Systematic functional analysis of the Com pilus in Streptococcus sanguinis: a minimalistic type 4 filament dedicated to DNA uptake in monoderm bacteria.

IMPORTANCE: Type 4 filaments (T4F) are nanomachines ubiquitous in prokaryotes, centered on filamentous polymers of type 4 pilins. T4F are exceptionally versatile and widespread virulence factors in bacterial pathogens. The mechanisms of filament assembly and the many functions they facilitate remain poorly understood because of the complexity of T4F machineries. This hinders the development of anti-T4F drugs. The significance of our research lies in characterizing the simplest known T4F-the Com pilus that mediates DNA uptake in competent monoderm bacteria-and showing that four protein components universally conserved in T4F are sufficient for filament assembly. The Com pilus becomes a model for elucidating the mechanisms of T4F assembly.

Noncanonical Streptococcus sanguinis ComCDE circuitry integrates environmental cues in transformation outcome decision.

Natural competence is the principal driver of streptococcal evolution. While acquisition of new traits could facilitate rapid fitness improvement for bacteria, entry into the competent state is a highly orchestrated event, involving an interplay between various pathways. We present a new type of competence-predation coordination mechanism in Streptococcus sanguinis. Unlike other streptococci that mediate competence through the ComABCDE regulon, several key components are missing in the S. sanguinis ComCDE circuitry. We assembled two synthetic biology devices linking competence-stimulating peptide (CSP) cleavage and export with a quantifiable readout to unravel the unique features of the S. sanguinis circuitry. Our results revealed the ComC precursor cleavage pattern and the two host ABC transporters implicated in the export of the S. sanguinis CSP. Moreover, we discovered a ComCDE-dependent bacteriocin locus. Overall, this study presents a mechanism for commensal streptococci to maximize transformation outcome in a fluid environment through extensive circuitry rewiring.

Amyloid Fibrils Produced by Streptococcus sanguinis Contribute to Biofilm Formation and Immune Evasion.

Bacterial surface proteins assembled into amyloids contribute to biofilm formation and host immune evasion. Streptococcus sanguinis, a pioneer colonizer of teeth commonly involved in cardiovascular infections, expresses about thirty-three proteins anchored to the cell wall by sortase A. Here, we characterized the production of amyloid in S. sanguinis strains differing in biofilm and immune evasion phenotypes and investigated the role of sortase A in amyloidogenesis. Amyloid was identified in biofilms formed by nine strains, using Congo red (CR) staining and cross-polarized light microscopy. Additionally, EGCG, an amyloid inhibitor, impaired biofilm maturation in a strain-specific fashion. The amounts of amyloid-like components quantified in culture fluids of nine strains using thioflavin T and fluorimetry negatively correlated with bacterial binding to complement-activating proteins (SAP, C1q), C3b deposition and rates of opsonophagocytosis in PMNs, implying amyloid production in immune evasion. The deletion of the sortase A gene (srtA) in strain SK36 compromised amyloid production and sucrose-independent biofilm maturation. The srtA mutant further showed increased susceptibility to C3b deposition and altered interactions with PMNs as well as reduced persistence in human blood. These findings highlight the contribution of amyloids to biofilm formation and host immune evasion in S. sanguinis strains, further indicating the participation of sortase A substrates in amyloidogenesis.

Antibacterial Activity of a Lysin LysP53 against Streptococcus mutans.

Dental caries is a common disease affecting quality of life globally. In the present study, we found that a bacteriophage lysin LysP53 against Acinetobacter baumannii possesses selective activity on Streptococcus mutans, the main etiological agent of dental caries, even in low pH caries microenvironments, whereas only minor LysP53 activity was detected against Streptococcus sanguinis, Streptococcus oralis, and Streptococcus mitis. Testing activity against S. mutans planktonic cells showed that 4 µM LysP53 could kill more than 84% of S. mutans within 1 min in buffer with optimal pHs ranging from 4.0 to 6.5. Daily application of LysP53 on biofilms formed in BHI medium supplemented or not with sucrose could reduce exopolysaccharides, expression of genes related to acid resistance and adhesion, and the number of live bacteria in the biofilms. LysP53 treatment also showed similar effects as 0.12% chlorhexidine in preventing enamel demineralization due to S. mutans biofilms, as well as effective removal of S. mutans colonization of tooth surfaces in mice without observed toxic effects. Because of its selective activity against main cariogenic bacteria and good activity in low pH caries microenvironments, it is advantageous to use LysP53 as an active agent for preventing caries.

