Post-translational modification by the Pgf glycosylation machinery modulates Streptococcus mutans OMZ175 physiology and virulence.

Streptococcus mutans is commonly associated with dental caries and the ability to form biofilms is essential for its pathogenicity. We recently identified the Pgf glycosylation machinery of S. mutans, responsible for the post-translational modification of the surface-associated adhesins Cnm and WapA. Since the four-gene pgf operon (pgfS-pgfM1-pgfE-pgfM2) is part of the S. mutans core genome, we hypothesized that the scope of the Pgf system goes beyond Cnm and WapA glycosylation. In silico analyses and tunicamycin sensitivity assays suggested a functional overlap between the Pgf machinery and the rhamnose-glucose polysaccharide synthesis pathway. Phenotypic characterization of pgf mutants (ΔpgfS, ΔpgfE, ΔpgfM1, ΔpgfM2, and Δpgf) revealed that the Pgf system is important for biofilm formation, surface charge, membrane stability, and survival in human saliva. Moreover, deletion of the entire pgf operon (Δpgf strain) resulted in significantly impaired colonization in a rat oral colonization model. Using Cnm as a model, we showed that Cnm is heavily modified with N-acetyl hexosamines but it becomes heavily phosphorylated with the inactivation of the PgfS glycosyltransferase, suggesting a crosstalk between these two post-translational modification mechanisms. Our results revealed that the Pgf machinery contributes to multiple aspects of S. mutans pathobiology that may go beyond Cnm and WapA glycosylation.

Catalase produced by Candida albicans protects Streptococcus mutans from H2O2 stress-one more piece in the cross-kingdom synergism puzzle.

Co-infection with Streptococcus mutans and Candida albicans is associated with dental caries, and their co-cultivation results in enhanced biofilm matrix production that contributes to increased virulence and caries risk. Moreover, the catalase-negative S. mutans demonstrates increased oxidative stress tolerance when co-cultivated in biofilms with C. albicans, a catalase-producing yeast. Here, we sought to obtain mechanistic insights into the increased H2O2 tolerance of S. mutans when co-cultivated with clinical isolates of Candida glabrata, Candida tropicalis, and C. albicans. Additionally, the C. albicans SC5314 laboratory strain, its catalase mutant (SC5314Δcat1), and S. mutans UA159 and its glucosyltransferase B/C mutant (UA159ΔgtfB/C) were grown as single- and dual-species biofilms. Time-kill assays revealed that upon acute H2O2 challenge, the survival rates of S. mutans in dual-species biofilms with the clinical isolates and C. albicans SC5314 were greater than when paired with SC5314Δcat1 or as a single-species biofilm. Importantly, this protection was independent of glucan production through S. mutans GtfB/C. Transwell assays and treatment with H2O2-pre-stimulated C. albicans SC5314 supernatant revealed that this protection is contact-dependent. Biofilm stability assays with sublethal H2O2 or peroxigenic Streptococcus A12 challenge resulted in biomass reduction of single-species S. mutans UA159 and dual-species with SC5314Δcat1 biofilms compared to UA159 biofilms co-cultured with C. albicans SC5314. S. mutans oxidative stress genes were upregulated in single-species biofilms when exposed to H2O2, but not when S. mutans was co-cultivated with C. albicans SC5314. Here, we uncovered a novel, contact-dependent, synergistic interaction in which the catalase of C. albicans protects S. mutans against H2O2. IMPORTANCE It is well established that co-infection with the gram-positive caries-associated bacterium Streptococcus mutans and the yeast pathobiont Candida albicans results in aggressive forms of caries in humans and animal models. Together, these microorganisms form robust biofilms through enhanced production of extracellular polysaccharide matrix. Further, co-habitation in a biofilm community appears to enhance these microbes' tolerance to environmental stressors. Here, we show that catalase produced by C. albicans protects S. mutans from H2O2 stress in a biofilm matrix-independent manner. Our findings uncovered a novel synergistic trait between these two microorganisms that could be further exploited for dental caries prevention and control.

Involvement of the Streptococcus mutans PgfE and GalE 4-epimerases in protein glycosylation, carbon metabolism, and cell division.

