Dissecting the Role of VicK Phosphatase in Aggregation and Biofilm Formation of Streptococcus mutans.

VicRK (WalRK or YycFG) is a conserved 2-component regulatory system (TCS) that regulates cell division, cell wall biosynthesis, and homeostasis in low-GC Gram-positive bacteria. VicRK is also associated with biofilm formation of Streptococcus mutans on the tooth surface as it directly regulates the extracellular polysaccharide (EPS) synthesis. Of the 2 components, VicK possesses both autokinase and phosphatase activities, which regulate the phosphorylation and dephosphorylation of the regulator VicR in response to environmental cues. However, the dual mechanism of VicK as the autokinase/phosphatase in regulating S. mutans' responses is not well elucidated. Previously, it has been shown that the phosphatase activity depends on the PAS domain and residues in the DHp domain of VicK in S. mutans. Specifically, mutating proline at 222 in the PAS domain inhibits VicK phosphatase activity. We generated a VicKP222A mutant to determine the level of VicR-P in the cytoplasm by Phos-tag sodium dodecyl sulfate polyacrylamide gel electrophoresis. We show that in VicKP222A phosphatase, attenuation increased phosphorylated VicR (VicR-P) that downregulated glucosyltransferases, gtfBC, thereby reducing the synthesis of water-insoluble polysaccharides (WIS-EPS) in the biofilm. In addition, VicKP222A presented as long-rod cells, reduced growth, and displayed asymmetrical division. A major adhesin of S. mutans, SpaP was downregulated in VicKP222A, making it unable to agglutinate in saliva. In summary, we have confirmed that VicK phosphatase activity is critical to maintain optimal phosphorylation status of VicR in S. mutans, which is important for cell growth, cell division, EPS synthesis, and bacterial agglutination in saliva. Hence, VicK phosphatase activity may represent a promising target to modulate S. mutans' pathogenicity.

Redirecting the immune response towards immunoprotective domains of a DNABII protein resolves experimental otitis media.

The chronicity and recurrence of many bacterial diseases is largely attributable to the presence of a biofilm, and eradication of these structures is confounded by an extracellular DNA-rich matrix. DNABII proteins, including integration host factor (IHF), are critical components of the matrix formed by all human pathogens tested to date. Whereas the natural adaptive immune response to IHF is against non-protective epitopes within the carboxyl-terminal region, antibodies against the DNA-binding "tips" induce biofilm collapse. We designed a "tip-chimer" immunogen to mimic the DNA-binding regions within the α-subunit and ß-subunit of IHF from nontypeable Haemophilus influenzae (IHFNTHi). Re-direction of the natural adaptive immune response toward immunoprotective domains disrupted NTHi biofilms in vitro and in an experimental model of otitis media. Our data support the rational design of a powerful therapeutic approach, and also that of a DNABII-directed vaccine antigen that would avoid augmentation of any pre-existing natural, but nonprotective, immune response.

Novel method for the depletion of cariogenic bacteria using dextranomer microspheres.

Streptococcus mutans is recognized as one of the key contributors to the dysbiotic state that results in dental caries. Existing treatment strategies reduce the incidence of tooth decay, but they also eliminate both the cariogenic and beneficial microbes. Here we introduce a novel treatment alternative using Sephadex, cross-linked dextranomer microspheres (DMs), typically used for gel filtration chromatography. In addition DM beads can be used for affinity purification of glucosyltransferases (GTFs) from S. mutans. In this study we take advantage of the native pathogenic mechanisms used by S. mutans to adhere, form a biofilm and induce dental caries through the expression of surface-associated GTFs. We demonstrate that planktonic and biofilm-grown (adhered to hydroxyapatite-coated pegs to mimic the tooth surface) S. mutans, specifically and competitively attach to DMs. Further investigation demonstrated that DMs are a specific affinity resin for S. mutans and other cariogenic/pathogenic oral streptococci, whereas other commensal and probiotic strains failed to readily adhere to DMs. Using antimicrobial cargo loaded into the DM lumen, we demonstrate that when in co-culture with non-binding to even modestly binding commensal species, S. mutans was selectively killed. This proof of concept study introduces a novel means to safely and effectively reduce the pool of S. mutans and other pathogenic streptococci in the oral cavity with limited disturbance of the necessary commensal (healthy) microbiota when compared with current oral healthcare products.

