, (ii) 22 genes with lower transcript levels in the DeltactsR strain, and (iii) at least 40 genes coregulated by both CtsR and sigma(B), including genes encoding proteins with confirmed or plausible roles in virulence and stress response. Our data demonstrate that interactions between CtsR and sigma(B) play an important role in L. monocytogenes stress resistance and virulence.
Project description:Mounting evidence suggests that sigma(B) and PrfA coregulate transcription of multiple genes in Listeria monocytogenes, therefore, the relative contributions of sigma(B) and PrfA to transcript levels of genes identified previously as differentially regulated by PrfA were measured. Group I genes are recognized virulence genes that are positively regulated by PrfA; group II genes were reported previously as negatively regulated by PrfA; and multiple group III genes were proposed to be coregulated by sigma(B) and PrfA. Transcript levels for selected genes were measured by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) in L. monocytogenes 10403S as well as in otherwise isogenic DeltasigB, DeltaprfA, and DeltasigBDeltaprfA strains grown under conditions demonstrated to induce either PrfA activity (0.2% activated charcoal) or both PrfA and sigma(B) activity (stationary phase). Although the Group I gene plcA was positively regulated by PrfA, transcript levels for the group II genes lmo0278 and lmo0178 were not affected by the prfA deletion. While the sigB deletion significantly affected transcript levels for the selected group III genes (i.e., lmo0596, lmo0654, bsh, and opuCA), with lower transcript levels in the DeltasigB strains under all conditions tested, transcript levels for these genes were not significantly affected by the prfA deletion. Our results suggest that the regulatory interactions between PrfA and sigma(B) contribute to PrfA's predominant role as a direct regulator of virulence genes critical for invasion and intracellular survival in L. monocytogenes 10403S, while sigma(B) regulates a wider range of virulence and stress response genes.
Project description:A total of 442 Listeria isolates, including 234 Listeria seeligeri, 80 L. monocytogenes, 74 L. welshimeri, 50 L. innocua, and 4 L. marthii isolates, were obtained from 1,805 soil, water, and other environmental samples collected over 2 years from four urban areas and four areas representing natural environments. Listeria spp. showed similar prevalences in samples from natural (23.4%) and urban (22.3%) environments. While L. seeligeri and L. welshimeri were significantly associated with natural environments (P ? 0.0001), L. innocua and L. monocytogenes were significantly associated with urban environments (P ? 0.0001). Sequencing of sigB for all isolates revealed 67 allelic types with a higher level of allelic diversity among isolates from urban environments. Some Listeria spp. and sigB allelic types showed significant associations with specific urban and natural areas. Nearest-neighbor analyses also showed that certain Listeria spp. and sigB allelic types were spatially clustered within both natural and urban environments, and there was evidence that these species and allelic types persisted over time in specific areas. Our data show that members of the genus Listeria not only are common in urban and natural environments but also show species- and subtype-specific associations with different environments and areas. This indicates that Listeria species and subtypes within these species may show distinct ecological preferences, which suggests (i) that molecular source-tracking approaches can be developed for Listeria and (ii) that detection of some Listeria species may not be a good indicator for L. monocytogenes.