Streptococcus mutans is a key pathogen associated with dental caries and is often implicated in infective endocarditis. This organism forms robust biofilms on tooth surfaces and can use collagen-binding proteins (CBPs) to efficiently colonize collagenous substrates, including dentin and heart valves. One of the best characterized CBPs of S. mutans is Cnm, which contributes to adhesion and invasion of oral epithelial and heart endothelial cells. These virulence properties were subsequently linked to post-translational modification (PTM) of the Cnm threonine-rich repeat region by the Pgf glycosylation machinery, which consists of 4 enzymes: PgfS, PgfM1, PgfE, and PgfM2. Inactivation of the S. mutans pgf genes leads to decreased collagen binding, reduced invasion of human coronary artery endothelial cells, and attenuated virulence in the Galleria mellonella invertebrate model. The present study aimed to better understand Cnm glycosylation and characterize the predicted 4-epimerase, PgfE. Using a truncated Cnm variant containing only 2 threonine-rich repeats, mass spectrometric analysis revealed extensive glycosylation with HexNAc2. Compositional analysis, complemented with lectin blotting, identified the HexNAc2 moieties as GlcNAc and GalNAc. Comparison of PgfE with the other S. mutans 4-epimerase GalE through structural modeling, nuclear magnetic resonance, and capillary electrophoresis demonstrated that GalE is a UDP-Glc-4-epimerase, while PgfE is a GlcNAc-4-epimerase. While PgfE exclusively participates in protein O-glycosylation, we found that GalE affects galactose metabolism and cell division. This study further emphasizes the importance of O-linked protein glycosylation and carbohydrate metabolism in S. mutans and identifies the PTM modifications of the key CBP, Cnm.

Quantitative Analysis of Biofilm Removal Following Instrumentation with TRUShape and Vortex Blue File Systems: Microbiological Study.

Mesial roots and isthmuses of mandibular molars are difficult areas to obtain adequate disinfection of root canal walls, and consequently microorganisms can survive treatment. The present study compared, through real-time polymerase chain reaction (qPCR), the effectiveness of TRUShape (TS) (Dentsply Tulsa Dental Specialties, Tulsa, OK) and Vortex Blue (VB) (Dentsply Tulsa Dental Specialties, Tulsa, OK) in removing Enterococcus faecalis (E. faecalis) from the mesial canals and isthmuses of mandibular molars. Fifty extracted human lower molars were inoculated with E. faecalis OG1RF for 14 days, and then an initial bacterial sample was collected with paper points from mesiobuccal and mesiolingual canals and isthmuses. The specimens were randomly divided into four groups (n = 10 teeth; 20 canals each), according to instrumentation system: TS 25/0.06, TS 30/0.06, VB 25/0.06 and VB 30/0.06. The remaining 10 teeth were divided between positive control, inoculated teeth without instrumentation or irrigation, and negative controls, teeth without inoculation. After instrumentation, the final sample was taken using paper points and DNA was isolated. Primers specific for E. faecalis were used for qPCR. The bacterial reduction between pre- and post-instrumentation was calculated. One-way analysis of variance (ANOVA) with Bonferroni's multiple-comparisons tests were for statistical analysis with significance of (p < 0.05). All file systems were able to reduce the load of E. faecalis from the prepared root canals, however, TS size 30 removed significantly more bacteria than size 25. Interestingly, regardless of the size, TS files removed significantly more E. faecalis biofilm (p < 0.05) than did VB files (63.7% vs 50.8% for size 25, and 69.5% vs 56% for size 30). In conclusion, when combined with irrigation, TS file system is more effective than VB in reducing E. faecalis biofilms from mesiobuccal and mesiolingual canals and the isthmuses of mandibular molars.

Amyloid Aggregates Are Localized to the Nonadherent Detached Fraction of Aging Streptococcus mutans Biofilms.

The number of bacterial species recognized to utilize purposeful amyloid aggregation within biofilms continues to grow. The oral pathogen Streptococcus mutans produces several amyloidogenic proteins, including adhesins P1 (also known as AgI/II, PAc) and WapA, whose truncation products, namely, AgII and AgA, respectively, represent the amyloidogenic moieties. Amyloids demonstrate common biophysical properties, including recognition by Thioflavin T (ThT) and Congo red (CR) dyes that bind to the cross ß-sheet quaternary structure of amyloid aggregates. Previously, we observed amyloid formation to occur only after 60 h or more of S. mutans biofilm growth. Here, we extend those findings to investigate where amyloid is detected within 1- and 5-day-old biofilms, including within tightly adherent compared with those in nonadherent fractions. CR birefringence and ThT uptake demonstrated amyloid within nonadherent material removed from 5-day-old cultures but not within 1-day-old or adherent samples. These experiments were done in conjunction with confocal microscopy and immunofluorescence staining with AgII- and AgA-reactive antibodies, including monoclonal reagents shown to discriminate between monomeric protein and amyloid aggregates. These results also localized amyloid primarily to the nonadherent fraction of biofilms. Lastly, we show that the C-terminal region of P1 loses adhesive function following amyloidogenesis and is no longer able to competitively inhibit binding of S. mutans to its physiologic substrate, salivary agglutinin. Taken together, our results provide new evidence that amyloid aggregation negatively impacts the functional activity of a widely studied S. mutans adhesin and are consistent with a model in which amyloidogenesis of adhesive proteins facilitates the detachment of aging biofilms. IMPORTANCE Streptococcus mutans is a keystone pathogen and causative agent of human dental caries, commonly known as tooth decay, the most prevalent infectious disease in the world. Like many pathogens, S. mutans causes disease in biofilms, which for dental decay begins with bacterial attachment to the salivary pellicle coating the tooth surface. Some strains of S. mutans are also associated with bacterial endocarditis. Amyloid aggregation was initially thought to represent only a consequence of protein mal-folding, but now, many microorganisms are known to produce functional amyloids with biofilm environments. In this study, we learned that amyloid formation diminishes the activity of a known S. mutans adhesin and that amyloid is found within the nonadherent fraction of older biofilms. This finding suggests that the transition from adhesin monomer to amyloid facilitates biofilm detachment. Knowing where and when S. mutans produces amyloid will help in developing therapeutic strategies to control tooth decay and other biofilm-related diseases.