A bacterial-biofilm-induced oral osteolytic infection can be successfully treated by immuno-targeting an extracellular nucleoid-associated protein.

Periodontal disease exemplifies a chronic and recurrent infection with a necessary biofilm component. Mucosal inflammation is a hallmark response of the host seen in chronic diseases, such as colitis, gingivitis, and periodontitis (and the related disorder peri-implantitis). We have taken advantage of our recently developed rat model of human peri-implantitis that recapitulates osteolysis, the requirement of biofilm formation, and the perpetuation of the bona fide disease state, to test a new therapeutic modality with two novel components. First we used hyperimmune antiserum directed against the DNABII family of proteins, now known to be a critical component of the extracellular matrix of bacterial biofilms. Second we delivered the antiserum as cargo in biodegradable microspheres to the site of the biofilm infection. We demonstrated that delivery of a single dose of anti-DNABII in poly(lactic-co-glycolic acid) (PLGA) microspheres induced significant resolution of experimental peri-implantitis, including marked reduction of inflammation. These data support the continued development of a DNABII protein-targeted therapeutic for peri-implantitis and other chronic inflammatory pathologies of the oral cavity in animals and humans.

Natural antigenic differences in the functionally equivalent extracellular DNABII proteins of bacterial biofilms provide a means for targeted biofilm therapeutics.

Bacteria that persist in the oral cavity exist within complex biofilm communities. A hallmark of biofilms is the presence of an extracellular polymeric substance (EPS), which consists of polysaccharides, extracellular DNA (eDNA), and proteins, including the DNABII family of proteins. The removal of DNABII proteins from a biofilm results in the loss of structural integrity of the eDNA and the collapse of the biofilm structure. We examined the role of DNABII proteins in the biofilm structure of the periodontal pathogen Porphyromonas gingivalis and the oral commensal Streptococcus gordonii. Co-aggregation with oral streptococci is thought to facilitate the establishment of P. gingivalis within the biofilm community. We demonstrate that DNABII proteins are present in the EPS of both S. gordonii and P. gingivalis biofilms, and that these biofilms can be disrupted through the addition of antisera derived against their respective DNABII proteins. We provide evidence that both eDNA and DNABII proteins are limiting in S. gordonii but not in P. gingivalis biofilms. In addition, these proteins are capable of complementing one another functionally. We also found that whereas antisera derived against most DNABII proteins are capable of binding a wide variety of DNABII proteins, the P. gingivalis DNABII proteins are antigenically distinct. The presence of DNABII proteins in the EPS of these biofilms and the antigenic uniqueness of the P. gingivalis proteins provide an opportunity to develop therapies that are targeted to remove P. gingivalis and biofilms that contain P. gingivalis from the oral cavity.

Regulation of bacteriocin production and cell death by the VicRK signaling system in Streptococcus mutans.

The VicRK two-component signaling system modulates biofilm formation, genetic competence, and stress tolerance in Streptococcus mutans. We show here that the VicRK modulates bacteriocin production and cell viability, in part by direct modulation of competence-stimulating peptide (CSP) production in S. mutans. Global transcriptome and real-time transcriptional analysis of the VicK-deficient mutant (SmuvicK) revealed significant modulation of several bacteriocin-related loci, including nlmAB, nlmC, and nlmD (P < 0.001), suggesting a role for the VicRK in producing mutacins IV, V, and VI. Bacteriocin overlay assays revealed an altered ability of the vic mutants to kill related species. Since a well-conserved VicR binding site (TGTWAH-N(5)-TGTWAH) was identified within the comC coding region, we confirmed VicR binding to this sequence using DNA footprinting. Overexpression of the vic operon caused growth-phase-dependent repression of comC, comDE, and comX. In the vic mutants, transcription of nlmC/cipB encoding mutacin V, previously linked to CSP-dependent cell lysis, as well as expression of its putative immunity factor encoded by immB, were significantly affected relative to the wild type (P < 0.05). In contrast to previous reports that proposed a hyper-resistant phenotype for the VicK mutant in cell viability, the release of extracellular genomic DNA was significantly enhanced in SmuvicK (P < 0.05), likely as a result of increased autolysis compared with the parent. The drastic influence of VicRK on cell viability was also demonstrated using vic mutant biofilms. Taken together, we have identified a novel regulatory link between the VicRK and ComDE systems to modulate bacteriocin production and cell viability of S. mutans.

