Nasal and pharyngeal mucosal immunity to poliovirus in children following routine immunization with inactivated polio vaccine (IPV) in the United States of America.

BACKGROUND: Although polioviruses (PVs) replicate in lymphoid tissue of both the pharynx and ileum, research on polio vaccine-induced mucosal immunity has predominantly focused on intestinal neutralizing and binding antibody levels measured in stool. METHODS: To investigate the extent to which routine immunization with intramuscularly injected inactivated polio vaccine (IPV) may induce nasal and pharyngeal mucosal immunity, we measured PV type-specific neutralization and immunoglobulin (Ig) G, IgA, and IgM levels in nasal secretions, adenoid cell supernatants, and sera collected from 12 children, aged 2 to 5 years, undergoing planned adenoidectomies. All participants were routinely immunized with IPV and had no known contact with live PVs. RESULTS: PV-specific mucosal neutralization was detected in nasal and adenoid samples, mostly from children who had previously received four IPV doses. Across the three PV serotypes, both nasal (Spearman's rho ≥ 0.87, p≤0.0003 for all) and adenoid (Spearman's rho ≥0.57, p≤0.05 for all) neutralization titers correlated with serum neutralization titers. In this small study sample, there was insufficient evidence to determine which Ig isotype(s) was correlated with neutralization. CONCLUSIONS: Our findings provide policy-relevant evidence that routine immunization with IPV may induce nasal and pharyngeal mucosal immunity. The observed correlations of nasal and pharyngeal mucosal neutralization with serum neutralization contrast with previous observations of distinct intestinal and serum responses to PV vaccines. Further research is warranted to determine which antibody isotype(s) correlate with polio vaccine-induced nasal and pharyngeal mucosal neutralizing activity and to understand the differences from intestinal mucosal immunity.

A Modified ToxT Inhibitor Reduces Vibrio cholerae Virulence in Vivo.

We have previously designed and synthesized small-molecule inhibitors that reduce Vibrio cholerae virulence in vitro by targeting the transcription factor ToxT. Here we report the synthesis and biological activity of derivatives of our previous bicyclic, fatty acid-like inhibitors. All of the synthesized derivatives show antivirulence activity in vitro. For the most potent compounds, a concentration of 5 µM completely inhibited ToxT-mediated tcpA expression as measured in the ß-galactosidase assay. One indole compound, 3-(1-butyl-1 H-indol-7-yl)propanoic acid (8), was also effective at inhibiting intestinal colonization in the infant mouse. These modified compounds may serve as good candidates for further anti-cholera drug development.

The Vibrio cholerae Minor Pilin TcpB Initiates Assembly and Retraction of the Toxin-Coregulated Pilus.

Type IV pilus (T4P) systems are complex molecular machines that polymerize major pilin proteins into thin filaments displayed on bacterial surfaces. Pilus functions require rapid extension and depolymerization of the pilus, powered by the assembly and retraction ATPases, respectively. A set of low abundance minor pilins influences pilus dynamics by unknown mechanisms. The Vibrio cholerae toxin-coregulated pilus (TCP) is among the simplest of the T4P systems, having a single minor pilin TcpB and lacking a retraction ATPase. Here we show that TcpB, like its homolog CofB, initiates pilus assembly. TcpB co-localizes with the pili but at extremely low levels, equivalent to one subunit per pilus. We used a micropillars assay to demonstrate that TCP are retractile despite the absence of a retraction ATPase, and that retraction relies on TcpB, as a V. cholerae tcpB Glu5Val mutant is fully piliated but does not induce micropillars movements. This mutant is impaired in TCP-mediated autoagglutination and TcpF secretion, consistent with retraction being required for these functions. We propose that TcpB initiates pilus retraction by incorporating into the growing pilus in a Glu5-dependent manner, which stalls assembly and triggers processive disassembly. These results provide a framework for understanding filament dynamics in more complex T4P systems and the closely related Type II secretion system.

The Fatty Acid Regulator FadR Influences the Expression of the Virulence Cascade in the El Tor Biotype of Vibrio cholerae by Modulating the Levels of ToxT via Two Different Mechanisms.

