Helena Sztajer

Autoinducer-2-regulated genes in Streptococcus mutans UA159 and global metabolic effect of the luxS mutation.

Autoinducer 2 (AI-2) is the only species-nonspecific autoinducer known in bacteria and is produced by both gram-negative and gram-positive organisms. Consequently, it is proposed to function as a universal quorum-sensing signal for interaction between bacterial species. AI-2 is produced as the by-product of a metabolic transformation carried out by the LuxS enzyme. To separate the metabolic function of the LuxS enzyme from the signaling role of AI-2, we carried out a global transcriptome analysis of a luxS null mutant culture of Streptococcus mutans UA159, an important cariogenic bacterium and a crucial component of the dental plaque biofilm community, in comparison to a luxS null mutant culture supplemented with chemically pure 4,5-dihydroxy-2,3-pentanedione, the precursor of AI-2. The data revealed fundamental changes in gene expression affecting 585 genes (30% of the genome) which could not be restored by the signal molecule AI-2 and are therefore not caused by quorum sensing but by lack of the transformation carried out by the LuxS enzyme in the activated methyl cycle. All functional classes of enzymes were affected, including genes known to be important for biofilm formation, bacteriocin synthesis, competence, and acid tolerance. At the same time, 59 genes were identified whose transcription clearly responded to the addition of AI-2. Some of them were related to protein synthesis, stress, and cell division. Three membrane transport proteins were upregulated which are not related to any of the known AI-2 transporters. Three transcription factors were identified whose transcription was stimulated repeatedly by AI-2 addition during growth. Finally, a global regulatory protein, the delta subunit of the RNA polymerase (rpoE), was induced 147-fold by AI-2, representing the largest differential gene expression observed. The data show that many phenotypes related to the luxS mutation cannot be ascribed to quorum sensing and have identified for the first time regulatory proteins potentially mediating AI-2-based signaling in gram-positive bacteria.

Streptococcus mutans inhibits Candida albicans hyphal formation by the fatty acid signaling molecule trans-2-decenoic acid (SDSF).

In the human mouth, fungi and several hundred species of bacteria coexist. Here we report a case of interkingdom signaling in the oral cavity: A compound excreted by the caries bacterium Streptococcus mutans inhibits the morphological transition from yeast to hyphae, an important virulence trait, in the opportunistic fungus Candida albicans. The compound excreted by S. mutans was originally studied because it inhibited signaling by the universal bacterial signal autoinducer‐2 (AI‐2), determined by the luminescence of a Vibrio harveyi sensor strain. The inhibitor was purified from cell‐free culture supernatants of S. mutans guided by its activity. Its chemical structure was elucidated by using NMR spectroscopy and GC‐MS and proved to be trans‐2‐decenoic acid. We show that trans‐2‐decenoic acid does not inhibit AI‐2‐specific signaling, but rather the luciferase reaction used for its detection. A potential biological role of trans‐2‐decenoic acid was then discovered. It is able to suppress the transition from yeast to hyphal morphology in the opportunistic human pathogen Candida albicans at concentrations that do not affect growth. The expression of HWP1, a hyphal‐specific signature gene of C. albicans, is abolished by trans‐2‐decenoic acid. trans‐2‐Decenoic acid is structurally similar to the diffusible signal factor (DSF) family of interkingdom‐signaling molecules and is the first member of this family from a Gram‐positive organism (Streptococcus DSF, SDSF). SDSF activity was also found in S. mitis, S. oralis, and S. sanguinis, but not in other oral bacteria. SDSF could be relevant in shaping multispecies Candida bacteria biofilms in the human body.

Cross-feeding and interkingdom communication in dual-species biofilms of Streptococcus mutans and Candida albicans.

Polymicrobial biofilms are of large medical importance, but relatively little is known about the role of interspecies interactions for their physiology and virulence. Here, we studied two human pathogens co-occuring in the oral cavity, the opportunistic fungus Candida albicans and the caries-promoting bacterium Streptococcus mutans. Dual-species biofilms reached higher biomass and cell numbers than mono-species biofilms, and the production of extracellular polymeric substances (EPSs) by S. mutans was strongly suppressed, which was confirmed by scanning electron microscopy, gas chromatography–mass spectrometry and transcriptome analysis. To detect interkingdom communication, C. albicans was co-cultivated with a strain of S. mutans carrying a transcriptional fusion between a green fluorescent protein-encoding gene and the promoter for sigX, the alternative sigma factor of S. mutans, which is induced by quorum sensing signals. Strong induction of sigX was observed in dual-species biofilms, but not in single-species biofilms. Conditioned media from mixed biofilms but not from C. albicans or S. mutans cultivated alone activated sigX in the reporter strain. Deletion of comS encoding the synthesis of the sigX-inducing peptide precursor abolished this activity, whereas deletion of comC encoding the competence-stimulating peptide precursor had no effect. Transcriptome analysis of S. mutans confirmed induction of comS, sigX, bacteriocins and the downstream late competence genes, including fratricins, in dual-species biofilms. We show here for the first time the stimulation of the complete quorum sensing system of S. mutans by a species from another kingdom, namely the fungus C. albicans, resulting in fundamentally changed virulence properties of the caries pathogen.

