Transcriptome analysis of Streptococcus mutans quorum sensing-induced cells
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ABSTRACT:
ORGANISM(S): Streptococcus mutans UA159
PROVIDER: GSE264670 | GEO | 2024/04/28
REPOSITORIES: GEO
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Project description:Persisters are a small fraction of growth-arrested phenotypic variants that can survive lethal concentrations of antibiotics but are able to resume growth once antibiotics are stopped. Their formation can be a stochastic process or triggered by environmental cues. In the human pathogen Streptococcus mutans, the canonical peptide-based quorum sensing system is an inducible DNA repair system that is pivotal for bacterial survival. Previous work showed that the CSP signaling peptide is a stress-signaling alarmone that promotes the formation of stress-induced persisters. In this study, we exposed S. mutans to the CSP pheromone to mimic DNA damage conditions and isolated the antibiotic persisters by treating the cultures with ofloxacin. Transcriptome analysis was then performed to evaluate the differential gene expression between the normal stationary phase cells and the persisters. RNA-sequencing revealed that triggered persistence was associated with the up-regulation of genes related to several stress-defense mechanisms, notably, multidrug efflux pumps, the arginine deaminase pathway, and the Opu/Opc system. In addition, we showed that inactivation of the VicK kinase of the YycFG essential two-component regulatory system abolished the formation of triggered persisters via the CSP pheromone. These data contribute to the understanding of the triggered persistence phenotype and may suggest new therapeutic strategies for treating persistent streptococcal infections
2023-09-29 | GSE244067 | GEO
Project description:The induction of genetic competence is a strategy used by bacteria to increase their genetic repertoire under stressful environmental conditions. Recently, Streptococcus pneumoniae has been shown to co-ordinate the uptake of transforming DNA with fratricide via increased expression of the peptide pheromone responsible for competence induction. Here, we document that environmental stress-induced expression of the peptide pheromone competence-stimulating peptide (CSP) in the oral pathogen Streptococcus mutans. We showed that CSP is involved in the stress response and determined the CSP-induced regulon in S. mutans by microarray analysis. Contrary to pneumococcus, S. mutans responds to increased concentrations of CSP by cell lysis in only a fraction of the population. We have focused on the mechanism of cell lysis and have identified a novel bacteriocin as the M-bM-^@M-^Xdeath effectorM-bM-^@M-^Y. Most importantly, we showed that this bacteriocin causes cell death via a novel mechanism of action: intracellular action against self. We have also identified the cognate bacteriocin immunity protein, which resides in a separate unlinked genetic locus to allow its differential regulation. The role of the lytic response in S. mutans competence is also discussed. Together, these findings reveal a novel autolytic pathway in S. mutans which may be involved in the dissemination of fitness-enhancing genes in the oral biofilm. Streptococcus mutans UA159 were grown with 2 uM CSP or without (uninduced control) to mid-log phase. Total RNA was extracted as described above. The cDNAs were prepared for hybridization using the PFGRC protocol. Microarray chips were scanned using a Gene Pix 4000B (Axon) and analyzed using the TM4 Microarray Software Suite (http://www.tm4.org/). Transcript levels were measured by cDNA hybridized to a fourfold redundant S. mutans microarray and averaged for three replicated hybridizations. Differential gene expression was based on a post-normalization cut-off of M-BM-1> twofold.
2010-05-29 | E-GEOD-22019 | biostudies-arrayexpress
Project description:In Streptococcus mutans, an oral colonizer associated with dental caries, competence for natural transformation can be triggered by both CSP and XIP pheromones. Competence induced by CSP is a late response that requires induction of the XIP encoding gene comS, but the mechanism(s) linking the two systems remains unknown. To learn how the CSP and XIP pheromone regulatory pathways are temporally linked, we mapped the global changes in gene expression at early and late phases of the CSP response and investigated the effect of deletion of comS on the S. mutans transcriptional profile. The early phase of the CSP response was characterized by an increase in gene expression at five loci associated with bacteriocin production and immunity. In the late phase, the up-regulated regions expanded to include a total of 27 loci, including comS and genes required for DNA uptake and recombination. In the absence of comS, no increase in expression of the genes up-regulated as a late response was observed in response to CSP, whereas expression of those regulated as an early response was maintained. These results indicate that the entire late response to CSP depends on the expression of comS and that the immediate transcriptional response to CSP, mediated by ComE, is restricted to just five bacteriocin-related loci To distinguish the immediate, and presumably direct, regulatory response to CSP from later, and presumably indirect, effects of exposure to this pheromone, we have assembled a comprehensive strand-specific microarray census of mRNA in strain UA159 at both early and late times in the CSP response as well as in a comS mutant.