PepO and CppA modulate Streptococcus sanguinis susceptibility to complement immunity and virulence.

Streptococcus sanguinis is a ubiquitous commensal species of the oral cavity commonly involved as an opportunistic pathogen in cardiovascular infections. In this study, we investigated the functions of endopeptidase O (PepO) and a C3-degrading protease (CppA) in the systemic virulence of S. sanguinis. Isogenic mutants of pepO and cppA obtained in strain SK36 showed increased susceptibility to C3b deposition and to opsonophagocytosis by human polymorphonuclear neutrophils (PMN). These mutants differ, however, in their profiles of binding to serum amyloid P component (SAP) and C1q, whereas both showed reduced interaction with C4b-binding protein (C4BP) and/or factor H (FH) regulators as compared to SK36. The two mutants showed defects in ex vivo persistence in human blood, serum-mediated invasion of HCAEC endothelial cells, and virulence in a Galleria mellonella infection model. The transcriptional activities of pepO and cppA, assessed by RT-qPCR in nine wild-type strains, further indicated strain-specific profiles of pepO/cppA expression. Moreover, non-conserved amino acid substitutions were detected among the strains, mostly in CppA. Phylogenetic comparisons with homologues of streptococcal species of the oral and oropharyngeal sites suggested that S. sanguinis PepO and CppA have independent ancestralities. Thus, this study showed that PepO and CppA are complement evasion proteins expressed by S. sanguinis in a strain-specific manner, which are required for multiple functions associated with cardiovascular virulence.

Crystal structure of the pilus-specific sortase from early colonizing oral Streptococcus sanguinis captures an active open-lid conformation.

Dental plaque is a complex microbial biofilm community of many species and a major cause of oral infections and infectious endocarditis. Plaque development begins when primary colonizers attach to oral tissues and undergo coaggregation. Primary colonizers facilitate cellular attachment and inter-bacterial interactions through sortase-dependent pili (or fimbriae) extending out from their cell surface. Consequently, the sortase enzyme is viewed as a potential drug target for controlling biofilm formation and avoiding infection. Streptococcus sanguinis is a primary colonizing bacterium whose pili consist of three different pilin subunits that are assembled together by the pilus-specific (C-type) SsaSrtC sortase. Here, we report on the crystal structure determination of the recombinant wild-type and active-site mutant forms of SsaSrtC. Interestingly, the SsaSrtC structure exhibits an open-lid conformation, although a conserved DPX motif is lacking in the lid. Based on molecular docking and structural analysis, we identified the substrate-binding residues essential for pilin recognition and pilus assembly. We also demonstrated that while recombinant SsaSrtC is enzymatically active toward the five-residue LPNTG sorting motif peptide of the pilins, this activity is significantly reduced by the presence of zinc. We further showed that rutin and α-crocin are potential candidate inhibitors of the SsaSrtC sortase via structure-based virtual screening and inhibition assays. The structural knowledge gained from our study will provide the means to develop new approaches that target pilus-mediated attachment, thereby preventing oral biofilm growth and infection.

Environmental influences on Streptococcus sanguinis membrane vesicle biogenesis.