FadR is a master regulator of fatty acid (FA) metabolism that coordinates the pathways of FA degradation and biosynthesis in enteric bacteria. We show here that a ΔfadR mutation in the El Tor biotype of Vibrio cholerae prevents the expression of the virulence cascade by influencing both the transcription and the posttranslational regulation of the master virulence regulator ToxT. FadR is a transcriptional regulator that represses the expression of genes involved in FA degradation, activates the expression of genes involved in unsaturated FA (UFA) biosynthesis, and also activates the expression of two operons involved in saturated FA (SFA) biosynthesis. Since FadR does not bind directly to the toxT promoter, we determined whether the regulation of any of its target genes indirectly influenced ToxT. This was accomplished by individually inserting a double point mutation into the FadR-binding site in the promoter of each target gene, thereby preventing their activation or repression. Although preventing FadR-mediated activation of fabA, which encodes the enzyme that carries out the first step in UFA biosynthesis, did not significantly influence either the transcription or the translation of ToxT, it reduced its levels and prevented virulence gene expression. In the mutant strain unable to carry out FadR-mediated activation of fabA, expressing fabA ectopically restored the levels of ToxT and virulence gene expression. Taken together, the results presented here indicate that V. cholerae FadR influences the virulence cascade in the El Tor biotype by modulating the levels of ToxT via two different mechanisms.IMPORTANCE Fatty acids (FAs) play important roles in membrane lipid homeostasis and energy metabolism in all organisms. In Vibrio cholerae, the causative agent of the acute intestinal disease cholera, they also influence virulence by binding into an N-terminal pocket of the master virulence regulator, ToxT, and modulating its activity. FadR is a transcription factor that coordinately controls the pathways of FA degradation and biosynthesis in enteric bacteria. This study identifies a new link between FA metabolism and virulence in the El Tor biotype by showing that FadR influences both the transcription and posttranslational regulation of the master virulence regulator ToxT by two distinct mechanisms.

Identification of a Small Molecule Activator for AphB, a LysR-Type Virulence Transcriptional Regulator in Vibrio cholerae.

AphB is a LysR-type transcriptional regulator (LTTR) that cooperates with a second transcriptional activator, AphA, at the tcpPH promoter to initiate expression of the virulence cascade in Vibrio cholerae. Because it is not yet known whether AphB responds to a natural ligand in V. cholerae that influences its ability to activate transcription, we used a computational approach to identify small molecules that influence its activity. In silico docking was used to identify potential ligands for AphB, and saturation transfer difference nuclear magnetic resonance was subsequently employed to access the validity of promising targets. We identified a small molecule, BP-15, that specifically binds the C-terminal regulatory domain of AphB and increases its activity. Interestingly, molecular docking predicts that BP-15 does not bind in the putative primary effector-binding pocket located at the interface of RD-I and RD-II as in other LTTRs, but rather at the dimerization interface. The information gained in this study helps us to further understand the mechanism by which transcriptional activation by AphB is regulated by suggesting that AphB has a secondary ligand binding site, as observed in other LTTRs. This study also lays the groundwork for the future design of inhibitory molecules to block the V. cholerae virulence cascade, thereby preventing the devastating symptoms of cholera infection.

Selection of positive controls and their impact on anti-drug antibody assay performance.

Development of assays to reliably identify and characterize anti-drug antibodies (ADAs) depends on positive control anti-idiotype (anti-id) reagents, which are used to demonstrate that the standards recommended by regulatory authorities are met. This work employs a set of therapeutic antibodies under clinical development and their corresponding anti-ids to investigate how different positive control reagent properties impact ADA assay development. Positive controls exhibited different response profiles and apparent assay analytical sensitivity values depending on assay format. Neither anti-id affinity for drug, nor sensitivity in direct immunoassays related to sensitivity in ADA assays. Anti-ids were differentially able to detect damage to drug conjugates used in bridging assays and were differentially drug tolerant. These parameters also failed to relate to assay sensitivity, further complicating selection of anti-ids for use in ADA assay development based on functional characteristics. Given this variability among anti-ids, alternative controls that could be employed across multiple antibody drugs were investigated as a more uniform means to define ADA detection sensitivity across drug products and assay protocols, which could help better relate assay results to clinical risks of ADA responses. Overall, this study highlights the importance of positive control selection to reliable detection and clinical interpretation of the presence and magnitude of ADA responses.

