ToxR activates the Vibrio cholerae virulence genes by tethering DNA to the membrane through versatile binding to multiple sites.

ToxR, a Vibrio cholerae transmembrane one-component signal transduction factor, lies within a regulatory cascade that results in the expression of ToxT, toxin coregulated pilus, and cholera toxin. While ToxR has been extensively studied for its ability to activate or repress various genes in V. cholerae, here we present the crystal structures of the ToxR cytoplasmic domain bound to DNA at the toxT and ompU promoters. The structures confirm some predicted interactions, yet reveal other unexpected promoter interactions with implications for other potential regulatory roles for ToxR. We show that ToxR is a versatile virulence regulator that recognizes diverse and extensive, eukaryotic-like regulatory DNA sequences, that relies more on DNA structural elements than specific sequences for binding. Using this topological DNA recognition mechanism, ToxR can bind both in tandem and in a twofold inverted-repeat-driven manner. Its regulatory action is based on coordinated multiple binding to promoter regions near the transcription start site, which can remove the repressing H-NS proteins and prepares the DNA for optimal interaction with the RNA polymerase.

Effect of Biosilicate® Addition on Physical-Mechanical and Biological Properties of Dental Glass Ionomer Cements.

This study investigated the influence of incorporating Biosilicate® on the physico-mechanical and biological properties of glass ionomer cement (GIC). This bioactive glass ceramic (23.75% Na2O, 23.75% CaO, 48.5% SiO2, and 4% P2O5) was incorporated by weight (5%, 10%, or 15%) into commercially available GICs (Maxxion R and Fuji IX GP). Surface characterization was made by SEM (n = 3), EDS (n = 3), and FTIR (n = 1). The setting and working (S/W time) times (n = 3) and compressive strength (CS) were analyzed (n = 10) according to ISO 9917-1:2007. The ion release (n = 6) was determined and quantified by ICP OES and by UV-Vis for Ca, Na, Al, Si, P, and F. To verify cell cytotoxicity, stem cells from the apical papilla (SCAP) were exposed to eluates (n = 3, at a ratio of 1.8 cm2/mL) and analyzed 24 h post-exposure. Antimicrobial activity against Streptococcus mutans (ATCC 25175, NCTC 10449) was analyzed by direct contact for 2 h (n = 5). The data were submitted for normality and lognormality testing. One-way ANOVA and Tukey's test were applied for the working and setting time, compressive strength, and ion release data. Data from cytotoxicity and antimicrobial activity were submitted for Kruskal-Wallis' testing and Dunn's post hoc test (α = 0.05). Among all experimental groups, only those with 5% (wt) of Biosilicate® showed better surface quality. Only M5% showed a comparable W/S time to the original material (p = 0.7254 and p = 0.5912). CS was maintained for all Maxxion R groups (p > 0.0001) and declined for Fuji IX experimental groups (p < 0.0001). The Na, Si, P, and F ions released were significantly increased for all Maxxion R and Fuji IX groups (p < 0.0001). Cytotoxicity was increased only for Maxxion R with 5% and 10% of Biosilicate®. A higher inhibition of S. mutans growth was observed for Maxxion R with 5% of Biosilicate® (less than 100 CFU/mL), followed by Maxxion R with 10% of Biosilicate® (p = 0.0053) and Maxxion R without the glass ceramic (p = 0.0093). Maxxion R and Fuji IX presented different behaviors regarding Biosilicate® incorporation. The impacts on physico-mechanical and biological properties were different depending on the GIC, but therapeutic ion release was increased for both materials.

Microbiology-Based Instruction during Prenatal Dental Visits Improves Perinatal Oral Health Literacy.