Membrane vesicles are produced by Gram-negative and Gram-positive bacteria. While membrane vesicles are potent elicitors of eukaryotic cells and involved in cell-cell communication, information is scarce about their general biology in the context of community members and the environment. Streptococcus sanguinis, a Gram-positive oral commensal, is prevalent in the oral cavity and well-characterized for its ability to antagonize oral pathobionts. We have found that production and dissemination of membrane vesicles by S. sanguinis is dependent on environmental and community factors. Co-culture with interacting commensal Corynebacterium durum, as well as with the periodontal pathobiont Filifactor alocis had no effect on S. sanguinis vesicle number and size, whereas the periodontal pathobiont Porphyromonas gingivalis abolished S. sanguinis vesicle production. Using both correlation and differential expression analyses to examine the transcriptomic changes underlying vesicle production, we found that differential expression of genes encoding proteins related to the cytoplasmic membrane and peptidoglycan correlate with the abundance of membrane vesicles. Proteomic characterizations of the vesicle cargo identified a variety of proteins, including those predicted to influence host interactions or host immune responses. Cell culture studies of gingival epithelial cells demonstrated that both crude and highly purified membrane vesicles could induce the expression of IL-8, TNF-α, IL-1ß, and Gro-α within 6 hours of inoculation at levels comparable to whole cells. Our findings suggest that production of membrane vesicles by S. sanguinis is heavily influenced by community and environmental factors and plays an important role in communication with host cells.

BacterAI maps microbial metabolism without prior knowledge.

Training artificial intelligence (AI) systems to perform autonomous experiments would vastly increase the throughput of microbiology; however, few microbes have large enough datasets for training such a system. In the present study, we introduce BacterAI, an automated science platform that maps microbial metabolism but requires no prior knowledge. BacterAI learns by converting scientific questions into simple games that it plays with laboratory robots. The agent then distils its findings into logical rules that can be interpreted by human scientists. We use BacterAI to learn the amino acid requirements for two oral streptococci: Streptococcus gordonii and Streptococcus sanguinis. We then show how transfer learning can accelerate BacterAI when investigating new environments or larger media with up to 39 ingredients. Scientific gameplay and BacterAI enable the unbiased, autonomous study of organisms for which no training data exist.

Stoichiometric models of sucrose and glucose fermentation by oral streptococci: Implications for free acid formation and enamel demineralization.

OBJECTIVES: The objective of this study is to develop stoichiometric models of sugar fermentation and cell biosynthesis for model cariogenic Streptococcus mutans and non-cariogenic Streptococcus sanguinis to better understand and predict metabolic product formation. METHODS: Streptococcus mutans (strain UA159) and Streptococcus sanguinis (strain DSS-10) were grown separately in bioreactors fed brain heart infusion broth supplemented with either sucrose or glucose at 37 °C. Cell mass concentration and fermentation products were measured at different hydraulic residence times (HRT) to determine cell growth yield. RESULTS: Sucrose growth yields were 0.080 ± 0.0078 g cell/g and 0.18 ± 0.031 g cell/g for S. sanguinis and S. mutans, respectively. For glucose, this reversed, with S. sanguinis having a yield of 0.10 ± 0.0080 g cell/g and S. mutans 0.053 ± 0.0064 g cell/g. Stoichiometric equations to predict free acid concentrations were developed for each test case. Results demonstrate that S. sanguinis produces more free acid at a given pH than S. mutans due to lesser cell yield and production of more acetic acid. Greater amounts of free acid were produced at the shortest HRT of 2.5 hr compared to longer HRTs for both microorganisms and substrates. SIGNIFICANCE: The finding that the non-cariogenic S. sanguinis produces greater amounts of free acids than S. mutans strongly suggests that bacterial physiology and environmental factors affecting substrate/metabolite mass transfer play a much greater role in tooth or enamel/dentin demineralization than acidogenesis. These findings enhance the understanding of fermentation production by oral streptococci and provide useful data for comparing studies under different environmental conditions.

vicR overexpression in Streptococcus mutans causes aggregation and affects interspecies competition.

Streptococcus mutans is considered to be a major causative agent of dental caries. VicRK is a two-component signal transduction system (TCSTS) of S. mutans, which can regulate the virulence of S. mutans, such as biofilm formation, exopolysaccharide production, acid production, and acid resistance. Meanwhile, it can also regulate the production of mutacins (nlmC) through the TCSTS ComDE. In this study, we found that the vicR-overexpressing strain was more likely to aggregate to form cell clusters, leading to the formation of abnormal biofilm; the overexpression of vicR increased the length of the chain of S. mutans. Furthermore, the expression of the mutacins in the vicR overexpression strain was increased under aerobic conditions. Compared with the control strain and the parental strain, the vicR overexpression strain was more competitive against Streptococcus gordonii. But there was no significant difference against Streptococcus sanguinis. In clinical strains, the expression level of vicR was positively correlated with their competitive ability against S. gordonii. Transcriptional profiling revealed 24 significantly upregulated genes in the vicR-overexpressing strain, including nlmA, nlmB, nlmC, and nlmD encoding mutacins. Electrophoretic mobility shift assays and DNase I footprinting assays confirmed that VicR can directly bind to the promoter sequence of nlmD. Taken together, our findings further demonstrate that VicRK, an important TCSTS of S. mutans, is involved in S. mutans cell morphology and biofilm formation. VicRK regulates the production of more mutacins in S. mutans in response to oxygen stimulation. VicR can bind to the promoter sequence of nlmD, thereby directly regulating the production of mutacins NlmD.

