Glycerol metabolism contributes to competition by oral streptococci through production of hydrogen peroxide.

As a biological byproduct from both humans and microbes, glycerol's contribution to microbial homeostasis in the oral cavity remains understudied. In this study, we examined glycerol metabolism by Streptococcus sanguinis, a commensal associated with oral health. Genetic mutants of glucose-PTS enzyme II (manL), glycerol metabolism (glp and dha pathways), and transcriptional regulators were characterized with regard to glycerol catabolism, growth, production of hydrogen peroxide (H2O2), transcription, and competition with Streptococcus mutans. Biochemical assays identified the glp pathway as a novel source for H2O2 production by S. sanguinis that is independent of pyruvate oxidase (SpxB). Genetic analysis indicated that the glp pathway requires glycerol and a transcriptional regulator, GlpR, for expression and is negatively regulated by PTS, but not the catabolite control protein, CcpA. Conversely, deletion of either manL or ccpA increased the expression of spxB and a second, H2O2-non-producing glycerol metabolic pathway (dha), indicative of a mode of regulation consistent with conventional carbon catabolite repression (CCR). In a plate-based antagonism assay and competition assays performed with planktonic and biofilm-grown cells, glycerol greatly benefited the competitive fitness of S. sanguinis against S. mutans. The glp pathway appears to be conserved in several commensal streptococci and actively expressed in caries-free plaque samples. Our study suggests that glycerol metabolism plays a more significant role in the ecology of the oral cavity than previously understood. Commensal streptococci, though not able to use glycerol as a sole carbohydrate source for growth, benefit from the catabolism of glycerol through production of both ATP and H2O2. IMPORTANCE: Glycerol is an abundant carbohydrate in the oral cavity. However, little is understood regarding the metabolism of glycerol by commensal streptococci, some of the most abundant oral bacteria. This was in part because most streptococci cannot grow on glycerol as the sole carbon source. In this study, we show that Streptococcus sanguinis, a commensal associated with dental health, can degrade glycerol for persistence and competition through two pathways, one of which generates hydrogen peroxide at levels capable of inhibiting Streptococcus mutans. Preliminary studies suggest that several additional commensal streptococci are also able to catabolize glycerol, and glycerol-related genes are actively expressed in human dental plaque samples. Our findings reveal the potential of glycerol to significantly impact microbial homeostasis, which warrants further exploration.

Dpr-mediated H2O2 resistance contributes to streptococcus survival in a cystic fibrosis airway model system.

The cystic fibrosis (CF) lung environment is conducive to the colonization of bacteria as polymicrobial biofilms, which are associated with poor clinical outcomes for persons with CF (pwCF). Streptococcus spp. are highly prevalent in the CF airway, but its role in the CF lung microbiome is poorly understood. Some studies have shown Streptococcus spp. to be associated with better clinical outcomes for pwCF, while others show that high abundance of Streptococcus spp. is correlated with exacerbations. Our lab previously reported a polymicrobial culture system consisting of four CF-relevant pathogens that can be used to study microbial behavior in a more clinically relevant setting. Here, we use this model system to identify genetic pathways that are important for Streptococcus sanguinis survival in the context of the polymicrobial community. We identified genes related to reactive oxygen species as differentially expressed in S. sanguinis monoculture versus growth of this microbe in the mixed community. Genetic studies identified Dpr as important for S. sanguinis survival in the community. We show that Dpr, a DNA-binding ferritin-like protein, and PerR, a peroxide-responsive transcriptional regulator of Dpr, are important for protecting S. sanguinis from phenazine-mediated toxicity in co-culture with Pseudomonas aeruginosa and when exposed to hydrogen peroxide, both of which mimic the CF lung environment. Characterizing such interactions in a clinically relevant model system contributes to our understanding of microbial behavior in the context of polymicrobial biofilm infections. IMPORTANCE: Streptococcus spp. are recognized as a highly prevalent pathogen in cystic fibrosis (CF) airway infections. However, the role of this microbe in clinical outcomes for persons with CF is poorly understood. Here, we leverage a polymicrobial community system previously developed by our group to model CF airway infections as a tool to investigate a Pseudomonas-Streptococcus interaction involving reactive oxygen species (ROS). We show that protection against ROS is required for Streptococcus sanguinis survival in a clinically relevant polymicrobial system. Using this model system to study interspecies interactions contributes to our broader understanding of the complex role of Streptococcus spp. in the CF lung.

