Steven D. Goodman

Mutation of luxS Affects Biofilm Formation in Streptococcus mutans

ABSTRACT Quorum sensing is a bacterial mechanism for regulating gene expression in response to changes in population density. Many bacteria are capable of acyl-homoserine lactone-based or peptide-based intraspecies quorum sensing and luxS-dependent interspecies quorum sensing. While there is good evidence about the involvement of intraspecies quorum sensing in bacterial biofilm, little is known about the role of luxS in biofilm formation. In this study, we report for the first time that luxS-dependent quorum sensing is involved in biofilm formation of Streptococcus mutans. S. mutans is a major cariogenic bacterium in the multispecies bacterial biofilm commonly known as dental plaque. An ortholog of luxS for S. mutans was identified using the data available in the S. mutans genome project (http://www.genome.ou.edu/smutans.html ). Using an assay developed for the detection of the LuxS-associated quorum sensing signal autoinducer 2 (AI-2), it was demonstrated that this ortholog was able to complement the luxS negative phenotype of Escherichia coli DH5α. It was also shown that AI-2 is indeed produced by S. mutans. AI-2 production is maximal during mid- to late-log growth in batch culture. Mutant strains devoid of the luxS gene were constructed and found to be defective in producing the AI-2 signal. There are also marked phenotypic differences between the wild type and the luxS mutants. Microscopic analysis of in vitro-grown biofilm structure revealed that the luxS mutant biofilms adopted a much more granular appearance, rather than the relatively smooth, confluent layer normally seen in the wild type. These results suggest that LuxS-dependent signal may play an important role in biofilm formation of S. mutans.

A VicRK Signal Transduction System in Streptococcus mutans Affects gtfBCD, gbpB, and ftf Expression, Biofilm Formation, and Genetic Competence Development

Bacteria exposed to transient host environments can elicit adaptive responses by triggering the differential expression of genes via two-component signal transduction systems. This study describes the vicRK signal transduction system in Streptococcus mutans. A vicK (putative histidine kinase) deletion mutant (SmuvicK) was isolated. However, a vicR (putative response regulator) null mutation was apparently lethal, since the only transformants isolated after attempted mutagenesis overexpressed all three genes in the vicRKX operon (Smuvic+). Compared with the wild-type UA159 strain, both mutants formed aberrant biofilms. Moreover, the vicK mutant biofilm formed in sucrose-supplemented medium was easily detachable relative to that of the parent. The rate of total dextran formation by this mutant was remarkably reduced compared to the wild type, whereas it was increased in Smuvic+. Based on real-time PCR, Smuvic+ showed increased gtfBCD, gbpB, and ftf expression, while a recombinant VicR fusion protein was shown to bind the promoter regions of the gtfB, gtfC, and ftf genes. Also, transformation efficiency in the presence or absence of the S. mutans competence-stimulating peptide was altered for the vic mutants. In vivo studies conducted using SmuvicK in a specific-pathogen-free rat model resulted in significantly increased smooth-surface dental plaque (Pearson-Filon statistic [PF], <0.001). While the absence of vicK did not alter the incidence of caries, a significant reduction in SmuvicK CFU counts was observed in plaque samples relative to that of the parent (PF, <0.001). Taken together, these findings support involvement of the vicRK signal transduction system in regulating several important physiological processes in 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.

Mechanistic Insights Revealed by the Crystal Structure of a Histidine Kinase with Signal Transducer and Sensor Domains

A crystal structure reveals an elegant mechanistic switch whereby helical bending and catalytic domain rotation allow self-activation of a histidine kinase during a bacterial stress response.

The Streptococcus mutans vicX gene product modulates gtfB/C expression, biofilm formation, genetic competence, and oxidative stress tolerance.

Streptococcus mutans is considered one of the primary etiologic agents of dental caries. Previously, we characterized the VicRK two-component signal transduction system, which regulates multiple virulence factors of S. mutans. In this study, we focused on the vicX gene of the vicRKX tricistronic operon. To characterize vicX, we constructed a nonpolar deletion mutation in the vicX coding region in S. mutans UA159. The growth kinetics of the mutant (designated SmuvicX) showed that the doubling time was longer and that there was considerable sensitivity to paraquat-induced oxidative stress. Supplementing a culture of the wild-type UA159 strain with paraquat significantly increased the expression of vicX (P < 0.05, as determined by analysis of variance [ANOVA]), confirming the role of this gene in oxidative stress tolerance in S. mutans. Examination of mutant biofilms revealed architecturally altered cell clusters that were seemingly denser than the wild-type cell clusters. Interestingly, vicX-deficient cells grown in a glucose-supplemented medium exhibited significantly increased glucosyltransferase B/C (gtfB/C) expression compared with the expression in the wild type (P < 0.05, as determined by ANOVA). Moreover, a sucrose-dependent adhesion assay performed using an S. mutans GS5-derived vicX null mutant demonstrated that the adhesiveness of this mutant was enhanced compared with that of the parent strain and isogenic mutants of the parent strain lacking gtfB and/or gtfC. Also, disruption of vicX reduced the genetic transformability of the mutant approximately 10-fold compared with that of the parent strain (P < 0.05, as determined by ANOVA). Collectively, these findings provide insight into important phenotypes controlled by the vicX gene product that can impact S. mutans pathogenicity.

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.