Characterization of BreR interaction with the bile response promoters breAB and breR in Vibrio cholerae.

The Vibrio cholerae BreR protein is a transcriptional repressor of the breAB efflux system operon, which encodes proteins involved in bile resistance. In a previous study (F. A. Cerda-Maira, C. S. Ringelberg, and R. K. Taylor, J. Bacteriol. 190:7441-7452, 2008), we used gel mobility shift assays to determine that BreR binds at two independent binding sites at the breAB promoter and a single site at its own promoter. Here it is shown, by DNase I footprinting and site-directed mutagenesis, that BreR is able to bind at a distal and a proximal site in the breAB promoter. However, only one of these sites, the proximal 29-bp site, is necessary for BreR-mediated transcriptional repression of breAB expression. In addition, it was determined that BreR represses its own expression by recognizing a 28-bp site at the breR promoter. These sites comprise regions of dyad symmetry within which residues critical for BreR function could be identified. The BreR consensus sequence AANGTANAC-N(6)-GTNTACNTT overlaps the -35 region at both promoters, implying that the repression of gene expression is achieved by interfering with RNA polymerase binding at these promoters.

The crystal structure of AphB, a virulence gene activator from Vibrio cholerae, reveals residues that influence its response to oxygen and pH.

Expression of the two critical virulence factors of Vibrio cholerae, toxin-coregulated pilus and cholera toxin, is initiated at the tcpPH promoter by the regulators AphA and AphB. AphA is a winged helix DNA-binding protein that enhances the ability of AphB, a LysR-type transcriptional regulator, to activate tcpPH expression. We present here the 2.2 Å X-ray crystal structure of full-length AphB. As reported for other LysR-type proteins, AphB is a tetramer with two distinct subunit conformations. Unlike other family members, AphB must undergo a significant conformational change in order to bind to DNA. We have found five independent mutations in the putative ligand-binding pocket region that allow AphB to constitutively activate tcpPH expression at the non-permissive pH of 8.5 and in the presence of oxygen. These findings indicate that AphB is responsive to intracellular pH as well as to anaerobiosis and that residues in the ligand-binding pocket of the protein influence its ability to respond to both of these signals. We have solved the structure of one of the constitutive mutants, and observe conformational changes that would allow DNA binding. Taken together, these results describe a pathway of conformational changes allowing communication between the ligand and DNA binding regions of AphB.

Characterization of Vibrio cholerae O1 El Tor biotype variant clinical isolates from Bangladesh and Haiti, including a molecular genetic analysis of virulence genes.

Vibrio cholerae serogroup O1, the causative agent of the diarrheal disease cholera, is divided into two biotypes: classical and El Tor. Both biotypes produce the major virulence factors toxin-coregulated pilus (TCP) and cholera toxin (CT). Although possessing genotypic and phenotypic differences, El Tor biotype strains displaying classical biotype traits have been reported and subsequently were dubbed El Tor variants. Of particular interest are reports of El Tor variants that produce various levels of CT, including levels typical of classical biotype strains. Here, we report the characterization of 10 clinical isolates from the International Centre for Diarrhoeal Disease Research, Bangladesh, and a representative strain from the 2010 Haiti cholera outbreak. We observed that all 11 strains produced increased CT (2- to 10-fold) compared to that of wild-type El Tor strains under in vitro inducing conditions, but they possessed various TcpA and ToxT expression profiles. Particularly, El Tor variant MQ1795, which produced the highest level of CT and very high levels of TcpA and ToxT, demonstrated hypervirulence compared to the virulence of El Tor wild-type strains in the infant mouse cholera model. Additional genotypic and phenotypic tests were conducted to characterize the variants, including an assessment of biotype-distinguishing characteristics. Notably, the sequencing of ctxB in some El Tor variants revealed two copies of classical ctxB, one per chromosome, contrary to previous reports that located ctxAB only on the large chromosome of El Tor biotype strains.