ZccE is a Novel P-type ATPase That Protects Streptococcus mutans Against Zinc Intoxication.

Zinc is a trace metal that is essential to all forms of life, but that becomes toxic at high concentrations. Because it has both antimicrobial and anti-inflammatory properties and low toxicity to mammalian cells, zinc has been used as a therapeutic agent for centuries to treat a variety of infectious and non-infectious conditions. While the usefulness of zinc-based therapies in caries prevention is controversial, zinc is incorporated into toothpaste and mouthwash formulations to prevent gingivitis and halitosis. Despite this widespread use of zinc in oral healthcare, the mechanisms that allow Streptococcus mutans, a keystone pathogen in dental caries and prevalent etiological agent of infective endocarditis, to overcome zinc toxicity are largely unknown. Here, we discovered that S. mutans is inherently more tolerant to high zinc stress than all other species of streptococci tested, including commensal streptococci associated with oral health. Using a transcriptome approach, we uncovered several potential strategies utilized by S. mutans to overcome zinc toxicity. Among them, we identified a previously uncharacterized P-type ATPase transporter and cognate transcriptional regulator, which we named ZccE and ZccR respectively, as responsible for the remarkable high zinc tolerance of S. mutans. In addition to zinc, we found that ZccE, which was found to be unique to S. mutans strains, mediates tolerance to at least three additional metal ions, namely cadmium, cobalt, and copper. Loss of the ability to maintain zinc homeostasis when exposed to high zinc stress severely disturbed zinc:manganese ratios, leading to heightened peroxide sensitivity that was alleviated by manganese supplementation. Finally, we showed that the ability of the ΔzccE strain to stably colonize the rat tooth surface after topical zinc treatment was significantly impaired, providing proof of concept that ZccE and ZccR are suitable targets for the development of antimicrobial therapies specifically tailored to kill S. mutans.

Amyloid Aggregation of Streptococcus mutans Cnm Influences Its Collagen-Binding Activity.

The cnm gene, coding for the glycosylated collagen- and laminin-binding surface adhesin Cnm, is found in the genomes of approximately 20% of Streptococcus mutans clinical isolates and is associated with systemic infections and increased caries risk. Other surface-associated collagen-binding proteins of S. mutans, such as P1 and WapA, have been demonstrated to form an amyloid quaternary structure with functional implications within biofilms. In silico analysis predicted that the ß-sheet-rich N-terminal collagen-binding domain (CBD) of Cnm has a propensity for amyloid aggregation, whereas the threonine-rich C-terminal domain was predicted to be disorganized. In this study, thioflavin-T fluorescence and electron microscopy were used to show that Cnm forms amyloids in either its native glycosylated or recombinant nonglycosylated form and that the CBD of Cnm is the main amyloidogenic unit of Cnm. We then performed a series of in vitro, ex vivo, and in vivo assays to characterize the amylogenic properties of Cnm. In addition, Congo red birefringence indicated that Cnm is a major amyloidogenic protein of S. mutans biofilms. Competitive binding assays using collagen-coated microtiter plates and dental roots, a substrate rich in collagen, revealed that Cnm monomers inhibit S. mutans binding to collagenous substrates, whereas Cnm amyloid aggregates lose this property. Thus, while Cnm contributes to recognition and initial binding of S. mutans to collagen-rich surfaces, amyloid formation by Cnm might act as a negative regulatory mechanism to modulate collagen-binding activity within S. mutans biofilms and warrants further investigation. IMPORTANCE Streptococcus mutans is a keystone pathogen that promotes caries by acidifying the dental biofilm milieu. The collagen- and laminin-binding glycoprotein Cnm is a virulence factor of S. mutans. Expression of Cnm by S. mutans is hypothesized to contribute to niche expansion, allowing colonization of multiple sites in the body, including collagen-rich surfaces such as dentin and heart valves. Here, we suggest that Cnm function might be modulated by its aggregation status. As a monomer, its primary function is to promote attachment to collagenous substrates via its collagen-binding domain (CBD). However, in later stages of biofilm maturation, the same CBD of Cnm could self-assemble into amyloid fibrils, losing the ability to bind to collagen and likely becoming a component of the biofilm matrix. Our findings shed light on the role of functional amyloids in S. mutans pathobiology and ecology.

c-di-AMP Is Essential for the Virulence of Enterococcus faecalis.

