Project description:Streptococcus mutans, the primary etiological agent of human dental caries, has developed multiple mechanisms to colonize and form biofilms on the tooth surface. The brpA gene codes for a predicted surface-associated protein with apparent roles in biofilm formation, autolysis, and cell division. In this study, we used two models to further characterize the biofilm-forming characteristics of a BrpA-deficient mutant, strain TW14. Compared to those of the parent strain, UA159, TW14 formed long chains and sparse microcolonies on hydroxylapatite disks but failed to accumulate and form three-dimensional biofilms when grown on glucose as the carbohydrate source. The biofilm formation defect was also readily apparent by confocal laser scanning microscopy when flow cells were used to grow biofilms. When subjected to acid killing at pH 2.8 for 45 min, the survival rate of strain TW14 was more than 1 log lower than that of the wild-type strain. TW14 was at least 3 logs more susceptible to killing by 0.2% hydrogen peroxide than was UA159. The expression of more than 200 genes was found by microarray analysis to be altered in cells lacking BrpA (P < 0.01). These results suggest that the loss of BrpA can dramatically influence the transcriptome and significantly affects the regulation of acid and oxidative stress tolerance and biofilm formation in S. mutans, which are key virulence attributes of the organism. RNA extraction. S. mutans strains were grown in 50 ml of BHI broth and harvested at an OD600 of [congruent with]0.5 by centrifugation at 3,800 × g at 4°C for 5 min. The pellets were quickly resuspended and treated with RNAprotect (QIAGEN, Inc., CA) by using the procedures recommended by the supplier. The treated cells were collected by centrifugation at 3,800 × g at 4°C for 10 min and stored at −80°C. RNA extractions were performed by using hot phenol as previously described (33). To remove all DNA, the purified RNAs were treated with DNaseI (Ambion, Inc., Austin, TX) and retrieved with the RNeasy purification kit (QIAGEN, Inc., CA). To prepare a reference RNA, S. mutans UA159 was grown in 3 liters of BHI until mid-exponential phase (OD600 [congruent with] 0.5) and total RNA was extracted as described above. The purified RNA was then aliquoted and stored at −80°C until use. cDNA synthesis, Cy dye coupling and array hybridization. Array analysis was performed by using the whole-genome S. mutans microarrays that were obtained from PFGRC at TIGR. The S. mutans genome array consisted of 70-mer oligonucleotides representing 1,960 open reading frames from strain UA159. The full 70-mer complement was printed four times on the surface of the slides. Experiments, including cDNA synthesis, Cy dye coupling, hybridization, and washing, were performed by using protocols from the PFGRC with minor modifications. Briefly, cDNA synthesis was carried out in a total volume of 45 μl by mixing 10 μg total RNA with 3 μg random hexamers (Invitrogen Life Technologies, CA), 9 μl 5× first-strand buffer, 4.5 μl 0.1 M dithiothreitol, 2 μl 12.5 mM dNTP/aa-UTP with a 1.5- to-1 ratio of aa-dUTP to dTTP, and 3 μl Superscript III reverse transcriptase. This mixture was incubated at 42°C for 16 h. Following the completion of cDNA synthesis, RNA was hydrolyzed with NaOH and the aminoallyl-labeled cDNA samples were purified by using the QIAGEN QIAquick PCR purification kit, followed by drying in a speed vacuum. The samples were then resuspended in 5 μl 0.1 M NaCO3 and incubated with 5 μl of Cy dye (resuspended as recommended by the manufacturer) for 2 h in the dark. For all experiments, the reference RNA samples were coupled to Cy5 and both wild-type and the BrpA-deficient mutant RNA samples were coupled to Cy3. Uncoupled Cy dyes were removed by the QIAquick PCR purification kit, and the coupled samples were eluted in 100 μl elution buffer. Each Cy3-labeled experimental sample was mixed with a Cy5-labeled reference sample, and the mixtures were allowed to dry in a speed vacuum. To hybridize, the samples were resuspended in 60 μl hybridization buffer and heated at 90°C for 10 min. Following a quick centrifugation, the Cy dye-coupled probes were applied to microarray slides and incubated at 42°C in a water bath for 17 h. Slides were then washed and scanned by using an Axon GenePix 4000B scanner (Axon Instruments, Foster City, CA). Array data normalization and statistical analysis. Data were collected from at least four separate array slides, which contained four copies of the genome, with RNA isolated from four independent experiments. The raw data were loaded into the TIGR Spotfinder program and further normalized with the TIGR microarray data analysis system using LOWESS iterative log mean centering parameters with the default settings, followed by in-slide replicate analysis. Spots that were missing or labeled as “bad” during the upstream processes in 50% of the slides were cut off in the output files. The ratios of channel A over channel B were then converted to log2 values and analyzed with BRB array tools (version 3.01, developed by Richard Simon and Amy Peng Lam, National Cancer Institute, Bethesda, MD). A pairwise Student's t test was used to analyze the mean log ratios of the BrpA-deficient strain and the wild type, and genes that were differentially expressed with the significance level of a P value of <0.001 were selected.

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).