To improve oral hygiene education, we evaluated the perception and potential impact of microbiology-focused oral hygiene instructions (OHI) given to pregnant patients. Dental hygienists provided this supplemental education and administered Saliva-Check Mutans (SCM) tests to pregnant patients (n = 188) in Obstetrics and Gynecology (OB/GYN) settings. Patients reported their self-perceived understanding of the relationship between oral bacteria and dental disease and returned postdelivery to receive a second SCM test and follow-up questionnaire (n = 47). Prior to the hygienist instruction, 84% of participants understood that bacteria caused tooth decay, while only 36% understood they could transfer these bacteria to their children. After instruction, patient understanding increased to 97% and 95%, respectively. Participants attributed these increases to the hygienist's explanation and SCM test. In postdelivery participants, >80% reported adherence to routine oral hygiene practices, and a significant decrease in patients with high-risk levels of salivary Streptococcus mutans was determined by SCM test (p = 0.0253). Participants agreed that the SCM test (89%) and microbiology explanation (95%) should be provided to every pregnant patient. Evaluation of patient perception of this intervention highlights how focused instruction on the infectious nature of dental disease can increase perinatal oral health literacy. Microbiology-focused education should be given to pregnant patients to reduce oral health disparities.

In vitro Streptococcus mutans adhesion and biofilm formation on different esthetic orthodontic archwires.

OBJECTIVES: To evaluate the ability of different esthetic archwires to retain oral biofilms in vitro. MATERIALS AND METHODS: Seven different brands of coated orthodontic archwires were tested: two epoxy coated, two polytetrafluoroethylene coated, two rhodium coated, and one silver plus polymer coated. Conventional uncoated metallic archwires were used as controls. Streptococus mutans adherence to archwires was quantified by colony count following 24 hours of biolfilm growth, and total wire-associated biofilm was measured using a crystal violet staining assay. For both tests, two conditions were used: 0% sucrose and 3% sucrose. For statistical analysis, P < .05 was considered as statistically significant. RESULTS: For S. mutans colony forming units per biofilm, there were no statistically significant differences among the various archwires (P = .795 for 0% sucrose; P = .905 for 3% sucrose). Regarding total biofilm formed on archwires in the 3% sucrose condition, there were statistically significant differences in crystal violet staining only for the comparison between Niti Micro Dental White and Copper Ni-Ti wires (P < .05). CONCLUSIONS: The clinical use of esthetic-coated orthodontic wires may be considered to have similar risks as uncoated archwires for biofilm retention.

Complement evasion mechanisms of the systemic pathogens Yersiniae and Salmonellae.

Pathogens that colonize deep tissues and spread systemically encounter the innate host resistance mechanism of complement-mediated lysis and complement opsonization leading to engulfment and degradation by phagocytic cells. Yersinia and Salmonella species have developed numerous strategies to block the antimicrobial effects of complement. These include recruitment of complement regulatory proteins factor H, C4BP, and vitronectin (Vn) as well as interference in late maturation events such as assembly of C9 into the membrane attack complex that leads to bacterial lysis. This review will discuss the contributions of various surface structures (proteins, lipopolysaccharide, and capsules) to evasion of complement-mediated immune clearance of the systemic pathogens Yersiniae and Salmonellae. Bacterial proteins required for recruitment of complement regulatory proteins will be described, including the details of their interaction with host regulatory proteins, where known. The potential role of the surface proteases Pla (Yersinia pestis) and PgtE (Salmonella species) on the activity of complement regulatory proteins will also be addressed. Finally, the implications of complement inactivation on host cell interactions and host cell targeting for type 3 secretion will be discussed.

Ail provides multiple mechanisms of serum resistance to Yersinia pestis.

Ail, a multifunctional outer membrane protein of Yersinia pestis, confers cell binding, Yop delivery and serum resistance activities. Resistance to complement proteins in serum is critical for the survival of Y. pestis during the septicemic stage of plague infections. Bacteria employ a variety of tactics to evade the complement system, including recruitment of complement regulatory factors, such as factor H, C4b-binding protein (C4BP) and vitronectin (Vn). Y. pestis Ail interacts with the regulatory factors Vn and C4BP, and Ail homologs from Y. enterocolitica and Y. pseudotuberculosis recruit factor H. Using co-sedimentation assays, we demonstrate that two surface-exposed amino acids, F80 and F130, are required for the interaction of Y. pestis Ail with Vn, factor H and C4BP. However, although Ail-F80A/F130A fails to interact with these complement regulatory proteins, it still confers 10,000-fold more serum resistance than a Δail strain and prevents C9 polymerization, potentially by directly interfering with MAC assembly. Using site-directed mutagenesis, we further defined this additional mechanism of complement evasion conferred by Ail. Finally, we find that at Y. pestis concentrations reflective of early-stage septicemic plague, Ail weakly recruits Vn and fails to recruit factor H, suggesting that this alternative mechanism of serum resistance may be essential during plague infection.