Second messenger nucleotides are produced by bacteria in response to environmental stimuli and play a major role in the regulation of processes associated with bacterial fitness, including but not limited to osmoregulation, envelope homeostasis, central metabolism, and biofilm formation. In this study, we uncovered the biological significance of c-di-AMP in the opportunistic pathogen Enterococcus faecalis by isolating and characterizing strains lacking genes responsible for c-di-AMP synthesis (cdaA) and degradation (dhhP and gdpP). Using complementary approaches, we demonstrated that either complete loss of c-di-AMP (ΔcdaA strain) or c-di-AMP accumulation (ΔdhhP, ΔgdpP, and ΔdhhP ΔgdpP strains) drastically impaired general cell fitness and virulence of E. faecalis. In particular, the ΔcdaA strain was highly sensitive to envelope-targeting antibiotics, was unable to multiply and quickly lost viability in human serum or urine ex vivo, and was virtually avirulent in an invertebrate (Galleria mellonella) and in two catheter-associated mouse infection models that recapitulate key aspects of enterococcal infections in humans. In addition to evidence linking these phenotypes to altered activity of metabolite and peptide transporters and inability to maintain osmobalance, we found that the attenuated virulence of the ΔcdaA strain also could be attributed to a defect in Ebp pilus production and activity that severely impaired biofilm formation under both in vitro and in vivo conditions. Collectively, these results demonstrate that c-di-AMP signaling is essential for E. faecalis pathogenesis and a desirable target for drug development.

Increased Oxidative Stress Tolerance of a Spontaneously Occurring perR Gene Mutation in Streptococcus mutans UA159.

The ability of bacteria, such as the dental pathogen Streptococcus mutans, to coordinate a response against damage-inducing oxidants is a critical aspect of their pathogenicity. The oxidative stress regulator SpxA1 has been demonstrated to be a major player in the ability of S. mutans to withstand both disulfide and peroxide stresses. While studying spontaneously occurring variants of an S. mutans ΔspxA1 strain, we serendipitously discovered that our S. mutans UA159 host strain bore a single-nucleotide deletion within the coding region of perR, resulting in a premature truncation of the encoded protein. PerR is a metal-dependent transcriptional repressor that senses and responds to peroxide stress such that loss of PerR activity results in activation of oxidative stress responses. To determine the impact of loss of PerR regulation, we obtained a UA159 isolate bearing an intact perR copy and created a clean perR deletion mutant. Our findings indicate that loss of PerR activity results in a strain that is primed to tolerate oxidative stresses in the laboratory setting. Interestingly, RNA deep sequencing (RNA-Seq) and targeted transcriptional expression analyses reveal that PerR offers a minor contribution to the ability of S. mutans to orchestrate a transcriptional response to peroxide stress. Furthermore, we detected loss-of-function perR mutations in two other commonly used laboratory strains of S. mutans, suggesting that this may be not be an uncommon occurrence. This report serves as a cautionary tale regarding the so-called domestication of laboratory strains and advocates for the implementation of more stringent strain authentication practices.IMPORTANCE A resident of the human oral biofilm, Streptococcus mutans is one of the major bacterial pathogens associated with dental caries. This report highlights a spontaneously occurring mutation within the laboratory strain S. mutans UA159 found in the coding region of perR, a gene encoding a transcriptional repressor associated with peroxide tolerance. Though perR mutant strains of S. mutans showed a distinct growth advantage and enhanced tolerance toward H2O2, a ΔperR deletion strain showed a small number of differentially expressed genes compared to the parent strain, suggesting few direct regulatory targets. In addition to characterizing the role of PerR in S. mutans, our findings serve as a warning to laboratory researchers regarding bacterial adaptation to in vitro growth conditions.

Detection of Streptococcus mutans in symptomatic and asymptomatic infected root canals.

