Characterization of a Signaling System in Streptococcus mitis That Mediates Interspecies Communication with Streptococcus pneumoniae.

Streptococcus mitis is found in the oral cavity and nasopharynx and forms a significant portion of the human microbiome. In this study, in silico analyses indicated the presence of an Rgg regulator and short hydrophobic peptide (Rgg/SHP) cell-to-cell communication system in S. mitis Although Rgg presented greater similarity to a repressor in Streptococcus pyogenes, autoinducing assays and genetic mutation analysis revealed that in S. mitis Rgg acts as an activator. Transcriptome analysis showed that in addition to shp, the system regulates two other downstream genes, comprising a segment of a putative lantibiotic gene cluster that is in a conjugative element locus in different members of the mitis group. Close comparison to a similar lantibiotic gene cluster in Streptococcus pneumoniae indicated that S. mitis lacked the full set of genes. Despite the potential of SHP to trigger a futile cycle of autoinduction, growth was not significantly affected for the rgg mutant under normal or antibiotic stress conditions. The S. mitis SHP was, however, fully functional in promoting cross-species communication and increasing S. pneumoniae surface polysaccharide production, which in this species is regulated by Rgg/SHP. The activity of SHPs produced by both species was detected in cocultures using a S. mitis reporter strain. In competitive assays, a slight advantage was observed for the rgg mutants. We conclude that the Rgg/SHP system in S. mitis regulates the expression of its own shp and activates an Rgg/SHP system in S. pneumoniae that regulates surface polysaccharide synthesis. Fundamentally, cross-communication of such systems may have a role during multispecies interactions.IMPORTANCE Bacteria secrete signal molecules into the environment which are sensed by other cells when the density reaches a certain threshold. In this study, we describe a communication system in Streptococcus mitis, a commensal species from the oral cavity, which we also found in several species and strains of streptococci from the mitis group. Further, we show that this system can promote cross-communication with S. pneumoniae, a closely related major human pathogen. Importantly, we show that this cross-communication can take place during coculture. While the genes regulated in S. mitis are likely part of a futile cycle of activation, the target genes in S. pneumoniae are potentially involved in virulence. The understanding of such complex communication networks can provide important insights into the dynamics of bacterial communities.

High-resolution profiles of the Streptococcus mitis CSP signaling pathway reveal core and strain-specific regulated genes.

BACKGROUND: In streptococci of the mitis group, competence for natural transformation is a transient physiological state triggered by competence stimulating peptides (CSPs). Although low transformation yields and the absence of a widespread functional competence system have been reported for Streptococcus mitis, recent studies revealed that, at least for some strains, high efficiencies can be achieved following optimization protocols. To gain a deeper insight into competence in this species, we used RNA-seq, to map the global CSP response of two transformable strains: the type strain NCTC12261T and SK321. RESULTS: All known genes induced by ComE in Streptococcus pneumoniae, including sigX, were upregulated in the two strains. Likewise, all sets of streptococcal SigX core genes involved in extracellular DNA uptake, recombination, and fratricide were upregulated. No significant differences in the set of induced genes were observed when the type strain was grown in rich or semi-defined media. Five upregulated operons unique to S. mitis with a SigX-box in the promoter region were identified, including two specific to SK321, and one specific to NCTC12261T. Two of the strain-specific operons coded for different bacteriocins. Deletion of the unique S. mitis sigX regulated genes had no effect on transformation. CONCLUSIONS: Overall, comparison of the global transcriptome in response to CSP shows the conservation of the ComE and SigX-core regulons in competent S. mitis isolates, as well as species and strain-specific genes. Although some S. mitis exhibit truncations in key competence genes, this study shows that in transformable strains, competence seems to depend on the same core genes previously identified in S. pneumoniae.

Exploring ex vivo biofilm dynamics: consequences of low ampicillin concentrations on the human oral microbiome.