2015-09-01 | E-GEOD-70067 | biostudies-arrayexpress
Project description:In Streptococcus mutans, an oral colonizer associated with dental caries, competence for natural transformation can be triggered by both CSP and XIP pheromones. Competence induced by CSP is a late response that requires induction of the XIP encoding gene comS, but the mechanism(s) linking the two systems remains unknown. To learn how the CSP and XIP pheromone regulatory pathways are temporally linked, we mapped the global changes in gene expression at early and late phases of the CSP response and investigated the effect of deletion of comS on the S. mutans transcriptional profile. The early phase of the CSP response was characterized by an increase in gene expression at five loci associated with bacteriocin production and immunity. In the late phase, the up-regulated regions expanded to include a total of 27 loci, including comS and genes required for DNA uptake and recombination. In the absence of comS, no increase in expression of the genes up-regulated as a late response was observed in response to CSP, whereas expression of those regulated as an early response was maintained. These results indicate that the entire late response to CSP depends on the expression of comS and that the immediate transcriptional response to CSP, mediated by ComE, is restricted to just five bacteriocin-related loci
2015-09-01 | GSE70067 | GEO
Project description:The induction of genetic competence is a strategy used by bacteria to increase their genetic repertoire under stressful environmental conditions. Recently, Streptococcus pneumoniae has been shown to co-ordinate the uptake of transforming DNA with fratricide via increased expression of the peptide pheromone responsible for competence induction. Here, we document that environmental stress-induced expression of the peptide pheromone competence-stimulating peptide (CSP) in the oral pathogen Streptococcus mutans. We showed that CSP is involved in the stress response and determined the CSP-induced regulon in S. mutans by microarray analysis. Contrary to pneumococcus, S. mutans responds to increased concentrations of CSP by cell lysis in only a fraction of the population. We have focused on the mechanism of cell lysis and have identified a novel bacteriocin as the ‘death effector’. Most importantly, we showed that this bacteriocin causes cell death via a novel mechanism of action: intracellular action against self. We have also identified the cognate bacteriocin immunity protein, which resides in a separate unlinked genetic locus to allow its differential regulation. The role of the lytic response in S. mutans competence is also discussed. Together, these findings reveal a novel autolytic pathway in S. mutans which may be involved in the dissemination of fitness-enhancing genes in the oral biofilm.
2010-05-29 | GSE22019 | GEO
Project description:Transcriptional profiling of the competence-stimulating peptide (CSP) response in Streptococcus mutans of FACS-separated subpopulations and mixed cells of a clonal culture. CSP-mediated competence development in S. mutans is a transient and biphasic process since only a subpopulation induces expression of ComX in the presence of CSP and activation of the DNA uptake machinery in this fraction shuts down ~3-4 hours post induction. Here we combine, for the first time in bacteria to our knowledge, flow cytometric sorting of cells and subpopulation-specific transcriptome analysis of both the competent and non-competent fractions of CSP-treated S. mutans cells. Sorting was guided by a ComX-GFP reporter and the transcriptome analysis demonstrated the successful combination of both methods because a strong enrichment of transcripts for comX and its downstream genes was achieved. Three two-component systems were expressed in the competent fraction, among them ComDE. Moreover, the recently identified regulator system ComR/S was expressed exclusively in the competent fraction. By contrast, expression of bacteriocin-related genes was at the same level in all cells. GFP reporter strains for ComE and mutacin V confirmed this expression pattern on the single cell level. Fluorescence microscopy revealed that some ComX-expressing cells committed autolysis in an early stage of competence initiation. In viable ComX-expressing cells, uptake of DNA could be shown on the single cell level. This study demonstrates that all cells in the population respond to CSP through activation of bacteriocin-related genes but that two subpopulations segregate, one becoming competent and another one that lyses, resulting in intrapopulation diversity of the clonal culture. Two conditions: induced and not induced with CSP. Three induced cell populations: competent subpopulation, incompetent subpopulation, all cells. Two biological replicates each population.