The LysR-type virulence activator AphB regulates the expression of genes in Vibrio cholerae in response to low pH and anaerobiosis.

AphB is a LysR-type activator that initiates the expression of the virulence cascade in Vibrio cholerae by cooperating with the quorum-sensing-regulated activator AphA at the tcpPH promoter on the Vibrio pathogenicity island (VPI). To identify the ancestral chromosomal genes in V. cholerae regulated by AphB, we carried out a microarray analysis and show here that AphB influences the expression of a number of genes that are not associated with the VPI. One gene strongly activated by AphB is cadC, which encodes the ToxR-like transcriptional activator responsible for activating the expression of lysine decarboxylase, which plays an important role in survival at low pH. Other genes activated by AphB encode a Na(+)/H(+) antiporter, a carbonic anhydrase, a member of the ClC family of chloride channels, and a member of the Gpr1/Fun34/YaaH family. AphB influences each of these genes directly by recognizing a conserved binding site within their promoters, as determined by gel mobility shift assays. Transcriptional lacZ fusions indicate that AphB activates the expression of these genes under aerobic conditions in response to low pH and also under anaerobic conditions at neutral pH. Further experiments show that the regulation of cadC by AphB in response to low pH and anaerobiosis is mirrored in the heterologous organism Escherichia coli, is independent of the global regulators Fnr and ArcAB, and depends upon the region of the promoter that contains the AphB binding site. These results raise the possibility that the activity of AphB is influenced by the pH and oxygen tension of the environment.

Structural basis for virulence regulation in Vibrio cholerae by unsaturated fatty acid components of bile.

The AraC/XylS-family transcriptional regulator ToxT is the master virulence activator of Vibrio cholerae, the gram-negative bacterial pathogen that causes the diarrheal disease cholera. Unsaturated fatty acids (UFAs) found in bile inhibit the activity of ToxT. Crystal structures of inhibited ToxT bound to UFA or synthetic inhibitors have been reported, but no structure of ToxT in an active conformation had been determined. Here we present the 2.5 Å structure of ToxT without an inhibitor. The structure suggests release of UFA or inhibitor leads to an increase in flexibility, allowing ToxT to adopt an active conformation that is able to dimerize and bind DNA. Small-angle X-ray scattering was used to validate a structural model of an open ToxT dimer bound to the cholera toxin promoter. The results presented here provide a detailed structural mechanism for virulence gene regulation in V. cholerae by the UFA components of bile and other synthetic ToxT inhibitors.

Integration host factor positively regulates virulence gene expression in Vibrio cholerae.

Virulence gene expression in Vibrio cholerae is dependent upon a complex transcriptional cascade that is influenced by both specific and global regulators in response to environmental stimuli. Here, we report that the global regulator integration host factor (IHF) positively affects virulence gene expression in V. cholerae. Inactivation of ihfA and ihfB, the genes encoding the IHF subunits, decreased the expression levels of the two main virulence factors tcpA and ctx and prevented toxin-coregulated pilus and cholera toxin production. IHF was found to directly bind to and bend the tcpA promoter region at an IHF consensus site centered at position -162 by using gel mobility shift assays and DNase I footprinting experiments. Deletion or mutation of the tcpA IHF consensus site resulted in the loss of IHF binding and additionally disrupted the binding of the repressor H-NS. DNase I footprinting revealed that H-NS protection overlaps with both the IHF and the ToxT binding sites at the tcpA promoter. In addition, disruption of ihfA in an hns or toxT mutant background had no effect on tcpA expression. These results suggest that IHF may function at the tcpA promoter to alleviate H-NS repression.

Architecture of the Vibrio cholerae toxin-coregulated pilus machine revealed by electron cryotomography.

