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.

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.

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.

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.

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.

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.

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.

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.

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.

Transcription of Oxidative Stress Genes Is Directly Activated by SpxA1 and, to a Lesser Extent, by SpxA2 in Streptococcus mutans.

UNLABELLED: The SpxA1 and SpxA2 (formerly SpxA and SpxB) transcriptional regulators of Streptococcus mutans are members of a highly conserved family of proteins found in Firmicutes, and they were previously shown to activate oxidative stress responses. In this study, we showed that SpxA1 exerts substantial positive regulatory influence over oxidative stress genes following exposure to H2O2, while SpxA2 appears to have a secondary regulatory role. In vitro transcription (IVT) assays using purified SpxA1 and/or SpxA2 showed that SpxA1 and, less often, SpxA2 directly activate transcription of some of the major oxidative stress genes. Addition of equimolar concentrations of SpxA1 and SpxA2 to the IVT reactions neither enhanced transcription of the tested genes nor disrupted the dominant role of SpxA1. Substitution of a conserved glycine residue (G52) present in both Spx proteins by arginine (SpxG52R) resulted in strains that phenocopied the Δspx strains. Moreover, addition of purified SpxA1G52R completely failed to activate transcription of ahpC, sodA, and tpx, further confirming that the G52 residue is critical for Spx functionality. IMPORTANCE: Streptococcus mutans is a pathogen associated with the formation of dental caries in humans. Within the oral cavity, S. mutans routinely encounters oxidative stress. Our previous data revealed that two regulatory proteins, SpxA1 and SpxA2 (formerly SpxA and SpxB), bear high homology to the Spx regulator that has been characterized as a critical activator of oxidative stress genes in Bacillus subtilis. In this report, we prove that Spx proteins of S. mutans directly activate transcription of genes involved in the oxidative stress response, though SpxA1 appears to have a more dominant role than SpxA2. Therefore, the Spx regulators play a critical role in the ability of S. mutans to thrive within the oral cavity.

The collagen binding protein Cnm contributes to oral colonization and cariogenicity of Streptococcus mutans OMZ175.

Streptococcus mutans is the etiological agent of dental caries and one of the many bacterial species implicated in infective endocarditis. The expression of the collagen-binding protein Cnm by S. mutans has been associated with extraoral infections, but its relevance for dental caries has only been theorized to date. Due to the collagenous composition of dentinal and root tissues, we hypothesized that Cnm may facilitate the colonization of these surfaces, thereby enhancing the pathogenic potential of S. mutans in advancing carious lesions. As shown for extraoral endothelial cell lines, Cnm mediates the invasion of oral keratinocytes and fibroblasts by S. mutans. In this study, we show that in the Cnm(+) native strain, OMZ175, Cnm mediates stringent adhesion to dentinal and root tissues as well as collagen-coated surfaces and promotes both cariogenicity and carriage in vivo. In vitro, ex vivo, and in vivo experiments revealed that while Cnm is not universally required for S. mutans cariogenicity, it contributes to (i) the invasion of the oral epithelium, (ii) enhanced binding on collagenous surfaces, (iii) implantation of oral biofilms, and (IV) the severity of caries due to a native Cnm(+) isolate. Taken together, our findings reveal that Cnm is a colonization factor that contributes to the pathogenicity of certain S. mutans strains in their native habitat, the oral cavity.

Modification of Streptococcus mutans Cnm by PgfS contributes to adhesion, endothelial cell invasion, and virulence.

Expression of the surface protein Cnm has been directly implicated in the ability of certain strains of Streptococcus mutans to bind to collagen and to invade human coronary artery endothelial cells (HCAEC) and in the killing of Galleria mellonella. Sequencing analysis of Cnm(+) strains revealed that cnm is located between the core genes SMU.2067 and SMU.2069. Reverse transcription-PCR (RT-PCR) analysis showed that cnm is cotranscribed with SMU.2067, encoding a putative glycosyltransferase referred to here as PgfS (protein glycosyltransferase of streptococci). Notably, Cnm contains a threonine-rich domain predicted to undergo O-linked glycosylation. The previously shown abnormal migration pattern of Cnm, the presence of the threonine-rich domain, and the molecular linkage of cnm with pgfS lead us to hypothesize that PgfS modifies Cnm. A ΔpgfS strain showed defects in several traits associated with Cnm expression, including collagen binding, HCAEC invasion, and killing of G. mellonella. Western blot analysis revealed that Cnm from the ΔpgfS mutant migrated at a lower molecular weight than that from the parent strain. In addition, Cnm produced by ΔpgfS was highly susceptible to proteinase K degradation, in contrast to the high-molecular-weight Cnm version found in the parent strain. Lectin-binding analyses confirmed the glycosylated nature of Cnm and strongly suggested the presence of N-acetylglucosamine residues attached to Cnm. Based on these findings, the phenotypes observed in ΔpgfS are most likely associated with defects in Cnm glycosylation that affects protein function, stability, or both. In conclusion, this study demonstrates that Cnm is a glycoprotein and that posttranslational modification mediated by PgfS contributes to the virulence-associated phenotypes linked to Cnm.

Streptococcus mutans: a new Gram-positive paradigm?