HMGA2 exhibits dRP/AP site cleavage activity and protects cancer cells from DNA-damage-induced cytotoxicity during chemotherapy

HMGA proteins are not translated in normal human somatic cells, but are present in high copy numbers in pluripotent embryonic stem cells and most neoplasias. Correlations between the degree of malignancy, patient prognostic index and HMGA levels have been firmly established. Intriguingly, HMGA2 is also found in rare tumor-inducing cells which are resistant to chemotherapy. Here, we demonstrate that HMGA1a/b and HMGA2 possess intrinsic dRP and AP site cleavage activities, and that lysines and arginines in the AT-hook DNA-binding domains function as nucleophiles. We also show that HMGA2 can be covalently trapped at genomic abasic sites in cancer cells. By employing a variety of cell-based assays, we provide evidence that the associated lyase activities promote cellular resistance against DNA damage that is targeted by base excision repair (BER) pathways, and that this protection directly correlates with the level of HMGA2 expression. In addition, we demonstrate an interaction between human AP endonuclease 1 and HMGA2 in cancer cells, which supports our conclusion that HMGA2 can be incorporated into the cellular BER machinery. Our study thus identifies an unexpected role for HMGA2 in DNA repair in cancer cells which has important clinical implications for disease diagnosis and therapy.

Structural Stability of Burkholderia cenocepacia Biofilms Is Reliant on eDNA Structure and Presence of a Bacterial Nucleic Acid Binding Protein

Cystic fibrosis (CF) is the most common lethal inherited genetic disorder affection Caucasians. Even with medical advances, CF is life-shortening with patients typically surviving only to age 38. Infection of the CF lung by Burkholderia cenocepacia presents exceptional challenges to medical management of these patients as clinically this microbe is resistant to virtually all antibiotics, is highly transmissible and infection of CF patients with this microbe renders them ineligible for lung transplant, often the last lifesaving option. Here we have targeted two abundant components of the B. cenocepacia biofilm for immune intervention: extracellular DNA and DNABII proteins, the latter of which are bacterial nucleic acid binding proteins. Treatment of B. cenocepacia biofilms with antiserum directed at one of these DNABII proteins (integration host factor or IHF) resulted in significant disruption of the biofilm. Moreover, when anti-IHF mediated destabilization of a B. cenocepacia biofilm was combined with exposure to traditional antibiotics, B. cenocepacia resident within the biofilm and thereby typically highly resistant to the action of antibiotics, were now rendered susceptible to killing. Pre-incubation of B. cenocepacia with anti-IHF serum prior to exposure to murine CF macrophages, which are normally unable to effectively degrade ingested B. cenocepacia, resulted in a statistically significant increase in killing of phagocytized B. cenocepacia. Collectively, these findings support further development of strategies that target DNABII proteins as a novel approach for treatment of CF patients, particularly those whose lungs are infected with B. cenocepacia.

CHARACTERIZATION OF DNA BINDING SITES OF THE COME RESPONSE REGULATOR FROM STREPTOCOCCUS MUTANS

In Streptococcus mutans, both competence and bacteriocin production are controlled by ComC and the ComED two-component signal transduction system. Recent studies of S. mutans suggested that purified ComE binds to two 11-bp direct repeats in the nlmC-comC promoter region, where ComE activates nlmC and represses comC. In this work, quantitative binding studies and DNase I footprinting analysis were performed to calculate the equilibrium dissociation constant and further characterize the binding site of ComE. We found that ComE protects sequences inclusive of both direct repeats, has an equilibrium dissociation constant in the nanomolar range, and binds to these two direct repeats cooperatively. Furthermore, similar direct repeats were found upstream of cslAB, comED, comX, ftf, vicRKX, gtfD, gtfB, gtfC, and gbpB. Quantitative binding studies were performed on each of these sequences and showed that only cslAB has a similar specificity and high affinity for ComE as that seen with the upstream region of comC. A mutational analysis of the binding sequences showed that ComE does not require both repeats to bind DNA with high affinity, suggesting that single site sequences in the genome may be targets for ComE-mediated regulation. Based on the mutational analysis and DNase I footprinting analysis, we propose a consensus ComE binding site, TCBTAAAYSGT.

Characterization of a Glutamate Transporter Operon, glnQHMP, in Streptococcus mutans and Its Role in Acid Tolerance

Glutamate contributes to the acid tolerance response (ATR) of many Gram-negative and Gram-positive bacteria, but its role in the ATR of the oral bacterium Streptococcus mutans is unknown. This study describes the discovery and characterization of a glutamate transporter operon designated glnQHMP (Smu.1519 to Smu.1522) and investigates its potential role in acid tolerance. Deletion of glnQHMP resulted in a 95% reduction in transport of radiolabeled glutamate compared to the wild-type UA159 strain. The addition of glutamate to metabolizing UA159 cells resulted in an increased production of acidic end products, whereas the glnQHMP mutant produced less lactic acid than UA159, suggesting a link between glutamate metabolism and acid production and possible acid tolerance. To investigate this possibility, we conducted a microarray analysis with glutamate and under pH 5.5 and pH 7.5 conditions which showed that expression of the glnQHMP operon was downregulated by both glutamate and mild acid. We also measured the growth kinetics of UA159 and its glnQHMP-negative derivative at pH 5.5 and found that the mutant doubled at a much slower rate than the parent strain but survived at pH 3.5 significantly better than the wild type. Taken together, these findings support the involvement of the glutamate transporter operon glnQHMP in the acid tolerance response in S. mutans.