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Streptococcus mutansis a keystone pathogen of dental caries, and the ability to form biofilms is essential for its pathogenicity. We identified a glycosylation machinery (Pgf) inS. mutansthat post-translationally modifies two surface-associated adhesins, Cnm and WapA. The fourpgfgenes (pgfS,pgfM1,pgfE,andpgfM2) are part ofS. mutanscore genome and we hypothesized that the scope of Pgf goes beyond Cnm and WapA. By inactivating eachpgfgene individually or creating a quadruplepgfmutant inS. mutansOMZ175, we showed that the Pgf machinery is important for biofilm formation. Compared to OMZ175, differences in surface charge, membrane stability, and genetic competence were also observed for most mutants. Importantly,in silicoanalyses and tunicamycin MIC assays suggest a functional redundancy between the Pgf machinery and the rhamnose-glucose polysaccharide synthesis pathway. Using a rat oral colonization model, we showed a 10-fold reduction in recovered CFUs for thepgfquadruple mutant compared to OMZ175. Finally, using Cnm as a model, we showed by glycoproteomics analyses that Cnm is heavily modified with N-acetyl hexosamine in OMZ175 whereas phosphorylations were observed for thepgfSmutant. Our findings indicate that the Pgf machinery participates in important aspects ofS. mutanspathobiology.Graphical AbstractAbbreviated summaryIn this study, we demonstrate that the Pgf glycosylation machinery ofStreptococcus mutans, a keystone pathogen of dental caries, regulates several aspects of bacterial pathophysiology that ultimately contribute toS. mutansfitness in oral colonization experiments. Using the heavily glycosylated Cnm adhesin as a model, we found that inactivation of the glycosyltransferase PgfS results in loss of Cnm glycosylation, but instead, Cnm became heavily phosphorylated, suggesting a crosstalk/competition between these two post-translational modification mechanisms.

SummaryTrace metals are essential to all domains of life but toxic when found at high concentrations. While the importance of iron in host-pathogen interactions is firmly established, contemporary studies indicate that other trace metals, including manganese and zinc, are also critical to the infectious process. In this study, we sought to identify and characterize the zinc uptake system(s) of S. mutans, a keystone pathogen in dental caries and a causative agent of bacterial endocarditis. Different than other pathogenic bacteria, including several streptococci, that encode multiple zinc import systems, bioinformatic analysis indicated that the S. mutans core genome encodes a single, highly conserved, zinc importer commonly known as AdcABC. Inactivation of the genes coding for the metal-binding AdcA (ΔadcA) or both AdcC ATPase and AdcB permease (ΔadcCB) severely impaired the ability of S. mutans to grow under zinc-depleted conditions. Intracellular metal quantifications revealed that both mutants accumulated less zinc when grown in the presence of a sub-inhibitory concentration of a zinc-specific chelator. Notably, the ΔadcCB strain displayed a severe colonization defect in a rat oral infection model. Both Δadc strains were hypersensitive to high concentrations of manganese, showed reduced peroxide tolerance, and formed less biofilm in sucrose-containing media when cultivated in the presence of the lowest amount of zinc that support their growth, but not when zinc was supplied in excess. Collectively, this study identifies AdcABC as the lone high affinity zinc importer of S. mutans and provides preliminary evidence that zinc is a growth-limiting factor within the dental biofilm.

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.

The Enterococci, mainly Enterococcus faecalis and E. faecium, are ubiquitous members of the human gastrointestinal tract consortia but also a leading cause of opportunistic infections. The global rise in human-associated enterococcal infections, often caused by multidrug resistant strains, highlights an urgent need to identify the bacterial factors contributing to its pathogenicity such that new therapies can be devised. The use of the Galleria mellonella (greater wax moth) larvae, commonly known as wax worm, as a model to study host-pathogen interactions has allowed the identification and characterization of numerous bacterial factors that contribute to disease in humans, serving both as an alternative and complementary approach to mammalian models. Here, we describe the methods for using G. mellonella to characterize the virulence factors of E. faecalis.

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.

Dental caries is often initiated by Streptococcus mutans , which establishes a biofilm and a low pH environment on tooth enamel surfaces. The current study has uncovered vulnerabilities of S. mutans mutant strains that are unable to produce the enzyme complex, acetoin dehydrogenase (Adh).