Functional Analysis of the Major Pilin Proteins of Type IV Pili in Streptococcus sanguinis CGMH010.

The pil gene cluster for Type IV pilus (Tfp) biosynthesis is commonly present and highly conserved in Streptococcus sanguinis. Nevertheless, Tfp-mediated twitching motility is less common among strains, and the factors determining twitching activity are not fully understood. Here, we analyzed the functions of three major pilin proteins (PilA1, PilA2, and PilA3) in the assembly and activity of Tfp in motile S. sanguinis CGMH010. Using various recombinant pilA deletion strains, we found that Tfp composed of different PilA proteins varied morphologically and functionally. Among the three PilA proteins, PilA1 was most critical in the assembly of twitching-active Tfp, and recombinant strains expressing motility generated more structured biofilms under constant shearing forces compared to the non-motile recombinant strains. Although PilA1 and PilA3 shared 94% identity, PilA3 could not compensate for the loss of PilA1, suggesting that the nature of PilA proteins plays an essential role in twitching activity. The single deletion of individual pilA genes had little effect on the invasion of host endothelia by S. sanguinis CGMH010. In contrast, the deletion of all three pilA genes or pilT, encoding the retraction ATPase, abolished Tfp-mediated invasion. Tfp- and PilT-dependent invasion were also detected in the non-motile S. sanguinis SK36, and thus, the retraction of Tfp, but not active twitching, was found to be essential for invasion.

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.

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.

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.

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.

Glycerol metabolism supports oral commensal interactions.

During oral biofilm development, interspecies interactions drive species distribution and biofilm architecture. To understand what molecular mechanisms determine these interactions, we used information gained from recent biogeographical investigations demonstrating an association of corynebacteria with streptococci. We previously reported that Streptococcus sanguinis and Corynebacterium durum have a close relationship through the production of membrane vesicle and fatty acids leading to S. sanguinis chain elongation and overall increased fitness supporting their commensal state. Here we present the molecular mechanisms of this interspecies interaction. Coculture experiments for transcriptomic analysis identified several differentially expressed genes in S. sanguinis. Due to its connection to fatty acid synthesis, we focused on the glycerol-operon. We further explored the differentially expressed type IV pili genes due to their connection to motility and biofilm adhesion. Gene inactivation of the glycerol kinase glpK had a profound impact on the ability of S. sanguinis to metabolize C. durum secreted glycerol and impaired chain elongation important for their interaction. Investigations on the effect of type IV pili revealed a reduction of S. sanguinis twitching motility in the presence of C. durum, which was caused by a decrease in type IV pili abundance on the surface of S. sanguinis as determined by SEM. In conclusion, we identified that the ability to metabolize C. durum produced glycerol is crucial for the interaction of C. durum and S. sanguinis. Reduced twitching motility could lead to a closer interaction of both species, supporting niche development in the oral cavity and potentially shaping symbiotic health-associated biofilm communities.

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.

Oral microbiome diversity: The curious case of Corynebacterium sp. isolation.

Oral microbiome sequencing efforts revealed the presence of hundreds of different microbes. Interindividual differences at strain and species resolution suggest that microbiome diversity could lead to mechanistically distinct gene regulation as well as species-related differences in phenotypes. Commonly, gene regulation and related phenotypes are studied in a few selected strains of a particular species with conclusions that are mostly generalized. The aim of this study was to isolate several species of Corynebacterium using an established protocol that led to the previous isolation of C. durum. Characterization of C. durum interspecies interactions revealed a specific mechanism for chain elongation in Streptococcus sanguinis that was the result of corynebacterial fatty acid production and secretion. While the protocol was successfully applied to isolate what we presumed to be additional Corynebacterium based on several phenotypic traits that seem to be identical to C. durum, genome sequencing of the newly isolated strains placed them closer to Actinomyces. Both Corynebacterium and Actinomyces are suborders of the Actinobacteridae and related species. Our study suggests to take several comprehensive strategies into consideration when taxonomically identifying closely related microorganisms. Furthermore, it seems to be important to test common core phenotypes in bacterial ecology to understand the behavior of specific groups of microbes, rather than simply relying upon genome sequence homology to establish relationships in the microbiome.