Prevalence of Type IV Pili-Mediated Twitching Motility in Streptococcus sanguinis Strains and Its Impact on Biofilm Formation and Host Adherence.

Type IV pili (Tfp) are known to mediate several biological activities, including surface-dependent twitching motility. Although a pil gene cluster for Tfp biosynthesis is found in all sequenced Streptococcus sanguinis strains, Tfp-mediated twitching motility is less commonly detected. Upon examining 81 clinical strains, 39 strains generated twitching zones on blood agar plates (BAP), while 27 strains displayed twitching on Todd-Hewitt (TH) agar. Although BAP appears to be more suitable for the development of twitching zones, 5 strains exhibited twitching motility only on TH agar, indicating that twitching motility is not only strain specific but also sensitive to growth media. Furthermore, different twitching phenotypes were observed in strains expressing comparable levels of pilT, encoding the retraction ATPase, suggesting that the twitching phenotype on agar plates is regulated by multiple factors. By using a PilT-null and a pilin protein-null derivative (CHW02) of twitching-active S. sanguinis CGMH010, we found that Tfp retraction was essential for biofilm stability. Further, biofilm growth was amplified in CHW02 in the absence of shearing force, indicating that S. sanguinis may utilize other ligands for biofilm formation in the absence of Tfp. Similar to SK36, Tfp from CGMH010 were required for colonization of host cells, but PilT only marginally affected adherence and only in the twitching-active strain. Taken together, the results suggest that Tfp participates in host cell adherence and that Tfp retraction facilitates biofilm stability. IMPORTANCE Although the gene clusters encoding Tfp are commonly present in Streptococcus sanguinis, not all strains express surface-dependent twitching motility on agar surfaces. Regardless of whether the Tfp could drive motility, Tfp can serve as a ligand for the colonization of host cells. Though many S. sanguinis strains lack twitching activity, motility can enhance biofilm stability in a twitching-active strain; thus, perhaps motility provides little or no advantage to the survival of bacteria within dental plaque. Rather, Tfp retraction could provide additional advantages for the bacteria to establish infections outside the oral cavity.

Contribution of a ZIP-family protein to manganese uptake and infective endocarditis virulence in Streptococcus sanguinis.

Streptococcus sanguinis is an important cause of infective endocarditis. In strain SK36, the ABC-family manganese transporter, SsaACB, is essential for virulence. We have now identified a ZIP-family protein, TmpA, as a secondary manganese transporter. A tmpA mutant had no phenotype, but a ΔssaACB ΔtmpA mutant was more attenuated for serum growth and for virulence in a rabbit model than its ΔssaACB parent. The growth of both mutants was restored by supplemental manganese, but the ΔssaACB ΔtmpA mutant required twenty-fold more and accumulated less. Although ZIP-family proteins are known for zinc and iron transport, TmpA-mediated transport of either metal was minimal. While ssaACB appears ubiquitous in St. sanguinis, tmpA was present in a majority of strains and a mntH gene encoding an NRAMP-family transporter was identified in relatively few, including VMC66. As in SK36, deletion of ssaACB greatly diminished VMC66 endocarditis virulence and serum growth, and deletion of tmpA from this mutant diminished virulence further. Virulence was not significantly altered by deletion of mntH from either VMC66 or its ΔssaACB mutant. This and the accompanying paper together suggest that SsaACB is of primary importance for endocarditis virulence while secondary transporters TmpA and MntH contribute to growth under differing conditions.