The agent largely responsible for initiating dental caries, Streptococcus mutans, produces acetoin dehydrogenase that is encoded by the adh operon. The operon consists of the adhA and B genes (E1 dehydrogenase), adhC (E2 lipoylated transacetylase), adhD (E3 dihydrolipoamide dehydrogenase), and lplA (lipoyl ligase). Evidence is presented that AdhC interacts with SpxA2, a redox-sensitive transcription factor functioning in cell wall and oxidative stress responses. In-frame deletion mutations of adh genes conferred oxygen-dependent sensitivity to slightly alkaline pH (pH 7.2–7.6), within the range of values observed in human saliva. Growth defects were also observed when glucose or sucrose served as major carbon sources. A deletion of the adhC orthologous gene, acoC gene of Streptococcus gordonii, did not result in pH sensitivity or defective growth in glucose and sucrose. The defects observed in adh mutants were partially reversed by addition of pyruvate. Unlike most 2-oxoacid dehydrogenases, the E3 AdhD subunit bears an N-terminal lipoylation domain nearly identical to that of E2 AdhC. Changing the lipoyl domains of AdhC and AdhD by replacing the lipoate attachment residue, lysine to arginine, caused no significant reduction in pH sensitivity but the adhDK43R mutation eliminating the lipoylation site resulted in an observable growth defect in glucose medium. The adh mutations were partially suppressed by a deletion of rex, encoding an NAD(+)/NADH-sensing transcription factor that represses genes functioning in fermentation. spxA2 adh double mutants show synthetic growth restriction at elevated pH and upon ampicillin treatment. These results suggest a role for Adh in stress management in S. mutans. IMPORTANCE Dental caries is often initiated by Streptococcus mutans, which establishes a biofilm and a low pH environment on tooth enamel surfaces. The current study has uncovered vulnerabilities of S. mutans mutant strains that are unable to produce the enzyme complex, acetoin dehydrogenase (Adh). Such mutants are sensitive to modest increases in pH to 7.2-7.6, within the range of human saliva, while a mutant of a commensal Streptococcal species is resistant. The S. mutans adh strains are also defective in carbohydrate utilization and are hypersensitive to a cell wall-acting antibiotic. The studies suggest that Adh could be a potential target for interfering with S. mutans colonization of the oral environment.

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 ΔdhhPΔgdpP 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 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 ΔcdaA could be also 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 reveal that c-di-AMP signaling is essential for E. faecalis pathogenesis and a desirable target for drug development.IMPORTANCEEnterococcus faecalis is an opportunistic pathogen and leading cause of multidrug resistant hospital-acquired infections. During the course of an infection, bacteria encounter multiple adverse (stress) conditions and understanding the adaptive mechanisms used by pathogens to survive these stresses can facilitate the development of new antimicrobial therapies. Here, we used in vitro, ex vivo and in vivo approaches to determine the importance of the second messenger nucleotide c-di-AMP, a global regulator essential for bacterial adaptation to osmotic stress, to E. faecalis pathophysiology. We demonstrated that either accumulation of c-di-AMP or complete loss of c-di-AMP impaired cell fitness and virulence of E. faecalis. Remarkably, the strain that was unable to produce c-di-AMP was avirulent in three animal infection models indicating that c-di-AMP signaling is essential for E. faecalis pathogenesis and a suitable antimicrobial target.

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.

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 proven 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 ofperR, 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 isolated 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-Seq and targeted transcriptional expression analyses reveal that PerR has 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 warning regarding the so-called domestication of laboratory strains and advocates for the implementation of more stringent strain authentication practices.ImportanceA 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 as 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.

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 H(2)O(2), 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.