Project description:Investigation of whole genome gene expression levels of P. gingivalis W83, F. nucleatum DSMZ 25586, S. sanguinis SK36, A. actinomycetemcomitans HK1651, S. mutans UA159 in an 24 h old culture. Additionally, whole genome gene expression level changes of S. mutans UA159 biofilm cells after co-cultivation with S. mitis ATCC 11843 were compared to its single species biofilm growth after 24 h. Aim: Demonstration of the usefulness of a five-species gene expression array. Multiple probes per gene enabled identification of single inter-species cross-hybridizing probes. The deletion of such probes lead almost not to the deletion of the whole gene. This was investigated and confirmed by a two-species biofilm expression analysis: The here described array was used for the identification of genes of S. mutans influenced by the presence of S. mitis. Materials and Methods: P. gingivalis W83, F. nucleatum DSMZ 25586, S. sanguinis SK36, A. actinomycetemcomitans HK1651,and S. mutans UA159 were grown in CDM/succrose or artificial saliva/galactose in a single-species culture for 24 h anaerobically resulting in biofilm structures or monolayers. Total RNA was isolated and used for microarray analysis. Probes were analysed for the presence of biological false positive signals caused by cross-hybridizing probes of one of the other species presented on the chip. Further, a simple procedure was developed for automatical identification and deletion of false positive signals caused by washing artefacts, resulting in a more reliable outcome. In the case of the S. mutans/S. mitis mixed-species biofilm, both species were cultured together for 24 h like previously described. The found gene regulations were verified by RT-PCR. Results: Experiments with cDNA from 24 h old single-species cultures allowed the identification of cross-species hybridizing probes on the array, which can be eliminated in mixed-species experimental settings without the need to exclude the whole genes from the analysis. Between 69 % and almost 100 % represented genomes on this array were found actively transcribed under the mono-species monolayer and biofilm conditions used here. S. mutans / S. mitis co-culture: Physiological investigations revealed an increase in S. mutans biofilm mass with a decrease in pH-value under the influence of S. mitis, thereby confirming previously published data. A stringent fold change cut-off of 2 (p<0.05) identified 19 S. mutans transcripts with increased abundance, and 11 with decreased abundance compared to a S. mutans mono-species biofilm. Many of the genes have previously been found differentially regulated under general and acid stress, thereby confirming the value of this array. Conclusions: Taken together, this new array allows transcriptome studies on multi-species oral biofilm interactions and could become an important asset in future oral biofilm and inhibitor/therapy studies. The chip study used pooled total RNA recovered from three biologically independent mono-species biofilms or adherent cells/monolayers of P. gingivalis W83, F. nucleatum DSMZ 25586, S. sanguinis SK36, A. actinomycetemcomitans HK1651, and S. mutans UA159. In the case of gene expression analysis of S. mutans/S.mitis biofilm structures compared to the single species biofilm of S. mutans three separate single and three separate two-species biofilm cultures were analysed. Each chip measured the expression level of all together 10186 genes (1883 genes of P. gingivalis W83, 1964 genes of F. nucleatum DSMZ 25586, 2244 genes of S. sanguinis SK36, 2168 genes of A. actinomycetemcomitans HK1651, 1927 genes of S. mutans UA159) with up to thirteen 60-mer probes per gene and with a three-fold technical redundancy.

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.

Project description:Investigation of whole genome gene expression levels of P. gingivalis W83, F. nucleatum DSMZ 25586, S. sanguinis SK36, A. actinomycetemcomitans HK1651, S. mutans UA159 in an 24 h old culture. Additionally, whole genome gene expression level changes of S. mutans UA159 biofilm cells after co-cultivation with S. mitis ATCC 11843 were compared to its single species biofilm growth after 24 h. Aim: Demonstration of the usefulness of a five-species gene expression array. Multiple probes per gene enabled identification of single inter-species cross-hybridizing probes. The deletion of such probes lead almost not to the deletion of the whole gene. This was investigated and confirmed by a two-species biofilm expression analysis: The here described array was used for the identification of genes of S. mutans influenced by the presence of S. mitis. Materials and Methods: P. gingivalis W83, F. nucleatum DSMZ 25586, S. sanguinis SK36, A. actinomycetemcomitans HK1651,and S. mutans UA159 were grown in CDM/succrose or artificial saliva/galactose in a single-species culture for 24 h anaerobically resulting in biofilm structures or monolayers. Total RNA was isolated and used for microarray analysis. Probes were analysed for the presence of biological false positive signals caused by cross-hybridizing probes of one of the other species presented on the chip. Further, a simple procedure was developed for automatical identification and deletion of false positive signals caused by washing artefacts, resulting in a more reliable outcome. In the case of the S. mutans/S. mitis mixed-species biofilm, both species were cultured together for 24 h like previously described. The found gene regulations were verified by RT-PCR. Results: Experiments with cDNA from 24 h old single-species cultures allowed the identification of cross-species hybridizing probes on the array, which can be eliminated in mixed-species experimental settings without the need to exclude the whole genes from the analysis. Between 69 % and almost 100 % represented genomes on this array were found actively transcribed under the mono-species monolayer and biofilm conditions used here. S. mutans / S. mitis co-culture: Physiological investigations revealed an increase in S. mutans biofilm mass with a decrease in pH-value under the influence of S. mitis, thereby confirming previously published data. A stringent fold change cut-off of 2 (p<0.05) identified 19 S. mutans transcripts with increased abundance, and 11 with decreased abundance compared to a S. mutans mono-species biofilm. Many of the genes have previously been found differentially regulated under general and acid stress, thereby confirming the value of this array. Conclusions: Taken together, this new array allows transcriptome studies on multi-species oral biofilm interactions and could become an important asset in future oral biofilm and inhibitor/therapy studies.

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Influence of BrpA on critical virulence attributes of Streptococcus mutans

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