Mechanistic studies of the cofactor assembly in class Ib ribonucleotide reductases and protein affinity for MnII and FeII.

Ribonucleotide reductase (RNR) is an essential enzyme found in all organisms. The function of RNR is to catalyze the conversion of nucleotides to deoxynucleotides. RNRs rely on metallocofactors to oxidize a conserved cysteine in the active site of the enzyme into a thiyl radical, which then initiates nucleotide reduction. The proteins required for MnIII2-Y• cluster formation in class Ib RNRs are NrdF (ß-subunit) and NrdI (flavodoxin). An oxidant is channeled from the FMN cofactor in NrdI to the dimanganese center in NrdF, where it oxidizes the dimanganese center and a tyrosyl radical (Y•) is formed. Both Streptococcus sanguinis and Escherichia coli MnII2-NrdF structures have a constriction in the channel immediately above the metal site. In E. coli, the constriction is formed by the side chain of S159, whereas in the S. sanguinis system it involves T158. This serine-to-threonine substitution was investigated using S. sanguinis and Streptococcus pneumoniae class Ib RNRs but it is also present in other pathogenic streptococci. Using stopped-flow kinetics, we investigate the role of this substitution in the mechanism of MnIII2-Y• cluster formation. In addition to different kinetics observed in the studied streptococci, we found that affinity constants of NrdF for MnII and FeII are about 1 µM and the previously reported preference for MnII could not be explained by affinity only.

Spontaneous Mutants of Streptococcus sanguinis with Defects in the Glucose-Phosphotransferase System Show Enhanced Post-Exponential-Phase Fitness.

Genetic truncations in a gene encoding a putative glucose-phosphotransferase system (PTS) protein (manL, EIIABMan) were identified in subpopulations of two separate laboratory stocks of Streptococcus sanguinis SK36; the mutants had reduced PTS activities on glucose and other monosaccharides. To understand the emergence of these mutants, we engineered deletion mutants of manL and showed that the ManL-deficient strain had improved bacterial viability in the stationary phase and was better able to inhibit the growth of the dental caries pathogen Streptococcus mutans. Transcriptional analysis and biochemical assays suggested that the manL mutant underwent reprograming of central carbon metabolism that directed pyruvate away from production of lactate, increasing production of hydrogen peroxide (H2O2) and excretion of pyruvate. Addition of pyruvate to the medium enhanced the survival of SK36 in overnight cultures. Meanwhile, elevated pyruvate levels were detected in the cultures of a small but significant percentage (∼10%) of clinical isolates of oral commensal bacteria. Furthermore, the manL mutant showed higher expression of the arginine deiminase system than the wild type, which enhanced the ability of the mutant to raise environmental pH when arginine was present. To our surprise, significant discrepancies in genome sequence were identified between strain SK36 obtained from ATCC and the sequence deposited in GenBank. As the conditions that are likely associated with the emergence of spontaneous manL mutations, i.e., excess carbohydrates and low pH, are those associated with caries development, we propose that glucose-PTS strongly influences commensal-pathogen interactions by altering the production of ammonia, pyruvate, and H2O2. IMPORTANCE A health-associated dental microbiome provides a potent defense against pathogens and diseases. Streptococcus sanguinis is an abundant member of a health-associated oral flora that antagonizes pathogens by producing hydrogen peroxide. There is a need for a better understanding of the mechanisms that allow bacteria to survive carbohydrate-rich and acidic environments associated with the development of dental caries. We report the isolation and characterization of spontaneous mutants of S. sanguinis with impairment in glucose transport. The resultant reprograming of the central metabolism in these mutants reduced the production of lactic acid and increased pyruvate accumulation; the latter enables these bacteria to better cope with hydrogen peroxide and low pH. The implications of these discoveries in the development of dental caries are discussed.

Post-translational modification of Streptococcus sanguinis SpxB influences protein solubility and H2 O2 production.