Despite the enormous contributions of the bacterial paradigms Escherichia coli and Bacillus subtilis to basic and applied research, it is well known that no single organism can be a perfect representative of all other species. However, given that some bacteria are difficult, or virtually impossible, to cultivate in the laboratory, that some are recalcitrant to genetic and molecular manipulation, and that others can be extremely dangerous to manipulate, the use of model organisms will continue to play an important role in the development of basic research. In particular, model organisms are very useful for providing a better understanding of the biology of closely related species. Here, we discuss how the lifestyle, the availability of suitable in vitro and in vivo systems, and a thorough understanding of the genetics, biochemistry and physiology of the dental pathogen Streptococcus mutans have greatly advanced our understanding of important areas in the field of bacteriology such as interspecies biofilms, competence development and stress responses. In this article, we provide an argument that places S. mutans, an organism that evolved in close association with the human host, as a novel Gram-positive model organism.

Psr is involved in regulation of glucan production, and double deficiency of BrpA and Psr is lethal in Streptococcus mutans.

Streptococcus mutans, the primary causative agent of dental caries, contains two paralogues of the LytR-CpsA-Psr family proteins encoded by brpA and psr, respectively. Previous studies have shown that BrpA plays an important role in cell envelope biogenesis/homeostasis and affects stress responses and biofilm formation by Strep. mutans, traits critical to cariogenicity of this bacterium. In this study, a Psr-deficient mutant, TW251, was constructed. Characterization of TW251 showed that deficiency of Psr did not have any major impact on growth rate. However, when subjected to acid killing at pH 2.8, the survival rate of TW251 was decreased dramatically compared with the parent strain UA159. In addition, TW251 also displayed major defects in biofilm formation, especially during growth with sucrose. When compared to UA159, the biofilms of TW251 were mainly planar and devoid of extracellular glucans. Real-time-PCR and Western blot analyses revealed that deficiency of Psr significantly decreased the expression of glucosyltransferase C, a protein known to play a major role in biofilm formation by Strep. mutans. Transmission electron microscopy analysis showed that deficiency of BrpA caused alterations in cell envelope and cell division, and the most significant defects were observed in TW314, a Psr-deficient and BrpA-down mutant. No such effects were observed with Psr mutant TW251 under similar conditions. These results suggest that while there are similarities in functions between BrpA and Psr, distinctive differences also exist between these two paralogues. Like Bacillus subtilis but different from Staphylococcus aureus, a functional BrpA or Psr is required for viability in Strep. mutans.

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.

The Spx regulator modulates stress responses and virulence in Enterococcus faecalis.

The ability to cope with endogenous or host-generated reactive oxygen species is considered a key virulence attribute of the opportunistic pathogen Enterococcus faecalis, a leading cause of hospital-acquired infections. In this study, we used in silico and mutational analyses to identify and characterize the role of the Spx global regulator in oxidative stress tolerance and virulence in E. faecalis. While the Δspx strain grew as well as the wild-type strain under anaerobic conditions, the mutant strain exhibited impaired growth under aerobic conditions and was highly sensitive to oxidative stress agents. The spx mutant strain was also sensitive to a variety of other stressful conditions, including antibiotic stress and killing by the mouse-derived macrophage cell line J774. Using a murine model of foreign body-associated peritonitis, we demonstrated that the ability of the Δspx strain to colonize the peritoneum and disseminate in the bloodstream was significantly reduced compared to that of the parent strain. Transcriptional analysis revealed that a large number of known oxidative stress genes are under positive control by Spx. Collectively, our results show that Spx is a major stress gene regulator and is implicated in the pathophysiology of E. faecalis. The relationship of Spx to other oxidative stress regulators is also discussed.

Global transcriptional analysis of the stringent response in Enterococcus faecalis.

In Enterococcus faecalis, production of guanosine tetraphosphate/guanosine pentaphosphate [(p)ppGpp], the effector molecule of the stringent response, is controlled by the bifunctional synthetase/hydrolase RelA and the monofunctional synthetase RelQ. Previously, the (p)ppGpp profiles of strains lacking relA, relQ or both genes indicated that RelA is the primary enzyme responsible for (p)ppGpp synthesis under stress conditions, while the contributions of RelQ to the stringent response and cell homeostasis remained elusive. Here, survival within the mouse-derived macrophage cell line J774A.1 and killing of Galleria mellonella supported initial evidence that virulence was attenuated in the (p)ppGpp(0) ΔrelAΔrelQ strain but not in the ΔrelA or ΔrelQ strains. We performed, for the first time to our knowledge, global transcriptome analysis in a documented (p)ppGpp(0) Gram-positive bacterium and provided the first insights into the role of a Gram-positive monofunctional (p)ppGpp synthetase in transcriptional regulation. Transcription profiling after mupirocin treatment confirmed that RelA is the major enzyme responsible for the (p)ppGpp-mediated transcriptional repression of genes associated with macromolecular biosynthesis, but also revealed that RelQ is required for full and timely stringent response induction. The delayed transcriptional response of ΔrelQ could not be correlated with reduced or slower production of (p)ppGpp, in part because RelA-dependent (p)ppGpp accumulation occurred very rapidly. Comparisons of the transcriptional responses of ΔrelA or ΔrelAΔrelQ strains with the parent strain under starvation conditions revealed upregulation of operons involved in energy metabolism in the (p)ppGpp(0) strain. Thus, while ΔrelA and ΔrelAΔrelQ cannot use (p)ppGpp to sense and respond to stresses, fitness of ΔrelAΔrelQ may be further impaired due to an unbalanced metabolism.

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.