Prolonged exposure to antibiotics at low concentration can promote processes associated with bacterial biofilm formation, virulence and antibiotic resistance. This can be of high relevance in microbial communities like the oral microbiome, where commensals and pathogens share a common habitat and where the total abundance of antibiotic resistance genes surpasses the abundance in the gut. Here, we used an ex vivo model of human oral biofilms to investigate the impact of ampicillin on biofilm viability. The ecological impact on the microbiome and resistome was investigated using shotgun metagenomics. The results showed that low concentrations promoted significant shifts in microbial taxonomic profile and could enhance biofilm viability by up to 1 to 2-log. For the resistome, low concentrations had no significant impact on antibiotic resistance gene (ARG) diversity, while ARG abundance decreased by up to 84%. A positive correlation was observed between reduced microbial diversity and reduced ARG abundance. The WHO priority pathogens Streptococcus pneumoniae and Staphylococcus aureus were identified in some of the samples, but their abundance was not significantly altered by ampicillin. Most of the antibiotic resistance genes that increased in abundance in the ampicillin group were associated with streptococci, including Streptococcus mitis, a well-known potential donor of ARGs to S. pneumoniae. Overall, the results highlight the potential of using the model to further our understanding of ecological and evolutionary forces driving antimicrobial resistance in oral microbiomes.

Complete Genome Sequence of Streptococcus pneumoniae Serotype 19F Strain EF3030.

We report the complete genome sequence of Streptococcus pneumoniae EF3030, a serotype 19F isolate that colonizes the nasopharynx of mice while being mostly noninvasive. Such attributes make this strain highly attractive in pneumococcal carriage studies. The availability of its complete genomic sequence is likely to advance studies in the field.

Biofilm formation and autoinducer-2 signaling in Streptococcus intermedius: role of thermal and pH factors.

BACKGROUND/AIM: Bacteria sense their population density using autoinducer (AI) signaling systems. The AI-2 signal is thought to mediate communication among and within bacterial species. Streptococcus intermedius is a commensal organism frequently associated with periodontitis and purulent infections. We investigated the role of AI-2 signaling in S. intermedius biofilm formation under temperatures and pH levels relevant to human physiology. METHODS: Bioluminescence was used to monitor the change in AI-2 levels at various temperatures. Growth and biofilm formation in S. intermedius and its luxS mutant SI006 were measured at 35, 37, 39, and 41 degrees C and in pH ranging from 5.7 to 7.5. To confirm the role of AI-2 signals in biofilm formation, the AI-2 precursor (S)-4,5-dihydroxy-2,3-pentanedione (DPD) was used to complement SI006 biofilm formation. RESULTS: S. intermedius AI-2 signals were detected at all growth temperatures but reached the highest levels at 37 degrees C. SI006 displayed significantly less biofilm formation than S. intermedius wild-type (WT); however, the role of AI-2 on biofilm formation was confined to 37 degrees C. DPD supplementation significantly increased SI006 biofilm formation to the S. intermedius WT level. The role of AI-2 in S. intermedius biofilm formation was not influenced by pH. High temperatures and low pH enhanced biofilm formation in both S. intermedius and its luxS mutant. CONCLUSIONS: High temperature and acidic conditions may favor biofilm formation by S. intermedius. The role of AI-2 in biofilm formation by S. intermedius, however, varies with physiological temperature changes. These results may assist in understanding possible behavior relative to health and disease.

Antibiotics alter the window of competence for natural transformation in streptococci.

Streptococcus pneumoniae transformation occurs within a short competence window, during which the alternative sigma factor X (SigX) is activated to orchestrate the expression of genes allowing extracellular DNA uptake and recombination. Importantly, antibiotic stress promotes transcriptional changes that may affect more than 20% of the S. pneumoniae genome, including competence genes. These can be activated or repressed, depending on the antibiotic agent. For most antibiotics, however, it remains unknown whether transcriptional effects on competence translate into altered transformability. Here we investigate the effect of antibiotic subinhibitory concentrations on sigX expression using a luciferase reporter, and correlate for the first time with transformation kinetics. Induction of sigX expression by ciprofloxacin and novobiocin correlated with increased and prolonged transformability in S. pneumoniae. The prolonged effect of ciprofloxacin on competence and transformation was also observed in the streptococcal relatives Streptococcus mitis and Streptococcus mutans. In contrast, tetracycline and erythromycin, which induced S. pneumoniae sigX expression, had either an inhibitory or a nonsignificant effect on transformation, whereas streptomycin and the ß-lactam ampicillin, inhibited both sigX expression and transformation. Thus, the results show that antibiotics may vary in their effects on competence, ranging from inhibitory to stimulatory effects, and that responses affecting transcription of sigX do not always correlate with the transformation outcomes. Antibiotics that increase or decrease transformation are of particular clinical relevance, as they may alter the ability of S. pneumoniae to escape vaccines and antibiotics.

Capsule expression in Streptococcus mitis modulates interaction with oral keratinocytes and alters susceptibility to human antimicrobial peptides.