Biofilms can be dispersed by focusing the immune system on a common family of bacterial nucleoid-associated proteins.

Bacteria that cause chronic and/or recurrent diseases often rely on a biofilm lifestyle. The foundation of the biofilm structure is the extracellular polymeric substance (EPS) that acts as a barrier to both effectors of the immune system and antimicrobial agents. Recent work has highlighted extracellular DNA (eDNA) as a key component common to many pathogenic biofilms. Here, we show that the DNABII family of proteins, well known for their strong structural influences on intracellular DNA, was also critical for the integrity of the EPS matrix of biofilms that contain eDNA. In fact, antisera derived against a purified Escherichia coli DNABII family member rapidly disrupts the biofilm EPS formed by multiple human pathogens in vitro. In addition, when a member of this family of proteins was used as an immunogen in an animal model in which the bacteria had already formed a robust biofilm at the site of infection, the resultant targeted immune response strongly ameliorated this biofilm disease in vivo. Finally, this methodology to debulk the biofilm of EPS was shown to work synergistically with otherwise ineffective traditional anti-microbial approaches in vitro. We discuss the prospects for targeting DNABII family members as a potential universal strategy for treating biofilm diseases.

A novel antimicrobial peptide significantly enhances acid-induced killing of Shiga toxin-producing Escherichia coli O157 and non-O157 serotypes.

Shiga toxin-producing Escherichia coli (STEC) colonizes the human intestine, causing haemorrhagic colitis and haemolytic uraemic syndrome (HUS). Treatment options are limited to intravenous fluids in part because sublethal doses of some antibiotics have been shown to stimulate increased toxin release and enhance the risk of progression to HUS. Preventative antimicrobial agents, especially those that build on the natural antimicrobial action of the host defence, may provide a better option. In order to survive the acid stress of gastric passage, STEC is equipped with numerous acid resistance and DNA repair mechanisms. Inhibition of acid-induced DNA repair may offer a strategy to target survival of ingested STEC. We report here that brief pretreatment with a novel antimicrobial peptide, which was previously shown to inhibit bacterial DNA repair, significantly and profoundly reduces survival of acid-stressed O157 : H7 and non-O157 : H7 STEC seropathotypes that are highly associated with HUS. Reduction in survival rates of STEC range from 3 to 5 log. We also show that peptide/acid treatment results in little or no increase in toxin production, thereby reducing the risk of progression to HUS. This study identifies the peptide wrwycr as a potential new candidate for a preventative antimicrobial for STEC infection.

Early detection of oxidized surfaces using Shewanella oneidensis MR-1 as a tool.

Corrosion is a natural global problem of immense importance. Oxidation of iron and steel not only compromises the structural stability of a widely used and versatile material but it also creates an abrasive compound (iron oxide) that can score the surfaces of metals, rendering them useless for the purpose for which they were designed. Clearly, the identification of corrosion in its nascent stages is a high priority for reasons that range from aesthetics to economics. Many bacteria in the facultatively aerobic genus Shewanella have the capacity to respire some metal oxides, such as iron oxide, by way of a variety of oxide-binding proteins lodged in their outer membrane. In this study, a rapid, cost-effective system for the specific early detection of a variety of oxidized steel surfaces is described, taking advantage of bacteria with natural affinities for iron oxides, to identify the sites of nascent corrosion.

Simultaneous interferometric measurement of corrosive or demineralizing bacteria and their mineral interfaces.

Here, we report simultaneous surface profile measurements of several bacterial species involved in microbially influenced corrosion and their solid-surface interfaces by using vertical scanning interferometry. The capacity to nondestructively quantify microscale topographic changes beneath a single bacterium without its removal offers a unique opportunity to examine in vivo microbe-surface interactions.

In search of the microbe/mineral interface: quantitative analysis of bacteria on metal surfaces using vertical scanning interferometry.