2011-02-01 | E-GEOD-25284 | biostudies-arrayexpress
Project description:Polymicrobial biofilms are of large medical importance, but little is known about their physiology and the underlying interspecies interactions. Here we studied two human pathogens, the opportunistic fungus Candida albicans and the caries promoting bacterium Streptococcus mutans. Both species formed biofilms in monoculture, with C. albicans growing mainly in the virulence-associated hyphae form, and S. mutans forming a thick layer of extracellular polymeric substances (EPS). Biofilm growth was enhanced in dual-species biofilms, which reached twice the biomass of monospecies biofilms and higher cell numbers of both S. mutans and C. albicans. EPS production by S. mutans was strongly suppressed in dual-species biofilms. Virulence traits of S. mutans, e.g. genetic competence, biofilm formation and bacteriocin synthesis are controlled by quorum sensing through activation of the alternative sigma factor SigX. SigX is induced by the pheromones CSP (competence stimulating factor) or XIP (sigX inducing peptide). Strong induction of sigX was observed in dual species biofilms indicated by fluorescence of a reporter strain for the sigX promoter, S. mutans PcomX-gfp, as well as by qRT-PCR of comX. The peak of sigX expression occurred after 10 h of biofilm growth. Conditioned media from mixed biofilms but not from C. albicans or S. mutans cultivated alone activated sigX in the reporter strain. Deletion mutants for the comC and comS genes encoding the precursors of CSP and XIP, respectively, were constructed. Conditioned media from mixed biofilms with S. mutans DcomS were unable to induce sigX in the reporter strain, while deletion of comC had no effect. These data show that synthesis of XIP was induced in S. mutans by coculture with C. albicans. Transcriptome analysis of S. mutans in single and mixed biofilms confirmed strong induction of comS, sigX, and the downstream late competence genes in dual-species biofilms. Among the late competence genes, fratricins were discovered for the first time. The comCDE operon and bacteriocin related genes were also induced, but much weaker. Genes related to oxidative stress, chaperones and glycosyltransferase genes required for EPS synthesis from sucrose were down-regulated, while glycogen synthesis genes were up-regulated, indicating that S. mutans was protected from oxidative stress and provided with excess sugar for storage polymer synthesis in mixed biofilms. The data show that in dual-species biofilms, C. albicans improves growth of S. mutans, suppresses its EPS formation and induces the complete quorum sensing signalling system, thus fundamentally changing the virulence properties of the caries pathogen, including its potential interactions with other members of the polymicrobial dental plaque community. Dual-species biofilms of S. mutans and C. albicans and single-species biofilms of S. mutans were cultivated in 24-well microtitre plates in YNBB medium. Transcriptional profiles of S. mutans in single- and dual-species biofilms were analysed at early (6 h) and late (10 h) logarithmic phase of the biofilm growth, as well as after 24 h when biofilms entered stationary phase. Transcriptional profiles of S. mutans grown in the dual-species biofilms were compared to profiles obtained for single-species biofilm from the same time point. Three biological and one to two technical replicas were used in the microarray study. RNA samples were labeled with Cy3 or Cy5 using the ULS fluorescent labeling kit (Kreatech, Germany). Seven hundred nanograms of Cy3 or Cy5 labeled RNA after fragmentation were hybridized to the microarray at 65M-BM-0C for 17 h using the Agilent hybridization chamber according to the manufacturer's instructions. The arrays were scanned using the Agilent DNA microarray scanner and the raw data were extracted using Agilent Feature Extraction software (v. 10.7).