Streptococcal pyruvate oxidase (SpxB) is a hydrogen peroxide-generating enzyme and plays a critical role in Streptococcus sanguinis interspecies interactions, but less is known about its biochemistry. We examined SpxB subcellular localization using protein fractionation and microscopy and found SpxB to be primarily cytoplasmic, but a small portion is also membrane associated. Potential post-translational modifications of SpxB were determined using coimmunoprecipitation and mass spectrometry. Two mutant strains were constructed to further validate the presence of predicted site-specific post-translational modifications. These site mutated SpxB proteins exhibited reduced solubility in vivo, which likely contributes to the observed phenotypic changes in colony morphology, bacterial growth, and H2 O2 production. Overall, our data suggest that SpxB post-translational modifications likely play a major role to regulate SpxB function in S. sanguinis.

Genome-wide identification of Streptococcus sanguinis fitness genes in human serum and discovery of potential selective drug targets.

Streptococcus sanguinis is a primary colonizer of teeth and is associated with oral health. When it enters the bloodstream, however, this bacterium may cause the serious illness infective endocarditis. The genes required for survival and proliferation in blood have not been identified. The products of these genes could provide a rich source of targets for endocarditis-specific antibiotics possessing greater efficacy for endocarditis, and also little or no activity against those bacteria that remain in the mouth. We previously created a comprehensive library of S. sanguinis mutants lacking every nonessential gene. We have now screened each member of this library for growth in human serum and discovered 178 mutants with significant abundance changes. The main biological functions disrupted in these mutants, including purine metabolism, were highlighted via network analysis. The components of an ECF-family transporter were required for growth in serum and were shown for the first time in any bacterium to be essential for endocarditis virulence. We also identified two mutants whose growth was reduced in serum but not in saliva. This strategy promises to enable selective targeting of bacteria based on their location in the body, in this instance, treating or preventing endocarditis while leaving the oral microbiome intact.

Intracellular Metal Speciation in Streptococcus sanguinis Establishes SsaACB as Critical for Redox Maintenance.

Streptococcus sanguinis is an oral commensal bacterium, but it can colonize pre-existing heart valve vegetations if introduced into the bloodstream, leading to infective endocarditis. Loss of Mn- or Fe-cofactored virulence determinants are thought to result in weakening of this bacterium. Indeed, intracellular Mn accumulation mediated by the lipoprotein SsaB, a component of the SsaACB transporter complex, has been shown to promote virulence for endocarditis and O2 tolerance. To delineate intracellular metal-ion abundance and redox speciation within S. sanguinis, we developed a protocol exploiting two spectroscopic techniques, Inductively coupled plasma-optical emission spectrometry (ICP-OES) and electron paramagnetic resonance (EPR) spectroscopy, to respectively quantify total intracellular metal concentrations and directly measure redox speciation of Fe and Mn within intact whole-cell samples. Addition of the cell-permeable siderophore deferoxamine shifts the oxidation states of accessible Fe and Mn from reduced-to-oxidized, as verified by magnetic moment calculations, aiding in the characterization of intracellular metal pools and metal sequestration levels for Mn2+ and Fe. We have applied this methodology to S. sanguinis and an SsaACB knockout strain (ΔssaACB), indicating that SsaACB mediates both Mn and Fe uptake, directly influencing the metal-ion pools available for biological inorganic pathways. Mn supplementation of ΔssaACB returns total intracellular Mn to wild-type levels, but it does not restore wild-type redox speciation or distribution of metal cofactor availability for either Mn or Fe. Our results highlight the biochemical basis for S. sanguinis oxidative resistance, revealing a dynamic role for SsaACB in controlling redox homeostasis by managing the intracellular Fe/Mn composition and distribution.

Synergism between Corynebacterium and Streptococcus sanguinis reveals new interactions between oral commensals.

The oral microbiome engages in a diverse array of highly sophisticated ecological interactions that are crucial for maintaining symbiosis with the host. Streptococci and corynebacteria are among the most abundant oral commensals and their interactions are critical for normal biofilm development. In this study, we discovered that Streptococcus sanguinis specifically responds to the presence of Corynebacterium durum by dramatically altering its chain morphology and improving its overall fitness. By employing gas chromatography-mass spectrometry (GC-MS) analysis, specific fatty acids were identified in C. durum supernatants that are responsible for the observed effect. Membrane vesicles (MVs) containing these fatty acids were isolated from C. durum supernatants and were able to replicate the chain morphology phenotype in S. sanguinis, suggesting MV as a mediator of interspecies interactions. Furthermore, S. sanguinis responds to C. durum lipids by decreasing the expression of key FASII genes involved in fatty acid synthesis. Several of these genes are also essential for the chain elongation phenotype, which implicates a regulatory connection between lipid metabolism and chain elongation. In addition, C. durum was found to affect the growth, cell aggregation, and phagocytosis of S. sanguinis, revealing a complex association of these species that likely supports oral commensal colonization and survival.