We report the whole genome sequence of the serotype e Cbm+ strain LAR01 of Streptococcus mutans, a dental pathogen frequently associated with extra-oral infections. The LAR01 genome is a single circular chromosome of 2.1 Mb with a GC content of 36.96%. The genome contains 15 phosphotransferase system gene clusters, seven cell wall-anchored (LPxTG) proteins, all genes required for the development of natural competence and genes coding for mutacins VI and K8. Interestingly, the cbm gene is genetically linked to a putative type VII secretion system that has been found in Mycobacteria and few other Gram-positive bacteria. When compared with the UA159 type strain, phenotypic characterization of LAR01 revealed increased biofilm formation in the presence of either glucose or sucrose but similar abilities to withstand acid and oxidative stresses. LAR01 was unable to inhibit the growth of Strpetococcus gordonii, which is consistent with the genomic data that indicate absence of mutacins that can kill mitis streptococci. On the other hand, LAR01 effectively inhibited growth of other S. mutans strains, suggesting that it may be specialized to outcompete strains from its own species. In vitro and in vivo studies using mutational and heterologous expression approaches revealed that Cbm is a virulence factor of S. mutans by mediating binding to extracellular matrix proteins and intracellular invasion. Collectively, the whole genome sequence analysis and phenotypic characterization of LAR01 provides new insights on the virulence properties of S. mutans and grants further opportunities to understand the genomic fluidity of this important human pathogen.

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., 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. mutans. IMPORTANCE 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.

As a major etiological agent of human dental caries, Streptococcus mutans resides primarily in biofilms that form on the tooth surfaces, also known as dental plaque. In addition to caries, S. mutans is responsible for cases of infective endocarditis with a subset of strains being indirectly implicated with the onset of additional extraoral pathologies. During the past 4 decades, functional studies of S. mutans have focused on understanding the molecular mechanisms the organism employs to form robust biofilms on tooth surfaces, to rapidly metabolize a wide variety of carbohydrates obtained from the host diet, and to survive numerous (and frequent) environmental challenges encountered in oral biofilms. In these areas of research, S. mutans has served as a model organism for ground-breaking new discoveries that have, at times, challenged long-standing dogmas based on bacterial paradigms such as Escherichia coli and Bacillus subtilis . In addition to sections dedicated to carbohydrate metabolism, biofilm formation, and stress responses, this article discusses newer developments in S. mutans biology research, namely, how S. mutans interspecies and cross-kingdom interactions dictate the development and pathogenic potential of oral biofilms and how next-generation sequencing technologies have led to a much better understanding of the physiology and diversity of S. mutans as a species.

Bacteria belonging to the genus Streptococcus are the first inhabitants of the oral cavity, which can be acquired right after birth and thus play an important role in the assembly of the oral microbiota. In this article, we discuss the different oral environments inhabited by streptococci and the species that occupy each niche. Special attention is given to the taxonomy of Streptococcus , because this genus is now divided into eight distinct groups, and oral species are found in six of them. Oral streptococci produce an arsenal of adhesive molecules that allow them to efficiently colonize different tissues in the mouth. Also, they have a remarkable ability to metabolize carbohydrates via fermentation, thereby generating acids as byproducts. Excessive acidification of the oral environment by aciduric species such as Streptococcus mutans is directly associated with the development of dental caries. However, less acid-tolerant species such as Streptococcus salivarius and Streptococcus gordonii produce large amounts of alkali, displaying an important role in the acid-base physiology of the oral cavity. Another important characteristic of certain oral streptococci is their ability to generate hydrogen peroxide that can inhibit the growth of S. mutans . Thus, oral streptococci can also be beneficial to the host by producing molecules that are inhibitory to pathogenic species. Lastly, commensal and pathogenic streptococci residing in the oral cavity can eventually gain access to the bloodstream and cause systemic infections such as infective endocarditis.

The Dps-like peroxide resistance protein (Dpr) is essential for H 2 O 2 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 H 2 O 2 . 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 Δ dpr strain, we observed the appearance of distinct colony variants, which sometimes lost the oxidative stress sensitivity typical of Δ dpr strains. Whole-genome sequencing of these phenotypically distinct Δ dpr 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 Δ dpr 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 Δ dpr 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. mutans . IMPORTANCE 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 Δ dpr 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 Δ dpr 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.

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 Δ covR and Δ vicK 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 Δ vicK 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 Δ covR 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 cnm . IMPORTANCE Streptococcus 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.