Type IV pili (T4P) are filamentous appendages found on many Bacteria and Archaea. They are helical fibres of pilin proteins assembled by a multi-component macromolecular machine we call the basal body. Based on pilin features, T4P are classified into type IVa pili (T4aP) and type IVb pili (T4bP)1,2. T4aP are more widespread and are involved in cell motility3, DNA transfer4, host predation5 and electron transfer6. T4bP are less prevalent and are mainly found in enteropathogenic bacteria, where they play key roles in host colonization7. Following similar work on T4aP machines8,9, here we use electron cryotomography10 to reveal the three-dimensional in situ structure of a T4bP machine in its piliated and non-piliated states. The specific machine we analyse is the Vibrio cholerae toxin-coregulated pilus machine (TCPM). Although only about half of the components of the TCPM show sequence homology to components of the previously analysed Myxococcus xanthus T4aP machine (T4aPM), we find that their structures are nevertheless remarkably similar. Based on homologies with components of the M. xanthus T4aPM and additional reconstructions of TCPM mutants in which the non-homologous proteins are individually deleted, we propose locations for all eight TCPM components within the complex. Non-homologous proteins in the T4aPM and TCPM are found to form similar structures, suggesting new hypotheses for their functions and evolutionary histories.

A new class of inhibitors of the AraC family virulence regulator Vibrio cholerae ToxT.

Vibrio cholerae is responsible for the diarrheal disease cholera that infects millions of people worldwide. While vaccines protecting against cholera exist, and oral rehydration therapy is an effective treatment method, the disease will remain a global health threat until long-term solutions such as improved sanitation and access to clean water become widely available. Because of this, there is a pressing need for potent therapeutics that can either mitigate cholera symptoms, or act prophylactically to prevent the virulent effects of a cholera infection. Here we report the design, synthesis, and characterization of a set of compounds that bind and inhibit ToxT, the transcription factor that directly regulates the two primary V. cholerae virulence factors. Using the folded structure of the monounsaturated fatty acid observed in the X-ray structure of ToxT as a template, we designed ten novel compounds that inhibit the virulence cascade to a greater degree than any known inhibitor. Our findings provide a structural and functional basis for the development of viable antivirulence therapeutics that combat cholera and, potentially, other forms of bacterial pathogenic disease.

Origins of pandemic Vibrio cholerae from environmental gene pools.

Some microorganisms can transition from an environmental lifestyle to a pathogenic one1-3. This ecological switch typically occurs through the acquisition of horizontally acquired virulence genes4,5. However, the genomic features that must be present in a population before the acquisition of virulence genes and emergence of pathogenic clones remain unknown. We hypothesized that virulence adaptive polymorphisms (VAPs) circulate in environmental populations and are required for this transition. We developed a comparative genomic framework for identifying VAPs, using Vibrio cholerae as a model. We then characterized several environmental VAP alleles to show that while some of them reduced the ability of clinical strains to colonize a mammalian host, other alleles conferred efficient host colonization. These results show that VAPs are present in environmental bacterial populations before the emergence of virulent clones. We propose a scenario in which VAPs circulate in the environment and become selected and enriched under certain ecological conditions, and finally a genomic background containing several VAPs acquires virulence factors that allow for its emergence as a pathogenic clone.

A selective review of glutamate pharmacological therapy in obsessive-compulsive and related disorders.

Glutamate, an excitatory central nervous system neurotransmitter, is emerging as a potential alternative pharmacological treatment when compared to gamma-aminobutyric acid (GABA)-, dopamine-, and serotonin-modulating treatments for neuropsychiatric conditions. The pathophysiology, animal models, and clinical trials of glutamate modulation are explored in disorders with underlying inhibitory deficits (cognitive, motor, behavioral) including obsessive-compulsive disorder, attention deficit hyperactivity disorder, Tourette syndrome, trichotillomania, excoriation disorder, and nail biting. Obsessive-compulsive disorder, attention deficit hyperactivity disorder, and grooming disorders (trichotillomania and excoriation disorder) have emerging positive data, although only scarce controlled trials are available. The evidence is less supportive for the use of glutamate modulators in Tourette syndrome. Glutamate-modulating agents show promise in the treatment of disorders of inhibition.

The 40-residue insertion in Vibrio cholerae FadR facilitates binding of an additional fatty acyl-CoA ligand.