Complete Genome Sequences of Cluster A Mycobacteriophages BobSwaget, Fred313, KADY, Lokk, MyraDee, Stagni, and StepMih.

Seven mycobacteriophages from distinct geographical locations were isolated, using Mycobacterium smegmatis mc2155 as the host, and then purified and sequenced. All of the genomes are related to cluster A mycobacteriophages, BobSwaget and Lokk in subcluster A2; Fred313, KADY, Stagni, and StepMih in subcluster A3; and MyraDee in subcluster A18, the first phage to be assigned to that subcluster.

Defining the Ail Ligand-Binding Surface: Hydrophobic Residues in Two Extracellular Loops Mediate Cell and Extracellular Matrix Binding To Facilitate Yop Delivery.

Yersinia pestis, the causative agent of plague, binds host cells to deliver cytotoxic Yop proteins into the cytoplasm that prevent phagocytosis and generation of proinflammatory cytokines. Ail is an eight-stranded ß-barrel outer membrane protein with four extracellular loops that mediates cell binding and resistance to human serum. Following the deletion of each of the four extracellular loops that potentially interact with host cells, the Ail-Δloop 2 and Ail-Δloop 3 mutant proteins had no cell-binding activity while Ail-Δloop 4 maintained cell binding (the Ail-Δloop 1 protein was unstable). Using the codon mutagenesis scheme SWIM (selection without isolation of mutants), we identified individual residues in loops 1, 2, and 3 that contribute to host cell binding. While several residues contributed to the binding of host cells and purified fibronectin and laminin, as well as Yop delivery, three mutations, F80A (loop 2), S128A (loop 3), and F130A (loop 3), produced particularly severe defects in cell binding. Combining these mutations led to an even greater reduction in cell binding and severely impaired Yop delivery with only a slight defect in serum resistance. These findings demonstrate that Y. pestis Ail uses multiple extracellular loops to interact with substrates important for adhesion via polyvalent hydrophobic interactions.

Formation of an Intramolecular Periplasmic Disulfide Bond in TcpP Protects TcpP and TcpH from Degradation in Vibrio cholerae.

UNLABELLED: TcpP and ToxR coordinately regulate transcription of toxT, the master regulator of numerous virulence factors in Vibrio cholerae. TcpP and ToxR are membrane-localized transcription factors, each with a periplasmic domain containing two cysteines. In ToxR, these cysteines form an intramolecular disulfide bond and a cysteine-to-serine substitution affects activity. We determined that the two periplasmic cysteines of TcpP also form an intramolecular disulfide bond. Disruption of this intramolecular disulfide bond by mutation of either cysteine resulted in formation of intermolecular disulfide bonds. Furthermore, disruption of the intramolecular disulfide bond in TcpP decreased the stability of TcpP. While the decreased stability of TcpP-C207S resulted in a nearly complete loss of toxT activation and cholera toxin (CT) production, the second cysteine mutant, TcpP-C218S, was partially resistant to proteolytic degradation and maintained ∼50% toxT activation capacity. TcpP-C218S was also TcpH independent, since deletion of tcpH did not affect the stability of TcpP-C218S, whereas wild-type TcpP was degraded in the absence of TcpH. Finally, TcpH was also unstable when intramolecular disulfides could not be formed in TcpP, suggesting that the single periplasmic cysteine in TcpH may assist with disulfide bond formation in TcpP by interacting with the periplasmic cysteines of TcpP. Consistent with this finding, a TcpH-C114S mutant was unable to stabilize TcpP and was itself unstable. Our findings demonstrate a periplasmic disulfide bond in TcpP is critical for TcpP stability and virulence gene expression. IMPORTANCE: The Vibrio cholerae transcription factor TcpP, in conjunction with ToxR, regulates transcription of toxT, the master regulator of numerous virulence factors in Vibrio cholerae. TcpP is a membrane-localized transcription factor with a periplasmic domain containing two cysteines. We determined that the two periplasmic cysteines of TcpP form an intramolecular disulfide bond and disruption of the intramolecular disulfide bond in TcpP decreased the stability of TcpP and reduced virulence gene expression. Normally TcpH, another membrane-localized periplasmic protein, protects TcpP from degradation. However, we found that TcpH was also unstable when intramolecular disulfides could not be formed in TcpP, indicating that the periplasmic cysteines of TcpP are required for functional interaction with TcpH and that this interaction is required for both TcpP and TcpH stability.