The SpxA1 and SpxA2 (formerly SpxA and SpxB) transcriptional regulators ofStreptococcus mutansare members of a highly conserved family of proteins found inFirmicutes, 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 vitrotranscription (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 Δspxstrains. Moreover, addition of purified SpxA1G52Rcompletely failed to activate transcription ofahpC,sodA, andtpx, further confirming that the G52 residue is critical for Spx functionality.IMPORTANCEStreptococcus mutansis a pathogen associated with the formation of dental caries in humans. Within the oral cavity,S. mutansroutinely 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 inBacillus subtilis. In this report, we prove that Spx proteins ofS. mutansdirectly 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 ofS. mutansto thrive within the oral cavity.

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. mutans.

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.

NADH oxidase (Nox, encoded by nox ) is a flavin-containing enzyme used by the oral pathogen Streptococcus mutans to reduce diatomic oxygen to water while oxidizing NADH to NAD + . The critical nature of Nox is 2-fold: it serves to regenerate NAD + , a carbon cycle metabolite, and to reduce intracellular oxygen, preventing formation of destructive reactive oxygen species (ROS). As oxygen and NAD + have been shown to modulate the activity of the global transcription factors Spx and Rex, respectively, Nox is potentially poised at a critical junction of two stress regulons. In this study, microarray data showed that either addition of oxygen or loss of nox resulted in altered expression of genes involved in energy metabolism and transport and the upregulation of genes encoding ROS-metabolizing enzymes. Loss of nox also resulted in upregulation of several genes encoding transcription factors and signaling molecules, including the redox-sensing regulator gene rex . Characterization of the nox promoter revealed that nox was regulated by oxygen, through SpxA, and by Rex. These data suggest a regulatory loop in which the roles of nox in reduction of oxygen and regeneration of NAD + affect the activity levels of Spx and Rex, respectively, and their regulons, which control several genes, including nox , crucial to growth of S. mutans under conditions of oxidative stress.

Biofilms are comprised of bacterial-clusters (microcolonies) enmeshed in an extracellular matrix. Streptococcus mutans can produce exopolysaccharides (EPS)-matrix and assemble microcolonies with acidic microenvironments that can cause tooth-decay despite the surrounding neutral-pH found in oral cavity. How the matrix influences the pH and bacterial activity locally remains unclear. Here, we simultaneously analyzed in situ pH and gene expression within intact biofilms and measured the impact of damage to the surrounding EPS-matrix. The spatiotemporal changes of these properties were characterized at a single-microcolony level following incubation in neutral-pH buffer. The middle and bottom-regions as well as inner-section within the microcolony 3D structure were resistant to neutralization (vs. upper and peripheral-region), forming an acidic core. Concomitantly, we used a green fluorescent protein (GFP) reporter to monitor expression of the pH-responsive atpB (PatpB::gfp) by S. mutans within microcolonies. The atpB expression was induced in the acidic core, but sharply decreased at peripheral/upper microcolony regions, congruent with local pH microenvironment. Enzymatic digestion of the surrounding matrix resulted in nearly complete neutralization of microcolony interior and down-regulation of atpB. Altogether, our data reveal that biofilm matrix facilitates formation of an acidic core within microcolonies which in turn activates S. mutans acid-stress response, mediating both the local environment and bacterial activity in situ.

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 Δ spx 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 Δ spx 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.

The ClpXP proteolytic complex is critical for maintaining cellular homeostasis, as well as expression of virulence properties. However, with the exception of the Spx global regulator, the molecular mechanisms by which the ClpXP complex exerts its influence in Streptococcus mutans are not well understood. Here, microarray analysis was used to provide novel insights into the scope of ClpXP proteolysis in S. mutans. In a ΔclpP strain, 288 genes showed significant changes in relative transcript amounts (P≤0.001, twofold cut-off) as compared with the parent. Similarly, 242 genes were differentially expressed by a ΔclpX strain, 113 (47 %) of which also appeared in the ΔclpP microarrays. Several genes associated with cell growth were downregulated in both mutants, consistent with the slow-growth phenotype of the Δclp strains. Among the upregulated genes were those encoding enzymes required for the biosynthesis of intracellular polysaccharides (glg genes) and malolactic fermentation (mle genes). Enhanced expression of glg and mle genes in ΔclpP and ΔclpX strains correlated with increased storage of intracellular polysaccharide and enhanced malolactic fermentation activity, respectively. Expression of several genes known or predicted to be involved in competence and mutacin production was downregulated in the Δclp strains. Follow-up transformation efficiency and deferred antagonism assays validated the microarray data by showing that competence and mutacin production were dramatically impaired in the Δclp strains. Collectively, our results reveal the broad scope of ClpXP regulation in S. mutans homeostasis and identify several virulence-related traits that are influenced by ClpXP proteolysis.

