Project description:Multispecies biofilms are the predominant form of bacterial growth in natural and human-associated environments. Although the pathways involved in monospecies biofilm have been well characterized, less is known about the metabolic pathways and emergent traits of a multispecies biofilm community. Here, we performed a transcriptome survey of the developmental stages of a 3-species biofilm community and combined it with quantitative imaging and growth experiments. We report the remodelling of central metabolism of two of the three species in this community. Specifically, we observed an increase in the expression of genes associated with glycolysis and pentose phosphate pathways in K. pneumoniae. Similarly, a decrease in the expression of the same pathways in P. protegens was observed along with an increase in expression of glyoxalate cycle genes when grown as a mixed species biofilm, suggesting reorganisation of metabolic pathways and metabolite sharing for the community biofilms. To test the possibility of cross-feeding for the community, planktonic growth experiments revealed that both the Pseudomonads grew well in TCA cycle intermediates, while K. pneumoniae grew poorly when given those carbon sources. Despite this poor growth in mono-culture, K. pneumoniae was still the dominant species in mixed species biofilms cultivated in TCA intermediates as the sole source of carbon. The biofilm growth data, combined with the transcriptomics data, suggests there is reorganisation of metabolism for the community members and may allow for cross-feeding that allows K. pneumoniae to dominate the community. We also demonstrated that sdsA1 of P. aeruginosa was induced upon exposure to the surfactant SDS and that this gene was essential in protecting mono and mixed species biofilms from surfactant stress. This also suggests that the community members can share defence mechanisms. Overall, this study describes a comprehensive transcriptomics level investigation of shared resources, metabolites and stress defence that may underpin the emergent properties of mixed species biofilm communities.

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.

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:Candida albicans, a major opportunistic fungal pathogen is frequently found together with Streptococcus mutans in dental biofilms associated with severe childhood tooth-decay, a prevalent pediatric oral disease. Previous studies have demonstrated that S. mutans and C. albicans synergizes virulence of plaque-biofilms in vivo. However, the nature of this bacterial-fungal relationship in this cross-kingdom biofilm remains largely uncharacterized. Using iTRAQ based quantitative proteomics, we found that proteins associated with carbohydrate metabolism such as alpha-1,4 glucan phosphorylase, Hexokinase-2, Isocitrate lyase and malate synthase were significantly upregulated in C. albicans in the mixed-species biofilms (P<0.05). C. albicans proteins associated with growth/morphogenesis such as pH-responsive protein-2, Fma1p and Hsp21 were also induced. Conversely, S. mutans proteins in the tricarboxylic acid cycle such as citrate synthase and in the pentose phosphate pathway such as Ribose-5-phosphate isomerase A as well as proteins associated with sugar transport systems were upregulated indicating enhanced carbohydrate metabolism. Interestingly mixed-species biofilm microenvironment had a lower pH than S. mutans single-species biofilms. This observation was supported by proteomics, wherein proteins associated with lactate and formate assimilation such as Glyoxalase and putative NADPH-dependent methylglyoxal reductase proteins were significantly upregulated in the mixed-species biofilms (P<0.05). Furthermore, we unexpectedly found that S. mutans derived glucosyltransferase B (GtfB), responsible for co-adhesion via glucans, can also contribute to C. albicans growth and carbohydrate metabolism by providing glucose and fructose from sucrose breakdown. These findings demonstrate synergistic bacterial-fungal interactions within mixed-species biofilms and a novel GtfB cross-feeding role. Taken together, quantitative proteomics provides new insights into this virulent cross-kingdom oral biofilm.

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Biofilm metagenome

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