To understand the development of biofilms on metal surfaces, analysis of initial bacterial attachment to surfaces is crucial. Here we present the results of a study, using Shewanella oneidensis MR-1 as a model organism, in which vertical scanning interferometry (VSI) was used to investigate the initial stages of cell attachment to glass, steel and aluminium surfaces. It was found that while VSI gave unambiguous results with opaque surfaces, when reflective surfaces were used, an artifact sometimes appeared, with the bacteria appearing as rod-shaped pits rather than as cells on the surface. When the bacteria were altered to increase opacity, this artifact disappeared, and upon further investigation, it was found that the observational artifact was the result of a conflict between light reflected from the bacteria and the light reflected from the bacteria-metal interface. These results suggest that not only can bacteria be measured on surfaces using VSI, but with some modifications to the analytical software, there may be a unique window for studying the bacterial/substrate interface that can be used for quantitative observations. Imaging and characterization of the bacteria-substrate interface in vivo (previously invisible) will provide new insights into the interactions that occur at this important juncture.

DNA bending and twisting properties of integration host factor determined by DNA cyclization.

The binding of many proteins to DNA is profoundly affected by DNA bending, twisting, and supercoiling. When protein binding alters DNA conformation, interaction between inherent and induced DNA conformation can affect protein binding affinity and specificity. Integration host factor (IHF), a sequence-specific, DNA-binding protein of Escherichia coli, strongly bends the DNA upon binding. To assess the influence of inherent DNA bending on IHF binding, we took advantage of the high degree of natural static curvature associated with an IHF site on a 163-bp minicircle and measured the binding affinity of IHF for its recognition site contained on this DNA in both circular and linear form. IHF showed a higher affinity for the circular form of the DNA when compared to the linear form. In addition, the presence of IHF during DNA cyclization changed the topology of cyclization products and their ability to bind IHF, consistent with IHF untwisting DNA. These results show that inherent DNA conformation anisotropy is an important determinant of IHF binding affinity and suggests a mechanism for modulation of IHF activity by local DNA conformation.

Characterization of the gtfB and gtfC promoters from Streptococcus mutans GS-5.

We have characterized the promoter regions of the gtfB and gtfC genes from Streptococcus mutans GS-5. Using a plasmid-based reporter system, we discovered that the gtfC promoter is an order of magnitude stronger than the gtfB promoter. In addition, we found that a variety of growth conditions failed to affect or discriminate between the expression of the two promoters. Only during the various phases of growth could we demonstrate significant changes in expression. We conclude that gtfB and gtfC promoters are coordinately expressed.

Replacement of integration host factor protein-induced DNA bending by flexible regions of DNA.

The Escherichia coli integration host factor (IHF) protein is required for site-specific recombination of bacteriophage lambda DNA. Previously, we had shown that alternative modules of static DNA curvature could partially replace IHF in recombination. Now we use regions of single-stranded DNA as a flexible tether to address whether the function of IHF in recombination is simply to reduce persistence length. Although we find that these modules clearly enhance recombination in the absence of IHF, they are not perfect replacements. In addition, evidence is presented that the efficacy of a flexibility swap is specific to a particular IHF site. This may indicate that additional functions beyond simple deformation of DNA are required of IHF. During the course of these experiments we discovered that these flexible sequences are still specific sites for IHF binding and function.

Global structure similarities of intact and nicked DNA complexed with IHF measured in solution by fluorescence resonance energy transfer.

We have analyzed the structure of two related protein-DNA complexes consisting of integration host factor (IHF) bound to two different versions of the H' site of bacteriophage lambda. Both DNA substrates were 55 bp in length. While one was native duplex the other possessed a nick in one strand at a crucial position within the IHF consensus at the same position as in the reported crystal structure of the DNA-IHF complex. By labeling the 5'-ends of these DNA molecules with donor and acceptor fluorescent dyes, we were able to measure the distance between the dyes by fluorescence resonance energy transfer (FRET) and model DNA distortion. The FRET efficiency decreased from 0.49 +/- 0.01 (nicked DNA) to 0.37 +/- 0.01 (intact DNA) when the gap in the DNA strand was closed. The measured dye-to-dye distance of IHF in complex with nicked DNA was in agreement with the expected value from the crystal structure. Although we found that the two structures were distinguishable, the global shape induced by IHF was retained between the two DNA molecules. Furthermore, our FRET and modeling techniques have sufficiently high resolution to distinguish subtle changes in nucleoprotein complexes with biological relevance.

Firefly luciferase as a reporter to study gene expression in Streptococcus mutans.