OBJECTIVES: To investigate the presence of Streptococcus mutans in root canals of symptomatic necrotic teeth (SNT) and their associated acute apical abscesses (AAA) and in the root canals of asymptomatic necrotic teeth (ANT). It also aimed to investigate the presence of the cnm and cbm genes in specimens that harbored S. mutans. MATERIALS AND METHODS: DNA was extracted from samples collected from 10 patients presenting pulpal necrosis associated with radiographic evidence of apical periodontitis (ANT) and from 10 patients in need of endodontic therapy due to the presence of pulpal necrosis (SNT) and AAA. The control group consisted of 10 patients with teeth with normal vital pulp and requiring endodontic treatment for prosthetic reasons. The presence of S. mutans was detected by quantitative real-time-PCR (qPCR) using species-specific primers. Samples harboring S. mutans were further evaluated for the presence of CBP genes by qPCR as well. RESULTS: All studied sites showed a high prevalence of S. mutans, except the control group. Specifically, 60% of ANT and 70% of AAA/SNT paired samples were positive for S. mutans. The cnm gene was detected positive for S. mutans only in ANT samples (66.6%). The cbm gene was not detected in any of the investigated sites. CONCLUSIONS: S. mutans was found in high prevalence in both asymptomatic and symptomatic endodontic infections, including in abscesses, but it was not detected in the root canals of teeth with normal vital pulp. Interestingly, cnm+ S. mutans was only detected in asymptomatic/chronic primary endodontic infections associated with apical lesion. Therefore, it appears that cnm, and possibly other CBPs, may play an underestimated role in chronic endodontic infections. CLINICAL RELEVANCE: A high prevalence of Streptococcus mutans cnm+ gene was detected only in asymptomatic primary endodontic infections associated with apical lesion. Therefore, it appears that this collagen-binding protein gene plays an underestimated role in asymptomatic/chronic endodontic infections.

Functional Analysis of the Collagen Binding Proteins of Streptococcus parasanguinis FW213.

Streptococcus parasanguinis is a dominant isolate of dental plaque and an opportunistic pathogen associated with subacute endocarditis. As the expression of collagen binding proteins (CBPs) could promote the establishment of S. parasanguinis in the host, the functions of three putative CBP-encoding loci, Spaf_0420, Spaf_1570, and Spaf_1573, were analyzed using isogenic mutant strains. It was revealed that S. parasanguinis FW213 bound effectively to fibronectin and type I collagen, but the strain's affinity for laminin and type IV collagen was quite low. By using various deletion derivatives, it was found that these three loci mediated the binding of S. parasanguinis to multiple extracellular matrix molecules, with type I collagen as the common substrate. Derivative strains with a deletion in any of the three loci expressed reduced binding to trypsin-treated swine heart valves. The deletion of these loci also reduced the viable count of S. parasanguinis bacteria within macrophages, especially the loss of Spaf_0420, but only strains with deletions in Spaf_0420 and Spaf_1570 expressed reduced virulence in the Galleria mellonella larva model. The deletion of Spaf_1570 and Spaf_1573 affected mainly the structure, but not the overall mass, of biofilm cultures in a flow cell system. Thus, CBPs are likely to be more critical for the initial colonization of S. parasanguinis on host tissues during the development of endocarditis.IMPORTANCE Bacteria generally can utilize multiple adhesins to establish themselves in the host. We found that Streptococcus parasanguinis, a dominant oral commensal and an opportunistic pathogen for subacute endocarditis, possesses at least three collagen-binding proteins that enable S. parasanguinis to successfully colonize damaged heart tissues and escape innate immune clearance. The binding specificities of these three proteins for extracellular matrix molecules differ, although all three proteins participate in biofilm formation by S. parasanguinis The "multiligand for multisubstrate" feature of these adhesins may explain the high adaptability of this microbe to different tissue sites.

Phenotypic and Genotypic Characterization of Streptococcus mutans Strains Isolated from Endodontic Infections.

Streptococcus mutans plays an important role in caries etiology and eventually in systemic infections. However, it is often found in infected root canals, but the pathophysiological characteristics of strains residing in this site are largely unknown. Here, we characterized strains of S. mutans isolated from root canals of primary (PI) and secondary/persistent (SI) endodontic infections in relation to serotype and genotype; presence of genes coding for collagen binding proteins (CBPs); collagen binding activity and biofilm formation capacity; ability to withstand environmental stresses; systemic virulence in Galleria mellonella; and invasion of human coronary artery endothelial cells and human dental pupal fibroblasts. Samples from 10 patients with PI and 10 patients with SI were collected, and a total of 14 S. mutans isolates, belonging to 3 genotypes, were obtained. Of these, 13 were serotype c, and 1 was serotype k. When compared with the reference strains, the clinical isolates were hypersensitive to hydrogen peroxide. Remarkably, all 14 strains harbored and expressed the CBP-encoding gene cbm, showing increased binding to collagen, enhanced systemic virulence in G. mellonella, and ability to invade human coronary artery endothelial cells and human dental pupal fibroblasts when compared with CBP-negative strains. Whole genome sequence analysis of PI and SI isolates revealed that these strains are phylogenetically related but genetically distinct from each other. Our findings highlight the importance of CBPs in facilitating colonization and persistence of S. mutans in collagenous substrates such as root canals and their potential role in the pathogenesis of endodontic infections.