Identification of Streptococcus sanguinis genes producing biofilm from gingivitis.

Streptococcus sanguinis is a teeth commensal frontier colonizer and among the most common species in the oral biofilm. Dental plaque, caries, and gingivitis/periodontitis are caused by dysbiosis of oral flora. A biofilm assay was developed to investigate biofilm formation in S. sanguinis using the microtiter plate, tube, and Congo red agar methods in order to identify causing bacteria and determine responsible genes. Three genes, including pur B, thr B, and pyre E, were suspected of playing a role in forming in vivo biofilms in S. sanguinis. The present study shows these genes to be responsible for increased biofilm formation in gingivitis patients.

Type IV Pili of Streptococcus sanguinis Contribute to Pathogenesis in Experimental Infective Endocarditis.

Streptococcus sanguinis is a common cause of infective endocarditis (IE). Efforts by research groups are aimed at identifying and characterizing virulence factors that contribute to the ability of this organism to cause IE. This Gram-positive pathogen causes heart infection by gaining access to the bloodstream, adhering to host extracellular matrix protein and/or platelets, colonizing the aortic endothelium, and incorporating itself into the aortic vegetation. While many virulence factors have been reported to contribute to the ability of S. sanguinis to cause IE, it is noteworthy that type IV pili (T4P) have not been described to be a virulence factor in this organism, although S. sanguinis strains typically encode these pili. Type IV pili are molecular machines that are capable of mediating diverse virulence functions and surface motility. T4P have been shown to mediate twitching motility in some strains of S. sanguinis, although in most strains it has been difficult to detect twitching motility. While we found that T4P are dispensable for direct in vitro platelet binding and aggregation phenotypes, we show that they are critical to the development of platelet-dependent biofilms representative of the cardiac vegetation. We also observed that T4P are required for in vitro invasion of S. sanguinis into human aortic endothelial cells, which indicates that S. sanguinis may use T4P to take advantage of an intracellular niche during infection. Importantly, we show that T4P of S. sanguinis are critical to disease progression (vegetation development) in a native valve IE rabbit model. The results presented here expand our understanding of IE caused by S. sanguinis and identify T4P as an important virulence factor for this pathogen. IMPORTANCE This work provides evidence that type IV pili produced by Streptococcus sanguinis SK36 are critical to the ability of these bacteria to attach to and colonize the aortic heart valve (endocarditis). We found that an S. sanguinis type IV pili mutant strain was defective in causing platelet-dependent aggregation in a 24-h infection assay but not in a 1-h platelet aggregation assay, suggesting that the type IV pili act at later stages of vegetation development. In a rabbit model of disease, a T4P mutant strain does not develop mature vegetations that form on the heart, indicating that this virulence factor is critical to disease and could be a target for IE therapy.

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.

Whole microbial community viability is not quantitatively reflected by propidium monoazide sequencing approach.

BACKGROUND: High-throughput sequencing provides a powerful window into the structural and functional profiling of microbial communities, but it is unable to characterize only the viable portion of microbial communities at scale. There is as yet not one best solution to this problem. Previous studies have established viability assessments using propidium monoazide (PMA) treatment coupled with downstream molecular profiling (e.g., qPCR or sequencing). While these studies have met with moderate success, most of them focused on the resulting "viable" communities without systematic evaluations of the technique. Here, we present our work to rigorously benchmark "PMA-seq" (PMA treatment followed by 16S rRNA gene amplicon sequencing) for viability assessment in synthetic and realistic microbial communities. RESULTS: PMA-seq was able to successfully reconstruct simple synthetic communities comprising viable/heat-killed Escherichia coli and Streptococcus sanguinis. However, in realistically complex communities (computer screens, computer mice, soil, and human saliva) with E. coli spike-in controls, PMA-seq did not accurately quantify viability (even relative to variability in amplicon sequencing), with its performance largely affected by community properties such as initial biomass, sample types, and compositional diversity. We then applied this technique to environmental swabs from the Boston subway system. Several taxa differed significantly after PMA treatment, while not all microorganisms responded consistently. To elucidate the "PMA-responsive" microbes, we compared our results with previous PMA-based studies and found that PMA responsiveness varied widely when microbes were sourced from different ecosystems but were reproducible within similar environments across studies. CONCLUSIONS: This study provides a comprehensive evaluation of PMA-seq exploring its quantitative potential in synthetic and complex microbial communities, where the technique was effective for semi-quantitative purposes in simple synthetic communities but provided only qualitative assessments in realistically complex community samples. Video abstract.