Streptococcus mitis is a colonizer of the oral cavity and the nasopharynx, and is closely related to Streptococcus pneumoniae. Both species occur in encapsulated and unencapsulated forms, but in S. mitis the role of the capsule in host interactions is mostly unknown. Therefore, the aim of this study was to examine how capsule expression in S. mitis can modulate interactions with the host with relevance for colonization. The S. mitis type strain, as well as two mutants of the type strain, an isogenic capsule deletion mutant, and a capsule switch mutant expressing the serotype 4 capsule of S. pneumoniae TIGR4, were used. Wild-type and capsule deletion strains of S. pneumoniae TIGR4 were included for comparison. We found that capsule production in S. mitis reduced adhesion to oral and lung epithelial cells. Further, exposure of oral epithelial cells to encapsulated S. mitis resulted in higher interleukin-6 and CXCL-8 transcription levels relative to the unencapsulated mutant. Capsule expression in S. mitis increased the sensitivity to human neutrophil peptide 1-3 but reduced the sensitivity to human ß-defensin-3 and cathelicidin. This was in contrast with S. pneumoniae in which capsule expression has been generally associated with increased sensitivity to human antimicrobial peptides (AMPs). Collectively, these findings indicate that capsule expression in S. mitis is important in modulating interactions with epithelial cells, and is associated with increased or reduced susceptibility to AMPs depending on the nature of the AMP.

Comprehensive Transcriptome Profiles of Streptococcus mutans UA159 Map Core Streptococcal Competence Genes.

In Streptococcus mutans, an oral colonizer associated with dental caries, development of competence for natural genetic transformation is triggered by either of two types of peptide pheromones, competence-stimulating peptides (CSPs) (18 amino acids [aa]) or SigX-inducing peptides (XIPs) (7 aa). Competence induced by CSP is a late response to the pheromone that requires the response regulator ComE and the XIP-encoding gene comS. XIP binds to ComR to allow expression of the alternative sigma factor SigX and the effector genes it controls. While these regulatory links are established, the precise set of effectors controlled by each regulator is poorly defined. To improve the definition of all three regulons, we used a high-resolution tiling array to map global changes in gene expression in the early and late phases of the CSP response. The early phase of the CSP response was limited to increased gene expression at four loci associated with bacteriocin production and immunity. In the late phase, upregulated regions expanded to a total of 29 loci, including comS and genes required for DNA uptake and recombination. The 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 four bacteriocin-related loci. Comparison of the new data with published transcriptome data permitted the identification of all of the operons in each regulon: 4 for ComE, 2 for ComR, and 21 for SigX. Finally, a core set of 27 panstreptococcal competence genes was identified within the SigX regulon by comparison of transcriptome data from diverse streptococcal species. IMPORTANCES. mutans has the hard surfaces of the oral cavity as its natural habitat, where it depends on its ability to form biofilms in order to survive. The comprehensive identification of S. mutans regulons activated in response to peptide pheromones provides an important basis for understanding how S. mutans can transition from individual to social behavior. Our study placed 27 of the 29 transcripts activated during competence within three major regulons and revealed a core set of 27 panstreptococcal competence-activated genes within the SigX regulon.

Genome editing by natural genetic transformation in Streptococcus mutans.

Classical mutagenesis strategies using selective markers linked to designed mutations are powerful and widely applicable tools for targeted mutagenesis via natural genetic transformation in bacteria and archaea. However, the markers that confer power are also potentially problematic as they can be cumbersome, risk phenotypic effects of the inserted genes, and accumulate as unwanted genes during successive mutagenesis cycles. Alternative mutagenesis strategies use temporary plasmid or cassette insertions and can in principle achieve equally flexible mutation designs, but design of suitable counter-selected markers can be complex. All these drawbacks are eased by use of direct genome editing. Here we describe a strategy for directly editing the genome of S. mutans, which is applied to the widely studied reference strain UA159 (ATCC 700610) and has the advantage of extreme simplicity, requiring construction of only one synthetic donor amplicon and a single transformation step, followed by a simple PCR screen among a few dozen clones to identify the desired mutant. The donor amplicon carries the mutant sequence and extensive flanking segments of homology, which ensure efficient and precise integration by the recombination machinery specific to competent cells. The recipients are highly competent cells, in a state achieved by treatment with a synthetic competence pheromone.

Identification of a functional capsule locus in Streptococcus mitis.

