RESUMO
Cell-cell adhesion between oral bacteria plays a key role in the development of polymicrobial communities such as dental plaque. Oral streptococci such as Streptococcus gordonii and Streptococcus oralis are important early colonizers of dental plaque and bind to a wide range of different oral microorganisms, forming multispecies clumps or "coaggregates." S. gordonii actively responds to coaggregation by regulating gene expression. To further understand these responses, we assessed gene regulation in S. gordonii and S. oralis following coaggregation in 25% human saliva. Coaggregates were formed by mixing, and after 30 min, RNA was extracted for dual transcriptome sequencing (RNA-Seq) analysis. In S. oralis, 18 genes (6 upregulated and 12 downregulated) were regulated by coaggregation. Significantly downregulated genes encoded functions such as amino acid and antibiotic biosynthesis, ribosome, and central carbon metabolism. In total, 28 genes were differentially regulated in Streptococcus gordonii (25 upregulated and 3 downregulated). Many genes associated with transporters and a two-component (NisK/SpaK) regulatory system were upregulated following coaggregation. Our comparative analyses of S. gordonii-S. oralis with different previously published S. gordonii pairings (S. gordonii-Fusobacterium nucleatum and S. gordonii-Veillonella parvula) suggest that the gene regulation is specific to each pairing, and responses do not appear to be conserved. This ability to distinguish between neighboring bacteria may be important for S. gordonii to adapt appropriately during the development of complex biofilms such as dental plaque. IMPORTANCE Dental plaque is responsible for two of the most prevalent diseases in humans, dental caries and periodontitis. Controlling the formation of dental plaque and preventing the transition from oral health to disease requires a detailed understanding of microbial colonization and biofilm development. Streptococci are among the most common colonizers of dental plaque. This study identifies key genes that are regulated when oral streptococci bind to one another, as they do in the early stages of dental plaque formation. We show that specific genes are regulated in two different oral streptococci following the formation of mixed-species aggregates. The specific responses of S. gordonii to coaggregation with S. oralis are different from those to coaggregation with other oral bacteria. Targeting the key genes that are upregulated during interspecies interactions may be a powerful approach to control the development of biofilm and maintain oral health.
Assuntos
Placa Dentária , Streptococcus gordonii , Streptococcus oralis , Transcriptoma , Placa Dentária/microbiologia , Humanos , RNA-Seq , Streptococcus gordonii/genética , Streptococcus oralis/genéticaRESUMO
Many oral bacteria form macroscopic clumps known as coaggregates when mixed with a different species. It is thought that these cell-cell interactions are critical for the formation of mixed-species biofilms such as dental plaque. Here, we assessed the impact of coaggregation between two key initial colonizers of dental plaque, Streptococcus gordonii and Veillonella parvula, on gene expression in each partner. These species were shown to coaggregate in buffer or human saliva. To monitor gene regulation, coaggregates were formed in human saliva and, after 30 minutes, whole-transcriptomes were extracted for sequencing and Dual RNA-Seq analysis. In total, 272 genes were regulated in V. parvula, including 39 genes in oxidoreductase processes. In S. gordonii, there was a high degree of inter-sample variation. Nevertheless, 69 genes were identified as potentially regulated by coaggregation, including two phosphotransferase system transporters and several other genes involved in carbohydrate metabolism. Overall, these data indicate that responses of V. parvula to coaggregation with S. gordonii are dominated by oxidative stress-related processes, whereas S. gordonii responses are more focussed on carbohydrate metabolism. We hypothesize that these responses may reflect changes in the local microenvironment in biofilms when S. gordonii or V. parvula immigrate into the system.