Pyruvate secretion by oral streptococci modulates hydrogen peroxide dependent antagonism.

Many commensal oral streptococci generate H2O2 via pyruvate oxidase (SpxB) to inhibit the growth of competing bacteria like Streptococcus mutans, a major cariogenic species. In Streptococcus sanguinis SK36 (SK36) and Streptococcus gordonii DL1 (DL1), spxB expression and H2O2 release are subject to carbon catabolite repression by the catabolite control protein A (CcpA). Surprisingly, ccpA deletion mutants of SK36 and DL1 fail to inhibit S. mutans despite their production of otherwise inhibitory levels of H2O2. Using H2O2-deficient spxB deletion mutants of SK36 and DL1, it was subsequently discovered that both strains confer protection in trans to other bacteria when H2O2 is added exogenously. This protective effect depends on the direct detoxification of H2O2 by the release of pyruvate. The pyruvate dependent protective effect is also present in other spxB-encoding streptococci, such as the pneumococcus, but is missing from spxB-negative species like S. mutans. Targeted and transposon-based mutagenesis revealed Nox (putative H2O-forming NADH dehydrogenase) as an essential component required for pyruvate release and oxidative protection, while other genes such as sodA and dps play minor roles. Furthermore, pyruvate secretion is only detectable in aerobic growth conditions at biofilm-like cell densities and is responsive to CcpA-dependent catabolite control. This ability of spxB-encoding streptococci reveals a new facet of the competitive interactions between oral commensals and pathobionts and provides a mechanistic basis for the variable levels of inhibitory potential observed among H2O2-producing strains of commensal oral streptococci.

Availability of Zinc Impacts Interactions between Streptococcus sanguinis and Pseudomonas aeruginosa in Coculture.

Airway infections associated with cystic fibrosis (CF) are polymicrobial. We reported previously that clinical isolates of Pseudomonas aeruginosa promote the growth of a variety of streptococcal species. To explore the mechanistic basis of this interaction, we performed a genetic screen to identify mutants of Streptococcus sanginuis SK36 whose growth was no longer enhanced by P. aeruginosa PAO1. Mutations in the zinc uptake systems of S. sanguinis SK36 reduced growth of these strains by 1 to 3 logs compared to that of wild-type S. sanguinis SK36 when grown in coculture with P. aeruginosa PAO1, and exogenous zinc (0.1 to 10 µM) rescued the coculture defect of zinc uptake mutants of S. sanguinis SK36. Zinc uptake mutants of S. sanguinis SK36 had no obvious growth defect in monoculture. Consistent with competition for zinc driving coculture dynamics, S. sanguinis SK36 grown in coculture with P. aeruginosa showed increased expression of zinc uptake genes compared to that of S. sanguinis grown alone. Strains of P. aeruginosa PAO1 defective in zinc transport also supported ∼2-fold more growth by S. sanguinis compared to that in coculture with wild-type P. aeruginosa PAO1. An analysis of 118 CF sputum samples revealed that total zinc levels varied from ∼5 to 145 µM. At relatively low zinc levels, Pseudomonas and Streptococcus spp. were found in approximately equal abundance; at higher zinc levels, we observed a decline in relative abundance of Streptococcus spp., perhaps as a result of increasing zinc toxicity. Together, our data indicate that the relative abundances of these microbes in the CF airway may be impacted by zinc levels.IMPORTANCE Polymicrobial infections in CF cases likely impact patient health, but the mechanism(s) underlying such interactions is poorly understood. Here, we show using an in vitro model system that interactions between Pseudomonas and Streptococcus are modulated by zinc availability, and clinical data are consistent with this model. Together with previous studies, our work supports a role for metal homeostasis as a key factor driving microbial interactions.