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.

The major subunit of widespread competence pili exhibits a novel and conserved type IV pilin fold.

Type IV filaments (T4F), which are helical assemblies of type IV pilins, constitute a superfamily of filamentous nanomachines virtually ubiquitous in prokaryotes that mediate a wide variety of functions. The competence (Com) pilus is a widespread T4F, mediating DNA uptake (the first step in natural transformation) in bacteria with one membrane (monoderms), an important mechanism of horizontal gene transfer. Here, we report the results of genomic, phylogenetic, and structural analyses of ComGC, the major pilin subunit of Com pili. By performing a global comparative analysis, we show that Com pili genes are virtually ubiquitous in Bacilli, a major monoderm class of Firmicutes. This also revealed that ComGC displays extensive sequence conservation, defining a monophyletic group among type IV pilins. We further report ComGC solution structures from two naturally competent human pathogens, Streptococcus sanguinis (ComGCSS) and Streptococcus pneumoniae (ComGCSP), revealing that this pilin displays extensive structural conservation. Strikingly, ComGCSS and ComGCSP exhibit a novel type IV pilin fold that is purely helical. Results from homology modeling analyses suggest that the unusual structure of ComGC is compatible with helical filament assembly. Because ComGC displays such a widespread distribution, these results have implications for hundreds of monoderm species.

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.

Rapid and Accurate Species Identification of Mitis Group Streptococci Using the MinION Nanopore Sequencer.

Differentiation between mitis group streptococci (MGS) bacteria in routine laboratory tests has become important for obtaining accurate epidemiological information on the characteristics of MGS and understanding their clinical significance. The most reliable method of MGS species identification is multilocus sequence analysis (MLSA) with seven house-keeping genes; however, because this method is time-consuming, it is deemed unsuitable for use in most clinical laboratories. In this study, we established a scheme for identifying 12 species of MGS (S. pneumoniae, S. pseudopneumoniae, S. mitis, S. oralis, S. peroris, S. infantis, S. australis, S. parasanguinis, S. sinensis, S. sanguinis, S. gordonii, and S. cristatus) using the MinION nanopore sequencer (Oxford Nanopore Technologies, Oxford, UK) with the taxonomic aligner "What's in My Pot?" (WIMP; Oxford Nanopore's cloud-based analysis platform) and Kraken2 pipeline with the custom database adjusted for MGS species identification. The identities of the species in reference genomes (n = 514), clinical isolates (n = 31), and reference strains (n = 4) were confirmed via MLSA. The nanopore simulation reads were generated from reference genomes, and the optimal cut-off values for MGS species identification were determined. For 31 clinical isolates (S. pneumoniae = 8, S. mitis = 17 and S. oralis = 6) and 4 reference strains (S. pneumoniae = 1, S. mitis = 1, S. oralis = 1, and S. pseudopneumoniae = 1), a sequence library was constructed via a Rapid Barcoding Sequencing Kit for multiplex and real-time MinION sequencing. The optimal cut-off values for the identification of MGS species for analysis by WIMP and Kraken2 pipeline were determined. The workflow using Kraken2 pipeline with a custom database identified all 12 species of MGS, and WIMP identified 8 MGS bacteria except S. infantis, S. australis, S. peroris, and S. sinensis. The results obtained by MinION with WIMP and Kraken2 pipeline were consistent with the MGS species identified by MLSA analysis. The practical advantage of whole genome analysis using the MinION nanopore sequencer is that it can aid in MGS surveillance. We concluded that MinION sequencing with the taxonomic aligner enables accurate MGS species identification and could contribute to further epidemiological surveys.