The polysaccharide capsule of Streptococcus pneumoniae is a hallmark for virulence in humans. In its close relative Streptococcus mitis, a common human commensal, analysis of the sequenced genomes of six strains revealed the presence of a putative capsule locus in four of them. We constructed an isogenic S. mitis mutant from the type strain that lacked the 19 open reading frames in the capsule locus (Δcps mutant), using a deletion strategy similar to previous capsule functional studies in S. pneumoniae. Transmission electron microscopy and atomic force microscopy revealed a capsule-like structure in the S. mitis type strain that was absent or reduced in the Δcps mutant. Since S. mitis are predominant oral colonizers of tooth surfaces, we addressed the relevance of the capsule locus for the S. mitis overall surface properties, autoaggregation and biofilm formation. The capsule deletion resulted in a mutant with approximately two-fold increase in hydrophobicity. Binding to the Stains-all cationic dye was reduced by 40%, suggesting a reduction in the overall negative surface charge of the mutant. The mutant exhibited also increased autoaggregation in coaggregation buffer, and up to six-fold increase in biofilm levels. The results suggested that the capsule locus is associated with production of a capsule-like structure in S. mitis and indicated that the S. mitis capsule-like structure may confer surface attributes similar to those associated with the capsule in S. pneumoniae.

Role of hyaluronidase in Streptococcus intermedius biofilm.

Streptococcus intermedius is found in biofilms on teeth and as a commensal member of the gastrointestinal and urinary floras, but may also be associated with deep-seated purulent infections and infective endocarditis. S. intermedius produces hyaluronidase, an enzyme that breaks down hyaluronan (HA), a major component of the extracellular matrix of connective tissue. We investigated the involvement of hyaluronidase in S. intermedius biofilm formation and dispersal as well as adhesion to human cells. The hyaluronidase activity and expression of the hyl gene were higher in growth media supplemented with HA. Inactivation of the S. intermedius hyaluronidase resulted in a mutant that formed up to 31 % more biofilm in media supplemented with HA. Hyaluronidase added to the medium caused dispersal of S. intermedius biofilm. Adhesion to epithelial cells was similar in the wild-type and the hyaluronidase mutant. We concluded that hyaluronidase may be important for S. intermedius detachment from biofilms but not for adhesion to epithelial cells. The ability of S. intermedius to detach from the surface and to spread may be crucial in the pathogenicity of this micro-organism.

LuxS and expression of virulence factors in Streptococcus intermedius.

BACKGROUND/AIMS: Autoinducer-2 (AI-2) is used by several bacteria in quorum-sensing signaling and is a product of LuxS. The aim was to investigate the effect of LuxS mutation on expression of Streptococcus intermedius virulence factors. METHODS: S. intermedius mutants were constructed by insertion inactivation or gene deletion. Real time RT-PCR was used to assess transcription of pas, ily and hyl. Hyaluronidase and intermedilysin activities were measured biochemically. RESULTS: The results indicated that disruption of luxS in S. intermedius may affect hyaluronidase and intermedilysin gene expressions. No difference in antigen I/II expression was observed. Biochemical methods showed a five-fold decrease in hemolytic activity of the luxS mutant; however, secreted hyaluronidase activity was unaffected. The AI-2 precursor 4,5-dihydroxy-2,3-pentanedione complemented lack of AI-2 production by the mutant thus restoring hemolytic activity. CONCLUSIONS: We suggest that AI-2 communication is involved in intermedilysin expression.

Synthetic bromated furanone inhibits autoinducer-2-mediated communication and biofilm formation in oral streptococci.

INTRODUCTION: Autoinducer-2 (AI-2) is a widespread communication-signal molecule that allows bacteria to sense and react to environmental factors. In some streptococci AI-2 is reported to be involved in virulence expression and biofilm formation. It has earlier been shown that the alga Delisea pulchra produces bromated furanones, which prevent bacterial colonization of the algae. METHODS AND RESULTS: We have previously published a novel and simple synthesis of (Z)-5-bromomethylene-2(5H)-furanone. In this study we showed that our synthesized furanone inhibited biofilm formation and bioluminescence induction by Streptococcus anginosus, Streptococcus intermedius, and Streptococcus mutans, as well as bioluminescence induction by Vibrio harveyi BB152. CONCLUSION: We suggest that the effect is linked to interference with the AI-2 signaling pathway because adding furanone to the medium had no effect on the ability of the AI-2-defective S. anginosus luxS and S. intermedius luxS mutants to form biofilms.