Identification, Quantification, and Determination of the Absolute Configuration of the Bacterial Quorum-Sensing Signal Autoinducer-2 by Gas Chromatography–Mass Spectrometry

Sensing the signal: A gas chromatography–mass spectrometry (GC–MS) method for the analysis of the quorum‐sensing autoinducer‐2 is described. It allows, for the first time, the direct analysis and accurate determination of this highly water soluble signaling compound, which exists in complex equilibria. The application on the caries‐causing bacterium Streptococcus mutans is described.

The Delta Subunit of RNA Polymerase, RpoE, Is a Global Modulator of Streptococcus mutans Environmental Adaptation

The delta subunit of RNA polymerase, RpoE, is widespread in low-G+C Gram-positive bacteria and is thought to play a role in enhancing transcriptional specificity by blocking RNA polymerase binding at weak promoter sites and stimulating RNA synthesis by accelerating core enzyme recycling. Despite the well-studied biochemical properties of RpoE, a role for this protein in vivo has not been defined in depth. In this study, we show that inactivation of rpoE in the human dental caries pathogen Streptococcus mutans causes impaired growth and loss of important virulence traits, including biofilm formation, resistance to antibiotics, and tolerance to environmental stresses. Complementation of the mutant with rpoE expressed in trans restored its phenotype to wild type. The luciferase fusion reporter showed that rpoE was highly transcribed throughout growth and that acid and hydrogen peroxide stresses repressed rpoE expression. Transcriptome profiling of wild-type and ΔrpoE cells in the exponential and early stationary phase of growth, under acid and hydrogen peroxide stress and under both stresses combined, revealed that genes involved in histidine synthesis, malolactic fermentation, biofilm formation, and antibiotic resistance were downregulated in the ΔrpoE mutant under all conditions. Moreover, the loss of RpoE resulted in dramatic changes in transport and metabolism of carbohydrates and amino acids. Interestingly, differential expression, mostly upregulation, of 330 noncoding regions was found. In conclusion, this study demonstrates that RpoE is an important global modulator of gene expression in S. mutans which is required for optimal growth and environmental adaptation.

Characterization of mleR, a positive regulator of malolactic fermentation and part of the acid tolerance response in Streptococcus mutans

BackgroundOne of the key virulence determinants of Streptococcus mutans, the primary etiological agent of human dental caries, is its strong acid tolerance. The acid tolerance response (ATR) of S. mutans comprises several mechanisms that are induced at low pH and allow the cells to quickly adapt to a lethal pH environment. Malolactic fermentation (MLF) converts L-malate to L-lactate and carbon dioxide and furthermore regenerates ATP, which is used to translocate protons across the membrane. Thus, MLF may contribute to the aciduricity of S. mutans but has not been associated with the ATR so far.ResultsHere we show that the malolactic fermentation (mle) genes are under the control of acid inducible promoters which are induced within the first 30 minutes upon acid shock in the absence of malate. Thus, MLF is part of the early acid tolerance response of S. mutans. However, acidic conditions, the presence of the regulator MleR and L-malate were required to achieve maximal expression of all genes, including mleR itself. Deletion of mleR resulted in a decreased capacity to carry out MLF and impaired survival at lethal pH in the presence of L-malate. Gel retardation assays indicated the presence of multiple binding sites for MleR. Differences in the retardation patterns occurred in the presence of L-malate, thus demonstrating its role as co-inducer for transcriptional regulation.ConclusionThis study shows that the MLF gene cluster is part of the early acid tolerance response in S. mutans and is induced by both low pH and L-malate.

High-Resolution Taxonomic Profiling of the Subgingival Microbiome for Biomarker Discovery and Periodontitis Diagnosis

ABSTRACT The oral microbiome plays a key role for caries, periodontitis, and systemic diseases. A method for rapid, high-resolution, robust taxonomic profiling of subgingival bacterial communities for early detection of periodontitis biomarkers would therefore be a useful tool for individualized medicine. Here, we used Illumina sequencing of the V1-V2 and V5-V6 hypervariable regions of the 16S rRNA gene. A sample stratification pipeline was developed in a pilot study of 19 individuals, 9 of whom had been diagnosed with chronic periodontitis. Five hundred twenty-three operational taxonomic units (OTUs) were obtained from the V1-V2 region and 432 from the V5-V6 region. Key periodontal pathogens like Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia could be identified at the species level with both primer sets. Principal coordinate analysis identified two outliers that were consistently independent of the hypervariable region and method of DNA extraction used. The linear discriminant analysis (LDA) effect size algorithm (LEfSe) identified 80 OTU-level biomarkers of periodontitis and 17 of health. Health- and periodontitis-related clusters of OTUs were identified using a connectivity analysis, and the results confirmed previous studies with several thousands of samples. A machine learning algorithm was developed which was trained on all but one sample and then predicted the diagnosis of the left-out sample (jackknife method). Using a combination of the 10 best biomarkers, 15 of 17 samples were correctly diagnosed. Training the algorithm on time-resolved community profiles might provide a highly sensitive tool to detect the onset of periodontitis.