FadR is a master regulator of fatty acid metabolism and influences virulence in certain members of Vibrionaceae. Among FadR homologues of the GntR family, the Vibrionaceae protein is unusual in that it contains a C-terminal 40-residue insertion. Here we report the structure of Vibrio cholerae FadR (VcFadR) alone, bound to DNA, and in the presence of a ligand, oleoyl-CoA. Whereas Escherichia coli FadR (EcFadR) contains only one acyl-CoA-binding site in each monomer, crystallographic and calorimetric data indicate that VcFadR has two. One of the binding sites resembles that of EcFadR, whereas the other, comprised residues from the insertion, has not previously been observed. Upon ligand binding, VcFadR undergoes a dramatic conformational change that would more fully disrupt DNA binding than EcFadR. These findings suggest that the ability to bind and respond to an additional ligand allows FadR from Vibrionaceae to function as a more efficient regulator.

Pulsatile and Steady-State Pressure Trends in Children: A Window into the Future?

The aorta has limited ability to accommodate increasing body size by remodeling. The dramatic rise in pediatric obesity threatens to overwhelm this intrinsic remodeling program and lead to abnormal aortic function. As hypothesized, pulse pressure, as an index of aortic function, has indeed risen dramatically in parallel with the rise of pediatric obesity, while at the same time mean arterial pressure, as an index of small resistance artery function, has fallen. These divergent large-artery-versus-small-artery indices may combine to explain the counterintuitive decrease in systolic blood pressure in children and adults during the global obesity pandemic. The pathophysiologic mechanisms underpinning these contrasting trends are not yet known.

The quorum sensing regulator HapR downregulates the expression of the virulence gene transcription factor AphA in Vibrio cholerae by antagonizing Lrp- and VpsR-mediated activation.

HapR is a quorum sensing-regulated transcription factor that represses the virulence cascade in Vibrio cholerae by binding to a specific site centred at -71 in the aphA promoter, ultimately preventing activation of the tcpPH promoter on the Vibrio pathogenicity island. In an effort to elucidate the mechanism by which HapR represses aphA expression, we identified two transcriptional regulators, Lrp and VpsR, both of which activate the aphA promoter. Lrp, the leucine-responsive regulatory protein, binds to a region between -136 and -123 in the promoter to initiate aphA expression. VpsR, the response regulator that controls biofilm formation, binds to a region between -123 and -73 to activate aphA expression. HapR represses aphA expression by antagonizing the functions of both of these activators. The HapR binding site at -71 lies downstream of the Lrp binding site and overlaps the VpsR binding site. HapR binding thus directly blocks access of VpsR to the promoter. A naturally occurring point mutation in the aphA promoter (G-77T), which has previously been shown to prevent HapR binding, also prevents VpsR binding. In the absence of HapR, either Lrp or VpsR is capable of achieving nearly full expression of the aphA promoter, but when present together their effects are to some degree additive. The aphA promoter is also negatively autoregulated and an AphA binding site is centred at -20. The results here provide a model for the dual activation of the aphA promoter by Lrp and VpsR as well as its dual repression by HapR and AphA.

Crystal structure of the Vibrio cholerae quorum-sensing regulatory protein HapR.

Quorum sensing in Vibrio cholerae involves signaling between two-component sensor protein kinases and the response regulator LuxO to control the expression of the master regulator HapR. HapR, in turn, plays a central role in regulating a number of important processes, such as virulence gene expression and biofilm formation. We have determined the crystal structure of HapR to 2.2-A resolution. Its structure reveals a dimeric, two-domain molecule with an all-helical structure that is strongly conserved with members of the TetR family of transcriptional regulators. The N-terminal DNA-binding domain contains a helix-turn-helix DNA-binding motif and alteration of certain residues in this domain completely abolishes the ability of HapR to bind to DNA, alleviating repression of both virulence gene expression and biofilm formation. The C-terminal dimerization domain contains a unique solvent accessible tunnel connected to an amphipathic cavity, which by analogy with other TetR regulators, may serve as a binding pocket for an as-yet-unidentified ligand.