Streptococcus mutans and Streptococcus sobrinus are considered the primary organisms responsible for human dental caries. The ability to generate acids and to adapt to low pH conditions is directly associated with the cariogenic potential of these bacteria. To survive acidic conditions, both species have been shown to mount an acid-tolerance response (ATR). However, previous characterization of the S. sobrinus ATR identified critical differences in the mechanisms of acid adaptation between S. mutans and S. sobrinus. Here, interspecies microarray and proteomic approaches were used to identify novel, previously unrecognized genes and pathways that participate in the S. sobrinus acid-stress response. The results revealed that, among other things, metabolic alterations that enhance energy generation and upregulation of the malolactic fermentation enzyme activity constitute important acid-resistance properties in S. sobrinus. Some of these acid adaptive traits are shared by S. mutans and might be considered optimal targets for therapeutic treatments designed to control dental caries.

The stringent response is a global bacterial response to stress that is mediated by accumulation of the alarmone (p)ppGpp. In this study, treatment with mupirocin was shown to induce high levels of (p)ppGpp production in Enterococcus faecalis , indicating that this nosocomial pathogen can mount a classic stringent response. In addition, (p)ppGpp was found to accumulate in cells subjected to heat shock, alkaline shock, and inhibitory concentrations of vancomycin. Sequence analysis of the E. faecalis genome indicated that (p)ppGpp synthesis is catalyzed by the bifunctional synthetase/hydrolase RelA and the RelQ small synthase. The (p)ppGpp profiles of Δ relA , Δ relQ , and Δ relAQ strains revealed that RelA is the major enzyme responsible for the accumulation of (p)ppGpp during antibiotic or physical stresses, while RelQ appears to be responsible for maintaining basal levels of alarmone during homeostatic growth. Compared to its parent, the Δ relA strain was more susceptible to several stress conditions, whereas complete elimination of (p)ppGpp in a Δ relAQ double mutant restored many of the stress-sensitive phenotypes of Δ relA . Interestingly, growth curves and time-kill studies indicated that tolerance to vancomycin is enhanced in the Δ relA strain but diminished in the Δ relQ and Δ relAQ strains. Finally, virulence of the Δ relAQ strain but not of the Δ relA or Δ relQ strain was significantly attenuated in the Caenorhabditis elegans model. Taken together, these results indicate that (p)ppGpp pools modulate environmental stress responses, vancomycin tolerance, and virulence in this important nosocomial pathogen.

Mutational analysis revealed that members of the Clp system, specifically the ClpL chaperone and the ClpXP proteolytic complex, modulate the expression of important virulence attributes of Streptococcus mutans . Compared to its parent, the Δ clpL strain displayed an enhanced capacity to form biofilms in the presence of sucrose, had reduced viability, and was more sensitive to acid killing. The Δ clpP and Δ clpX strains displayed several phenotypes in common: slow growth, tendency to aggregate in culture, reduced autolysis, and reduced ability to grow under stress, including acidic pH. Unexpectedly, the Δ clpP and Δ clpX mutants were more resistant to acid killing and demonstrated enhanced viability in long-term survival assays. Biofilm formation by the Δ clpP and Δ clpX strains was impaired when grown in glucose but enhanced in sucrose. In an animal study, the average number of S. mutans colonies recovered from the teeth of rats infected with the Δ clpP or Δ clpX strain was slightly lower than that of the parent strain. In Bacillus subtilis , the accumulation of the Spx global regulator, a substrate of ClpXP, has accounted for the Δ clpXP phenotypes. Searching the S. mutans genome, we identified two putative spx genes, designated spxA and spxB . The inactivation of either of these genes bypassed phenotypes of the clpP and clpX mutants. Western blotting demonstrated that Spx accumulates in the Δ clpP and Δ clpX strains. Our results reveal that the proteolysis of ClpL and ClpXP plays a role in the expression of key virulence traits of S. mutans and indicates that the underlying mechanisms by which ClpXP affect virulence traits are associated with the accumulation of two Spx orthologues.