Involvement of antigen I/II surface proteins in Streptococcus mutans and Streptococcus intermedius biofilm formation.

BACKGROUND/AIM: Dental diseases are caused by microorganisms organized in biofilms. Streptococcus mutans and Streptococcus intermedius are commensals of the human oral cavity. S. mutans is associated with caries, whereas S. intermedius is associated with purulent infections. Oral streptococci including S. mutants and S. intermedius express a family of surface proteins termed antigen I/II (Ag I/II). Ag I/II is implicated in adhesion; however, its role in biofilm formation has not yet been investigated. METHODS: By using isogenic Ag I/II-deficient mutants of S. mutans and S. intermedius we studied the influence of Ag I/II on in vitro biofilm formation. Biofilm was quantified in polystyrene microtiter plates and visualized by scanning electron microscopy. Ag I/II expression in planktonic and biofilm cells, as well as in the presence or absence of saliva was investigated by immunoblotting. RESULTS: In the presence of saliva, the Ag I/II-deficient mutants formed 65% less biofilm than the wild-types. In the absence of saliva, no difference was observed in S. mutans, whereas the S. intermedius Ag I/II mutant formed 41% less biofilm. Ag I/II expression was reduced in the presence of saliva. No differences in expression were observed between biofilm and planktonic cells. CONCLUSION: The results indicated that Ag I/II may be important during biofilm formation particularly in the presence of saliva. These findings may provide useful information regarding the importance of Ag I/II in biofilm formation and in the search of new strategies to control biofilm-mediated infections.

Genetic transformation in Streptococcus mutans requires a peptide secretion-like apparatus.

Competence for genetic transformation in Streptococcus pneumoniae and Streptococcus gordonii involves the ComAB secretion apparatus, which is thought to export the competence-stimulating peptide. Homologous secretory systems are also used for the export of certain bacteriocins and bacteriocin-like peptides. In this study, a similar secretory apparatus was found in the Streptococcus mutans genome, and its role in transformation was investigated. Gene inactivation resulted in a mutant deficient in transformability. We suggest that secretion of a peptide, possibly the competence-stimulating peptide itself, is involved in competence induction also in S. mutans.

Functional variation of the antigen I/II surface protein in Streptococcus mutans and Streptococcus intermedius.

Although Streptococcus intermedius and Streptococcus mutans are regarded as members of the commensal microflora of the body, S. intermedius is often associated with deep-seated purulent infections, whereas S. mutans is frequently associated with dental caries. In this study, we investigated the roles of the S. mutans and S. intermedius antigen I/II proteins in adhesion and modulation of cell surface characteristics. By using isogenic mutants, we show that the antigen I/II in S. mutans, but not in S. intermedius, was involved in adhesion to a salivary film under flowing conditions, as well as in binding to rat collagen type I. Binding to human fibronectin was a common function associated with the S. mutans and S. intermedius antigen I/II. Adhesion of S. mutans or S. intermedius to human collagen types I or IV was negligible. Hydrophobicity, as measured by water contact angles, and zeta potentials were unaltered in the S. intermedius mutant. The S. mutans isogenic mutants, on the other hand, exhibited more positive zeta potentials at physiological pH values than did the wild type. The results indicate common and species-specific roles for the antigen I/II in mediating the attachment of S. mutans and S. intermedius to host components and in determining cell surface properties.

Expression and functional properties of the Streptococcus intermedius surface protein antigen I/II.

Streptococcus intermedius is associated with deep-seated purulent infections. In this study, we investigated expression and functional activities of antigen I/II in S. intermedius. The S. intermedius antigen I/II appeared to be cell surface associated, with a molecular mass of approximately 160 kDa. Northern blotting indicated that the S. intermedius NCTC 11324 antigen I/II gene was transcribed as a monocistronic message. Maximum expression was seen during the early exponential phase. Insertional inactivation of the antigen I/II gene resulted in reduced hydrophobicity during early exponential phase, whereas no effect was detected during mid- and late exponential phases. Binding to human fibronectin and laminin was reduced in the isogenic mutant, whereas binding to human collagen types I and IV and to rat collagen type I was not significant for either the wild type or the mutant. Compared to the wild type, the capacity of the isogenic mutant to induce interleukin 8 (IL-8) release by THP-1 monocytic cells was significantly reduced. The results indicate that the S. intermedius antigen I/II is involved in adhesion to human receptors and in IL-8 induction.