PepO is a target of the two-component systems VicRK and CovR required for systemic virulence of Streptococcus mutans.

Streptococcus mutans, a cariogenic species, is often associated with cardiovascular infections. Systemic virulence of specific S. mutans serotypes has been associated with the expression of the collagen- and laminin-binding protein Cnm, which is transcriptionally regulated by VicRK and CovR. In this study, we characterized a VicRK- and CovR-regulated gene, pepO, coding for a conserved endopeptidase. Transcriptional and protein analyses revealed that pepO is highly expressed in S. mutans strains resistant to complement immunity (blood isolates) compared to oral isolates. Gel mobility assay, transcriptional, and Western blot analyses revealed that pepO is repressed by VicR and induced by CovR. Deletion of pepO in the Cnm+ strain OMZ175 (OMZpepO) or in the Cnm- UA159 (UApepO) led to an increased susceptibility to C3b deposition, and to low binding to complement proteins C1q and C4BP. Additionally, pepO mutants showed diminished ex vivo survival in human blood and impaired capacity to kill G. mellonella larvae. Inactivation of cnm in OMZ175 (OMZcnm) resulted in increased resistance to C3b deposition and unaltered blood survival, although both pepO and cnm mutants displayed attenuated virulence in G. mellonella. Unlike OMZcnm, OMZpepO could invade HCAEC endothelial cells. Supporting these phenotypes, recombinant proteins rPepO and rCnmA showed specific profiles of binding to C1q, C4BP, and to other plasma (plasminogen, fibronectin) and extracellular matrix proteins (type I collagen, laminin). Therefore this study identifies a novel VicRK/CovR-target required for immune evasion and host persistence, pepO, expanding the roles of VicRK and CovR in regulating S. mutans virulence.

Regulatory circuits controlling Spx levels in Streptococcus mutans.

Spx is a major regulator of stress responses in Firmicutes. In Streptococcus mutans, two Spx homologues, SpxA1 and SpxA2, were identified as mediators of oxidative stress responses but the regulatory circuits controlling their levels and activity are presently unknown. Comparison of SpxA1 and SpxA2 protein sequences revealed differences at the C-terminal end, with SpxA1 containing an unusual number of acidic residues. Here, we showed that a green fluorescence protein (GFP) reporter becomes unstable when fused to the last 10 amino acids of SpxA2 but remained stable when fused to the C-terminal acidic tail of SpxA1. Inactivation of clpP or simultaneous inactivation of clpC and clpE stabilized the GFP::SpxA2tail fusion protein. Addition of acidic amino acids to the GFP::SpxA2tail chimera stabilized GFP, while deletion of the acidic residues destabilized GFP::SpxA1tail . Promoter reporter fusions revealed that spxA1 transcription is co-repressed by the metalloregulators PerR and SloR while spxA2 transcription is largely dependent on the envelope stress regulator LiaFSR. In agreement with spxA2 being part of the LiaR regulon, SpxA2 was found to be critical for the growth of S. mutans under envelope stress conditions. Finally, we showed that redox sensing is essential for SpxA1-dependent activation of oxidative stress responses but dispensable for SpxA2-mediated envelope stress responses.

Manganese Uptake, Mediated by SloABC and MntH, Is Essential for the Fitness of Streptococcus mutans.

Early epidemiological studies implicated manganese (Mn) as a possible caries-promoting agent, while laboratory studies have indicated that manganese stimulates the expression of virulence-related factors in the dental pathogen Streptococcus mutans To better understand the importance of manganese homeostasis to S. mutans pathophysiology, we first used RNA sequencing to obtain the global transcriptional profile of S. mutans UA159 grown under Mn-restricted conditions. Among the most highly expressed genes were those of the entire sloABC operon, encoding a dual iron/manganese transporter, and an uncharacterized gene, here mntH, that codes for a protein bearing strong similarity to Nramp-type transporters. While inactivation of sloC, which encodes the lipoprotein receptor of the SloABC system, or of mntH alone had no major consequence for the overall fitness of S. mutans, simultaneous inactivation of sloC and mntH (ΔsloC ΔmntH) impaired growth and survival under Mn-restricted conditions, including in human saliva or in the presence of calprotectin. Further, disruption of Mn transport resulted in diminished stress tolerance and reduced biofilm formation in the presence of sucrose. These phenotypes were markedly improved when cells were provided with excess Mn. Metal quantifications revealed that the single mutant strains contained intracellular levels of Mn similar to those seen with the parent strain, whereas Mn was nearly undetectable in the ΔsloC ΔmntH strain. Collectively, these results reveal that SloABC and MntH work independently and cooperatively to promote cell growth under Mn-restricted conditions and that maintenance of Mn homeostasis is essential for the expression of major virulence attributes in S. mutansIMPORTANCE As transition biometals such as manganese (Mn) are essential for all forms of life, the ability to scavenge biometals in the metal-restricted host environment is an important trait of successful cariogenic pathobionts. Here, we showed that the caries pathogen Streptococcus mutans utilizes two Mn transport systems, namely, SloABC and MntH, to acquire Mn from the environment and that the ability to maintain the cellular levels of Mn is important for the manifestation of characteristics that associate S. mutans with dental caries. Our results indicate that the development of strategies to deprive S. mutans of Mn hold promise in the combat against this important bacterial pathogen.