2014-09-01 | E-GEOD-52543 | biostudies-arrayexpress
Project description:Transcriptional profiling of the competence-stimulating peptide (CSP) response in Streptococcus mutans of FACS-separated subpopulations and mixed cells of a clonal culture. CSP-mediated competence development in S. mutans is a transient and biphasic process since only a subpopulation induces expression of ComX in the presence of CSP and activation of the DNA uptake machinery in this fraction shuts down ~3-4 hours post induction. Here we combine, for the first time in bacteria to our knowledge, flow cytometric sorting of cells and subpopulation-specific transcriptome analysis of both the competent and non-competent fractions of CSP-treated S. mutans cells. Sorting was guided by a ComX-GFP reporter and the transcriptome analysis demonstrated the successful combination of both methods because a strong enrichment of transcripts for comX and its downstream genes was achieved. Three two-component systems were expressed in the competent fraction, among them ComDE. Moreover, the recently identified regulator system ComR/S was expressed exclusively in the competent fraction. By contrast, expression of bacteriocin-related genes was at the same level in all cells. GFP reporter strains for ComE and mutacin V confirmed this expression pattern on the single cell level. Fluorescence microscopy revealed that some ComX-expressing cells committed autolysis in an early stage of competence initiation. In viable ComX-expressing cells, uptake of DNA could be shown on the single cell level. This study demonstrates that all cells in the population respond to CSP through activation of bacteriocin-related genes but that two subpopulations segregate, one becoming competent and another one that lyses, resulting in intrapopulation diversity of the clonal culture.
2011-02-01 | GSE25284 | GEO
Project description:S. mutans UA159 was cultivated in chemically defined medium. Changes in the transcriptome in response to the autoinducing peptides CSP and XIP were monitored for 30 minutes.
2015-06-01 | GSE65982 | GEO
Project description:Polymicrobial biofilms are of large medical importance, but little is known about their physiology and the underlying interspecies interactions. Here we studied two human pathogens, the opportunistic fungus Candida albicans and the caries promoting bacterium Streptococcus mutans. Both species formed biofilms in monoculture, with C. albicans growing mainly in the virulence-associated hyphae form, and S. mutans forming a thick layer of extracellular polymeric substances (EPS). Biofilm growth was enhanced in dual-species biofilms, which reached twice the biomass of monospecies biofilms and higher cell numbers of both S. mutans and C. albicans. EPS production by S. mutans was strongly suppressed in dual-species biofilms. Virulence traits of S. mutans, e.g. genetic competence, biofilm formation and bacteriocin synthesis are controlled by quorum sensing through activation of the alternative sigma factor SigX. SigX is induced by the pheromones CSP (competence stimulating factor) or XIP (sigX inducing peptide). Strong induction of sigX was observed in dual species biofilms indicated by fluorescence of a reporter strain for the sigX promoter, S. mutans PcomX-gfp, as well as by qRT-PCR of comX. The peak of sigX expression occurred after 10 h of biofilm growth. Conditioned media from mixed biofilms but not from C. albicans or S. mutans cultivated alone activated sigX in the reporter strain. Deletion mutants for the comC and comS genes encoding the precursors of CSP and XIP, respectively, were constructed. Conditioned media from mixed biofilms with S. mutans DcomS were unable to induce sigX in the reporter strain, while deletion of comC had no effect. These data show that synthesis of XIP was induced in S. mutans by coculture with C. albicans. Transcriptome analysis of S. mutans in single and mixed biofilms confirmed strong induction of comS, sigX, and the downstream late competence genes in dual-species biofilms. Among the late competence genes, fratricins were discovered for the first time. The comCDE operon and bacteriocin related genes were also induced, but much weaker. Genes related to oxidative stress, chaperones and glycosyltransferase genes required for EPS synthesis from sucrose were down-regulated, while glycogen synthesis genes were up-regulated, indicating that S. mutans was protected from oxidative stress and provided with excess sugar for storage polymer synthesis in mixed biofilms. The data show that in dual-species biofilms, C. albicans improves growth of S. mutans, suppresses its EPS formation and induces the complete quorum sensing signalling system, thus fundamentally changing the virulence properties of the caries pathogen, including its potential interactions with other members of the polymicrobial dental plaque community.
2014-09-01 | GSE52543 | GEO