The utility of firefly luciferase as a reporter was tested in Streptococcus mutans. Under control of an endogenous promoter, the luciferase coding sequence was strongly expressed, while a promoterless version was indistinguishable from the background. Luciferase activity was easily extracted and the assay rapid and reproducible. In addition, the half-life of luciferase activity was found to be comparable to those of other frequently used reporters. Thus, firefly luciferase can readily be used as a reporter in S. mutans, a useful alternative to methods requiring radioactive isotopes.

In vitro selection of integration host factor binding sites.

Integration host factor (IHF) is a bacterial protein that binds and severely bends a specific DNA target. IHF binding sites are approximately 30 to 35 bp long and are apparently divided into two domains. While the 3' domain is conserved, the 5' domain is degenerate but is typically AT rich. As a result of physical constraints that IHF must impose on DNA in order to bind, it is believed that this 5' domain must possess structural characteristics conducive for both binding and bending with little regard for specific contacts between the protein and the DNA. We have examined the sequence requirements of the 5' binding domain of the IHF binding target. Using a SELEX procedure, we randomized and selected variants of a natural IHF site. We then analyzed these variants to determine how the 5' binding domain affects the structure, affinity, and function of an IHF-DNA complex in a native system. Despite finding individual sequences that varied over 100-fold in affinity for IHF, we found no apparent correlation between affinity and function.

Growth phase-dependent regulation of nuoA-N expression in Escherichia coli K-12 by the Fis protein: upstream binding sites and bioenergetic significance.

The expression of the nuoA-N operon of Escherichia coli K-12, which encodes the proton-pumping NADH dehydrogenase I is modulated by growth phase-dependent regulation. Under respiratory growth conditions, expression was stimulated in early exponential, and to a lesser extent in late exponential and stationary growth phases. The stimulation in the early exponential growth phase was not observed in fis mutants, which are deficient for the growth phase-responsive regulator Fis. Neither the alternative sigma factor RpoS nor the integration host factor (IHF) are involved in growth phase-dependent regulation of this operon. When incubated with nuo promoter DNA, isolated Fis protein formed three retarded complexes in gel mobility experiments. DNase I footprinting identified three distinct binding sites for Fis, 237 bp (fis1), 197 bp (fis2) and 139 bp (fis3) upstream of the start of the major transcript of nuoA-N, T1. The protein concentrations required for half-maximal binding to fis1, fis2 and fis3 were about 20 nM, 40 nM and 100 nM Fis, respectively. The IHF protein bound 82 bp upstream of the start of transcript T2 with a half-maximal concentration for binding of 50 nM. Due to the growth phase-dependent regulation by Fis, the synthesis of the coupling NADH dehydrogenase I is increased relative to that of the noncoupling NADH dehydrogenase II during early exponential growth. This ensures higher ATP yields under conditions where large amounts of ATP are required.

Knowledge-based model of a glucosyltransferase from the oral bacterial group of mutans streptococci.

Mutans streptococci glucosyltransferases catalyze glucosyl transfer from sucrose to a glucan chain. We previously identified an aspartyl residue that participates in stabilizing the glucosyl transition state. The sequence surrounding the aspartate was found to have substantial sequence similarity with members of alpha-amylase family. Because little is known of the protein structure beyond the amino acid sequence, we used a knowledge-based interactive algorithm, MACAW, which provided significant level of homology with alpha-amylases and glucosyltransferase from Streptococcus downei gtfI (GTF). The significance of GTF similarity is underlined by GTF/alpha-amylase residues conserved in all but one alpha-amylase invariant residues. Site-directed mutagenesis of the three GTF catalytic residues are homologous with the alpha-amylase catalytic triad. The glucosyltransferases are members of the 4/7-superfamily that have a (beta/alpha)8-barrel structure and belong to family 13 of the glycohydralases.

The complete nucleotide sequence of bacteriophage HP1 DNA.

The complete nucleotide sequence of the temperate phage HP1 of Haemophilus influenzae was determined. The phage contains a linear, double-stranded genome of 32 355 nt with cohesive termini. Statistical methods were used to identify 41 probable protein coding segments organized into five plausible transcriptional units. Regions encoding proteins involved in recombination, replication, transcriptional control, host cell lysis and phage production were identified. The sizes of proteins in the mature HP1 particle were determined to assist in identifying genes for structural proteins. Similarities between HP1 coding sequences and those in databases, as well as similar gene organizations and control mechanisms, suggest that HP1 is a member of the P2-like phage family, with strong similarities to coliphages P2 and 186 and some similarity to the retronphage Ec67.