CovR and VicRKX Regulate Transcription of the Collagen Binding Protein Cnm of Streptococcus mutans.

Cnm is a surface-associated protein present in a subset of Streptococcus mutans strains that mediates binding to extracellular matrices, intracellular invasion, and virulence. Here, we showed that cnm transcription is controlled by the global regulators CovR and VicRKX. In silico analysis identified multiple putative CovR- and VicR-binding motifs in the regulatory region of cnm as well as in the downstream gene pgfS, which is associated with the posttranslational modification of Cnm. Electrophoretic mobility shift assays revealed that CovR and VicR specifically and independently bind to the cnm and pgfS promoter regions. Quantitative real-time PCR and Western blot analyses of ΔcovR and ΔvicK strains as well as of a strain overexpressing vicRKX revealed that CovR functions as a positive regulator of cnm, whereas VicRKX acts as a negative regulator. In agreement with the role of VicRKX as a repressor, the ΔvicK strain showed enhanced binding to collagen and laminin and higher intracellular invasion rates. Overexpression of vicRKX was associated with decreased rates of intracellular invasion but did not affect collagen or lamin binding activities, suggesting that this system controls additional genes involved in binding to these extracellular matrix proteins. As expected, based on the role of CovR in cnm regulation, the ΔcovR strain showed decreased intracellular invasion rates, but, unexpectedly collagen and laminin binding activities were increased in this mutant strain. Collectively, the results presented here expand the repertoire of virulence-related genes regulated by CovR and VicRKX to include the core gene pgfS and the noncore gene cnmIMPORTANCEStreptococcus mutans is a major pathogen associated with dental caries and also implicated in systemic infections, in particular, infective endocarditis. The Cnm adhesin of S. mutans is an important virulence factor associated with systemic infections and caries severity. Despite its role in virulence, the regulatory mechanisms governing cnm expression are poorly understood. Here, we describe the identification of two independent regulatory systems controlling the transcription of cnm and the downstream pgfS-pgfM1-pgfE-pgfM2 operon. A better understanding of the mechanisms controlling expression of virulence factors like Cnm can facilitate the development of new strategies to treat bacterial infections.

Disruption of a Novel Iron Transport System Reverses Oxidative Stress Phenotypes of a dpr Mutant Strain of Streptococcus mutans.

The Dps-like peroxide resistance protein (Dpr) is essential for H2O2 stress tolerance and aerobic growth of the oral pathogen Streptococcus mutans Dpr accumulates during oxidative stress, protecting the cell by sequestering iron ions and thereby preventing the generation of toxic hydroxyl radicals that result from the interaction of iron with H2O2 Previously, we reported that the SpxA1 and SpxA2 regulators positively regulate expression of dpr in S. mutans Using an antibody raised against S. mutans Dpr, we confirmed at the protein level the central and cooperative nature of SpxA1 and SpxA2 regulation in Dpr production. During phenotypic characterization of the S. mutans Δdpr strain, we observed the appearance of distinct colony variants, which sometimes lost the oxidative stress sensitivity typical of Δdpr strains. Whole-genome sequencing of these phenotypically distinct Δdpr isolates revealed that a putative iron transporter operon, smu995-smu998, was a genomic hot spot with multiple single nucleotide polymorphisms identified within the different isolates. Deletion of smu995 or the entire smu995-smu998 operon in the Δdpr background strain completely reversed the oxidative stress-sensitive phenotypes associated with dpr inactivation. Conversely, inactivation of genes encoding the ferrous iron transport system FeoABC did not alleviate phenotypes of the Δdpr strain. Preliminary characterization of strains lacking smu995-smu998, feoABC, and the iron/manganese transporter gene sloABC revealed the interactive nature of these three systems in iron transport but also indicated that there may be additional iron uptake systems in S. mutansIMPORTANCE The dental caries-associated pathogen Streptococcus mutans routinely encounters oxidative stress within the human plaque biofilm. Previous studies revealed that the iron-binding protein Dpr confers protection toward oxidative stress by limiting free iron availability, which is associated with the generation of toxic hydroxyl radicals. Here, we report the identification of spontaneously occurring mutations within Δdpr strains. Several of those mutations were mapped to the operon smu995-smu998, revealing a previously uncharacterized system that appears to be important in iron acquisition. Disruption of the smu995-smu998 operon resulted in reversion of the stress-sensitive phenotype typical of a Δdpr strain. Our data suggest that the Smu995-Smu998 system works along with other known metal transport systems of S. mutans, i.e., FeoABC and SloABC, to coordinate iron uptake.