Assuntos
Interações Microbianas , Streptococcus gordonii/genética , Transcriptoma , Veillonella/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Metabolismo dos Carboidratos , Humanos , Saliva/microbiologia , Streptococcus gordonii/metabolismo , Streptococcus gordonii/fisiologia , Veillonella/metabolismo , Veillonella/fisiologiaRESUMO
The ability of microorganisms to regulate gene expression is thought to be critical for survival and growth during the development of polymicrobial biofilms such as dental plaque. The commensal dental plaque colonizer, Streptococcus gordonii, responds to cell--cell contact (coaggregation) with Actinomyces oris by regulating >20 genes, including those involved in arginine biosynthesis. We hypothesized that an S. gordonii extracellular protease is critical for sensing by providing amino acids that modulate gene expression. S. gordonii coaggregated strongly with A. oris in buffer, saliva or chemically defined medium (CDM). In wild-type S. gordonii, expression of arginine biosynthesis genes argC and argG increased within two hours' growth in CDM in monocultures, but not following coaggregation with A. oris. By contrast, coaggregation of A. oris with an S. gordonii mutant lacking sgc, encoding the extracellular protease Challisin, resulted in increases in argC and argG gene expression that were similar to monocultures. Genetic complementation of sgc restored the ability of S. gordonii to sense coaggregation with A. oris. Coaggregation enabled growth of S. gordonii in low/no arginine and disruption of sgc did not affect this ability. We propose that extracellular bacterial proteases may be key mediators of cell--cell contact sensing by diverse microbial species.
Assuntos
Actinomyces/fisiologia , Proteínas de Bactérias/metabolismo , Peptídeo Hidrolases/metabolismo , Streptococcus gordonii/enzimologia , Arginina/metabolismo , Proteínas de Bactérias/genética , Placa Dentária/microbiologia , Humanos , Peptídeo Hidrolases/genética , Saliva/microbiologia , Streptococcus gordonii/genética , Streptococcus gordonii/metabolismoRESUMO
Cell-cell interactions between genetically distinct bacteria, known as coaggregation, are important for the formation of mixed-species biofilms such as dental plaque. Interactions lead to gene regulation in the partner organisms that may be critical for adaptation and survival in mixed-species biofilms. Here, gene regulation responses to coaggregation between Streptococcus gordonii and Fusobacterium nucleatum were studied using dual RNA-Seq. Initially, S. gordonii was shown to coaggregate strongly with F. nucleatum in buffer or human saliva. Using confocal laser scanning microscopy and transmission electron microscopy, cells of different species were shown to be evenly distributed throughout the coaggregate and were closely associated with one another. This distribution was confirmed by serial block face sectioning scanning electron microscopy, which provided high resolution three-dimensional images of coaggregates. Cell-cell sensing responses were analysed 30 minutes after inducing coaggregation in human saliva. By comparison with monocultures, 16 genes were regulated following coaggregation in F. nucleatum whereas 119 genes were regulated in S. gordonii. In both species, genes involved in amino acid and carbohydrate metabolism were strongly affected by coaggregation. In particular, one 8-gene operon in F. nucleatum encoding sialic acid uptake and catabolism was up-regulated 2- to 5-fold following coaggregation. In S. gordonii, a gene cluster encoding functions for phosphotransferase system-mediated uptake of lactose and galactose was down-regulated up to 3-fold in response to coaggregation. The genes identified in this study may play key roles in the development of mixed-species communities and represent potential targets for approaches to control dental plaque accumulation.
Assuntos
Adesinas Bacterianas/metabolismo , Aderência Bacteriana , Biofilmes/crescimento & desenvolvimento , Fusobacterium nucleatum/fisiologia , Streptococcus gordonii/fisiologia , Adesinas Bacterianas/genética , Placa Dentária/microbiologia , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Humanos , Microscopia Confocal , Microscopia Eletrônica , Óperon , Saliva/microbiologiaRESUMO
Simbiotics is a spatially explicit multiscale modeling platform for the design, simulation and analysis of bacterial populations. Systems ranging from planktonic cells and colonies, to biofilm formation and development may be modeled. Representation of biological systems in Simbiotics is flexible, and user-defined processes may be in a variety of forms depending on desired model abstraction. Simbiotics provides a library of modules such as cell geometries, physical force dynamics, genetic circuits, metabolic pathways, chemical diffusion and cell interactions. Model defined processes are integrated and scheduled for parallel multithread and multi-CPU execution. A virtual lab provides the modeler with analysis modules and some simulated lab equipment, enabling automation of sample interaction and data collection. An extendable and modular framework allows for the platform to be updated as novel models of bacteria are developed, coupled with an intuitive user interface to allow for model definitions with minimal programming experience. Simbiotics can integrate existing standards such as SBML, and process microscopy images to initialize the 3D spatial configuration of bacteria consortia. Two case studies, used to illustrate the platform flexibility, focus on the physical properties of the biosystems modeled. These pilot case studies demonstrate Simbiotics versatility in modeling and analysis of natural systems and as a CAD tool for synthetic biology.