Linking changes in community composition and function under climate change.

Climate change is expected to directly alter the composition of communities and the functioning of ecosystems across the globe. Improving our understanding of links between biodiversity and ecosystem functioning across large spatial scales and rapid global change is a major priority to help identify management responses that will retain diverse, functioning systems. Here we address this challenge by linking projected changes in plant community composition and functional attributes (height, leaf area, seed mass) under climate change across Tasmania, Australia. Using correlative community-level modeling, we found that projected changes in plant community composition were not consistently related to projected changes in community mean trait values. In contrast, we identified specific mechanisms through which alternative combinations of projected functional and compositional change across Tasmania could be realized, including loss/replacement of functionally similar species (lowland grasslands/grassy woodlands) and loss of a small number of functionally unique species (lowland forests). Importantly, we demonstrate how these linked projections of change in community composition and functional attributes can be utilized to inform specific management actions that may assist in maintaining diverse, functioning ecosystems under climate change.

A small unstructured region in Vibrio cholerae ToxT mediates the response to positive and negative effectors and ToxT proteolysis.

Vibrio cholerae is the causative agent of the severe diarrheal disease cholera. The production of the virulence factors that are required for human disease is controlled by a complex network of transcriptional and posttranscriptional regulators. ToxT is the transcription regulator that directly controls the production of the two major virulence factors, toxin-coregulated pilus (TCP) and cholera toxin (CT). The solved crystal structure of ToxT revealed an unstructured region in the N-terminal domain between residues 100 and 110. This region and the surrounding amino acids have been previously implicated in ToxT proteolysis, resistance to inhibition by negative effectors, and ToxT dimerization. To better characterize this region, site-directed mutagenesis was performed to assess the effects on ToxT proteolysis and bile sensitivity. This analysis identified specific mutations within this unstructured region that prevent ToxT proteolysis and other mutations that reduce inhibition by bile and unsaturated fatty acids. In addition, we found that mutations that affect the sensitivity of ToxT to bile also affect the sensitivity of ToxT to its positive effector, bicarbonate. These results suggest that a small unstructured region in the ToxT N-terminal domain is involved in multiple aspects of virulence gene regulation and response to human host signals.

Bicarbonate increases binding affinity of Vibrio cholerae ToxT to virulence gene promoters.

The major Vibrio cholerae virulence gene transcription activator, ToxT, is responsible for the production of the diarrhea-inducing cholera toxin (CT) and the major colonization factor, toxin coregulated pilus (TCP). In addition to the two primary virulence factors mentioned, ToxT is responsible for the activation of accessory virulence genes, such as aldA, tagA, acfA, acfD, tcpI, and tarAB. ToxT activity is negatively modulated by bile and unsaturated fatty acids found in the upper small intestine. Conversely, previous work identified another intestinal signal, bicarbonate, which enhances the ability of ToxT to activate production of CT and TCP. The work presented here further elucidates the mechanism for the enhancement of ToxT activity by bicarbonate. Bicarbonate was found to increase the activation of ToxT-dependent accessory virulence promoters in addition to those that produce CT and TCP. Bicarbonate is taken up into the V. cholerae cell, where it positively affects ToxT activity by increasing DNA binding affinity for the virulence gene promoters that ToxT activates regardless of toxbox configuration. The increase in ToxT binding affinity in the presence of bicarbonate explains the elevated level of virulence gene transcription.