Comparing the cariogenic species Streptococcus sobrinus and S. mutans on whole genome level

Background Two closely related species of mutans streptococci, namely Streptococcus mutans and Streptococcus sobrinus, are associated with dental caries in humans. Their acidogenic and aciduric capacity is directly associated with the cariogenic potential of these bacteria. To survive acidic and temporarily harsh conditions in the human oral cavity with hundreds of other microbial co-colonizers as competitors, both species have developed numerous mechanisms for adaptation. Objectives The recently published novel genome information for both species is used to elucidate genetic similarities but especially differences and to discuss the impact on cariogenicity of the corresponding phenotypic properties including adhesion, carbohydrate uptake and fermentation, acid tolerance, signaling by two component systems, competence, and oxidative stress resistance. Conclusions S. sobrinus can down-regulate the SpaA-mediated adherence to the pellicle. It has a smaller number of two-component signaling systems and bacteriocin-related genes than S. mutans, but all or even more immunity proteins. It lacks the central competence genes comC, comS, and comR. There are more genes coding for glucosyltransferases and a novel energy production pathway formed by lactate oxidase, which is not found in S. mutans. Both species show considerable differences in the regulation of fructan catabolism. However, both S. mutans and S. sobrinus share most of these traits and should therefore be considered as equally virulent with regard to dental caries.

Genetic variability of mutans streptococci revealed by wide whole-genome sequencing

BackgroundMutans streptococci are a group of bacteria significantly contributing to tooth decay. Their genetic variability is however still not well understood.ResultsGenomes of 6 clinical S. mutans isolates of different origins, one isolate of S. sobrinus (DSM 20742) and one isolate of S. ratti (DSM 20564) were sequenced and comparatively analyzed. Genome alignment revealed a mosaic-like structure of genome arrangement. Genes related to pathogenicity are found to have high variations among the strains, whereas genes for oxidative stress resistance are well conserved, indicating the importance of this trait in the dental biofilm community. Analysis of genome-scale metabolic networks revealed significant differences in 42 pathways. A striking dissimilarity is the unique presence of two lactate oxidases in S. sobrinus DSM 20742, probably indicating an unusual capability of this strain in producing H2O2 and expanding its ecological niche. In addition, lactate oxidases may form with other enzymes a novel energetic pathway in S. sobrinus DSM 20742 that can remedy its deficiency in citrate utilization pathway.Using 67 S. mutans genomes currently available including the strains sequenced in this study, we estimates the theoretical core genome size of S. mutans, and performed modeling of S. mutans pan-genome by applying different fitting models. An “open” pan-genome was inferred.ConclusionsThe comparative genome analyses revealed diversities in the mutans streptococci group, especially with respect to the virulence related genes and metabolic pathways. The results are helpful for better understanding the evolution and adaptive mechanisms of these oral pathogen microorganisms and for combating them.

Functional biomarkers for chronic periodontitis and insights into the roles of Prevotella nigrescens and Fusobacterium nucleatum ; a metatranscriptome analysis

Background/Objectives:Periodontitis is the most prevalent inflammatory disease worldwide and is caused by a dysbiotic subgingival biofilm. Here we used metatranscriptomics to determine the functional shift from health to periodontitis, the response of individual species to dysbiosis and to discover biomarkers.Methods:Sixteen individuals were studied, from which six were diagnosed with chronic periodontitis. Illumina sequencing of the total messenger RNA (mRNA) yielded ~42 million reads per sample. A total of 324 human oral taxon phylotypes and 366,055 open reading frames from the HOMD database reference genomes were detected.Results:The transcriptionally active community shifted from Bacilli and Actinobacteria in health to Bacteroidia, Deltaproteobacteria, Spirochaetes and Synergistetes in periodontitis. Clusters of orthologous groups (COGs) related to carbohydrate transport and catabolism dominated in health, whereas protein degradation and amino acid catabolism dominated in disease. The LEfSe, random forest and support vector machine methods were applied to the 2,000 most highly expressed genes and discovered the three best functional biomarkers, namely haem binding protein HmuY from Porphyromonas gingivalis, flagellar filament core protein FlaB3 from Treponema denticola, and repeat protein of unknown function from Filifactor alocis. They predicted the diagnosis correctly for 14 from 16 individuals, and when applied to an independent study misclassified one out of six subjects only. Prevotella nigrescens shifted from commensalism to virulence by upregulating the expression of metalloproteases and the haem transporter. Expression of genes for the synthesis of the cytotoxic short-chain fatty acid butyrate was observed by Fusobacterium nucleatum under all conditions. Four additional species contributed to butyrate synthesis in periodontitis and they used an additional pathway.Conclusion:Gene biomarkers of periodontitis are highly predictive. The pro-inflammatory role of F. nucelatum is not related to butyrate synthesis.