Characterization of the pgf operon involved in the posttranslational modification of Streptococcus mutans surface proteins.

Protein glycosylation has been described as the most abundant and complex post-translational modification occurring in nature. Recent studies have enhanced our view of how this modification occurs in bacteria highlighting the role of protein glycosylation in various processes such as biofilm formation, virulence and host-microbe interactions. We recently showed that the collagen- and laminin-binding adhesin Cnm of the dental pathogen Streptococcus mutans is post-translationally modified by the PgfS glycosyltransferase. Following this initial identification of Cnm as a glycoprotein, we have now identified additional genes (pgfM1, pgfE and pgfM2) that are also involved in the posttranslational modification of Cnm. Similar to the previously characterized ΔpgfS strain, inactivation of pgfM1, pgfE or pgfM2 directly impacts Cnm by altering its migration pattern, proteolytic stability and function. In addition, we identified the wall-associated protein A (WapA) as an additional substrate of Pgf-dependent modification. We conclude that the pgS-pgfM1-pgfE-pgfM2 operon encodes for a protein machinery that can modify, likely through the addition of glycans, both core and non-core gene products in S. mutans.

Transcriptome responses of Streptococcus mutans to peroxide stress: identification of novel antioxidant pathways regulated by Spx.

The oxidative stress regulator Spx is ubiquitously found among Gram-positive bacteria. Previously, we reported identification of two Spx proteins in Streptococcus mutans - SpxA1 was the primary activator of oxidative stress genes whereas SpxA2 served a backup role. Here, we used RNA sequencing to uncover the scope of the H2O2 (peroxide)-stress regulon and to further explore the significance of Spx regulation in S. mutans. The transcriptome data confirmed the relationship between Spx and genes typically associated with oxidative stress, but also identified novel genes and metabolic pathways controlled by Spx during peroxide stress. While individual inactivation of newly identified peroxide stress genes had modest or no obvious consequences to bacterial survival, a phenotype enhancement screen using the ∆spxA1 strain as background for creation of double mutants revealed that four of the five genes inactivated were required for stress survival. Physiological and biochemical assays validated, at least in part, the transcriptome data indicating that SpxA1 coordinates transcriptional changes during peroxide stress that modify global metabolism and facilitate production of antioxidants. Collectively, our findings unraveled the scope of the peroxide stress regulon and expand the repertoire of oxidative stress genes in S. mutans, shedding new light on the role of Spx regulation.

Pro-inflammatory Analysis of Macrophages in Contact with Titanium Particles and Porphyromonas gingivalis.

During insertion of titanium dental implants, particles may shear from the implant to the periimplant region causing osteolysis, and their association with bacteria can exacerbate the inflammatory reaction. However, the association of a high invasive bacterium from the oral cavity, Porphyromonas gingivalis (Pg), and titanium particles remains unknown. This study evaluated pro-inflammatory reaction of human macrophages in contact with micro and nanoparticles of titanium associated with Porphyromonas gingivalis lipopolysaccharide (PgLPS). THP-1 cell were used and treated for 12, 24 and 48 h following 6 groups: Control(C), PgLPS (L); Microparticles (M); Nanoparticles (N); PgLPS and microparticles (LM); PgLPS and nanoparticles (LN). The following assays were carried out: i) cell viability using MTS, ii) cell morphology by SEM and iii) expression of tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1ß) and interleukin-6 (IL-6) by qRT-PCR and ELISA. For statistics two-way ANOVA followed by Tukey's test was used (p<0.05). After treatment, cells presented similar viability and morphology demonstrating that the treatments were not able to induce cell death. Gene expression was significantly higher for TNF-α and IL1-ß after 12 h, and for IL-6 after 24 h in the N and LN groups. Cytokine production over time was an ascending curve for TNF-α with the peak at 48 h and IL1-ß and IL-6 had a straight line among the time points, although cells from N group presented a significant production of IL-6 at 48 h. In conclusion, these results suggest that titanium nanoparticles stimulate stronger pro-inflammatory response in macrophages, independent of their association with LPS from P.gingivalis.