In oral biofilms, two of the major environmental challenges encountered by the dental pathogen Streptococcus mutans are acid and oxidative stresses. Previously, we showed that the S. mutans transcriptional regulators SpxA1 and SpxA2 (formerly SpxA and SpxB, respectively) are involved in stress survival by activating the expression of classic oxidative stress genes such as dpr, nox, sodA and tpx. We reasoned that some of the uncharacterized genes under SpxA1/A2 control are potentially involved in oxidative stress management. Therefore, the goal of this study was to use Spx-regulated genes as a tool to identify novel oxidative stress genes in S. mutans. Quantitative real-time PCR was used to evaluate the responses of ten Spx-regulated genes during H2O2 stress in the parent and Δspx strains. Transcription activation of the H2O2-induced genes (8 out of 10) was strongly dependent on SpxA1 and, to a lesser extent, SpxA2. In vitro transcription assays revealed that one or both Spx proteins directly regulate three of these genes. The gene encoding the FeoB ferrous permease was slightly repressed by H2O2 but constitutively induced in strains lacking SpxA1. Nine genes were selected for downstream mutational analysis but inactivation of smu127, encoding a subunit of the acetoin dehydrogenase was apparently lethal. In vitro and in vivo characterization of the viable mutants indicated that, in addition to the transcriptional activation of reducing and antioxidant pathways, Spx performs an important role in iron homeostasis by regulating the intracellular availability of free iron. In particular, inactivation of the genes encoding the Fe-S biogenesis SUF system and the previously characterized iron-binding protein Dpr resulted in impaired growth under different oxidative stress conditions, increased sensitivity to iron and lower infectivity in rats. These results serve as an entryway into the characterization of novel genes and pathways that allow S. mutans to cope with oxidative stress.

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.

Lactic acid bacteria (LAB) are a diverse group of Gram-positive microbes that ferment carbohydrates to organic acids, primarily lactic acid. LAB include organisms vitally important to the production of foods, bread, and wine, and they include major human pathogens for diseases of the oropharyneal space, lungs, mouth, and skin. For all of these organs, the production of lactic acid rapidly and substantially lowers the pH of their external environments. Because bacterial membranes are essentially porous to protons, these bacteria are at risk of damaging cellular constituents to the extent that growth ceases, and eventually they do not survive. Thus, the LAB have evolved a broad range of mechanisms to protect themselves from acidification, to repair cellular damage, and to use low-pH environments to outcompete other bacteria. In this chapter, we describe the acid-stress-responsive mechanisms of representative LAB, which have provided a framework of how these organisms respond to, and prosper in, acidic environments.

Enterococcus faecalis is an opportunistic nosocomial pathogen that is highly resistant to a variety of environmental insults, including an intrinsic tolerance to antimicrobials that target the cell wall (CW). With the goal of determining the CW-stress stimulon of E. faecalis, the global transcriptional profile of E. faecalis OG1RF exposed to ampicillin, bacitracin, cephalotin or vancomycin was obtained via microarrays. Exposure to the β-lactams ampicillin and cephalotin resulted in the fewest transcriptional changes with 50 and 192 genes differentially expressed 60 min after treatment, respectively. On the other hand, treatment with bacitracin or vancomycin for 60 min affected the expression of, respectively, 377 and 297 genes. Despite the differences in the total number of genes affected, all antibiotics induced a very similar gene expression pattern with an overrepresentation of genes encoding hypothetical proteins, followed by genes encoding proteins associated with cell envelope metabolism as well as transport and binding proteins. In particular, all drug treatments, most notably bacitracin and vancomycin, resulted in an apparent metabolic downshift based on the repression of genes involved in translation, energy metabolism, transport and binding. Only 19 genes were up-regulated by all conditions at both the 30 and 60 min time points. Among those 19 genes, 4 genes encoding hypothetical proteins (EF0026, EF0797, EF1533 and EF3245) were inactivated and the respective mutant strains characterized in relation to antibiotic tolerance and virulence in the Galleria mellonella model. The phenotypes obtained for two of these mutants, ΔEF1533 and ΔEF3245, support further characterization of these genes as potential candidates for the development of novel preventive or therapeutic approaches.