The toxbox: specific DNA sequence requirements for activation of Vibrio cholerae virulence genes by ToxT

The Gram-negative, curved rod Vibrio cholerae causes the severe diarrhoeal disease cholera. The two major virulence factors produced by V. cholerae during infection are the cholera toxin (CT) and the toxin-coregulated pilus (TCP). Transcription of the genes encoding both CT and the components of the TCP is directly activated by ToxT, a transcription factor in the AraC/XylS family. ToxT binds upstream of the ctxAB genes, encoding CT, and upstream of tcpA , the first gene in a large operon encoding the components of the TCP. The DNA sequences upstream of ctxAB and tcpA that contain ToxT binding sites do not have any significant similarity other than being AT-rich. Extensive site-directed mutagenesis was performed on the region upstream of tcpA previously shown to be protected by ToxT, and we identified specific base pairs important for activation of tcpA transcription by ToxT. This genetic approach was complemented by copper-phenanthroline footprinting experiments that showed protection by ToxT of the base pairs identified as most important for transcription activation in the mutagenesis experiments. Based on this new information and on previous work, we propose the presence of a ToxT-binding motif – the ‘toxbox’– in promoters regulated by ToxT. At tcpA , two toxbox elements are present in a direct repeat configuration and both are required for activation of transcription by ToxT. The identity of only a few of the base pairs within the toxbox is important for activation by ToxT, and we term these the core toxbox elements. Lastly, we examined ToxT binding to a mutant having 5 bp inserted between the two toxboxes at tcpA and found that occupancy of both binding sites is retained regardless of the positions of the binding sites relative to each other on the face of the DNA. This suggests that ToxT binds independently as a monomer to each toxbox in the tcpA direct repeat, in accordance with what we observed previously with the inverted repeat ToxT sites between acfA and acfD .Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/75755/1/j.1365-2958.2006.05053.x.pd

Risk assessment of EDCs in Europe based on human biomonitoring data

A major advantage of human biomonitoring (HBM) data is that they provide an integrated overview of the body burden to xenobiotics that an individual is exposed to. However, quantification of exposure based on HBM data poses significant challenges that are worth facing, given the opportunities that HBM provides in terms of informing and effectively supporting risk assessment. Based on the above, the aim of this within the HBM4EU project was to derive EU-wide external exposure estimates starting from HBM data and to derive and risk characterization ratio (RCR) by comparing these estimates with existing regulatory thresholds. For the reconstruction of exposure the INTEGRA computational platform was properly parameterised for the compounds of interest, namely bisphenol-A (BPA), phthalates (DEHP, DiNP and DnBP) and DINCH, emerging flame retardants (TCEP) and Perfluorinated compounds (PFOA and PFOS). The results indicated that for the majority of the examined compounds, daily intake levels are below the existing regulatory thresholds. For BPA, mean daily intake is almost 2 orders of magnitude below the respective threshold proposed by EFSA. For phthalates, daily intake estimates are usually one or two orders of magnitude below the respective TDI, with the exception of BBzP, for which intake estimates of the upper part of the exposure distribution is close to the threshold of 10 μg/kg_bw/d. Regarding TCEP, which is a typical emerging flame retardant, the mean daily intake estimate is below 0.1 μg/kg_bw/d, which is far below the calculated ‘provisional’ TDI of 13 μg/kg_bw/d, however, at the moment very few HBM data were available and these exposure levels are rather indicative than representative of the European countries. Finally, regarding the estimated intakes of PFCs, intake levels of PFOS are very close to TDI (0.15 μg/ kg_bw/d proposed by the CONTAM Panel), while the calculated levels for PFOA are one order of magnitude below the respective TDI of 1.5 μg/kg_bw/d

Biomarkers of exposure in environment-wide association studies – Opportunities to decode the exposome using human biomonitoring data

Background: The European Union's 7th Framework Programme (EU's FP7) project HEALS – Health and Environment-wide Associations based on Large Population Surveys – aims a refinement of the methodology to elucidate the human exposome. Human biomonitoring (HBM) provides a valuable tool for understanding the magnitude of human exposure from all pathways and sources. However, availability of specific biomarkers of exposure (BoE) is limited. Objectives: The objective was to summarize the availability of BoEs for a broad range of environmental stressors and exposure determinants and corresponding reference and exposure limit values and biomonitoring equivalents useful for unraveling the exposome using the framework of environment-wide association studies (EWAS). Methods: In a face-to-face group discussion, scope, content, and structure of the HEALS deliverable “Guidelines for appropriate BoE selection for EWAS studies” were determined. An expert-driven, distributed, narrative review process involving around 30 individuals of the HEALS consortium made it possible to include extensive information targeted towards the specific characteristics of various environmental stressors and exposure determinants. From the resulting 265 page report, targeted information about BoE, corresponding reference values (e.g., 95th percentile or measures of central tendency), exposure limit values (e.g., the German HBM I and II values) and biomonitoring equivalents (BEs) were summarized and updated. Results: 64 individual biological, chemical, physical, psychological and social environmental stressors or exposure determinants were included to fulfil the requirements of EWAS. The list of available BoEs is extensive with a number of 135; however, 12 of the stressors and exposure determinants considered do not leave any measurable specific substance in accessible body specimens. Opportunities to estimate the internal exposure stressors not (yet) detectable in human specimens were discussed. Conclusions: Data about internal exposures are useful to decode the exposome. The paper provides extensive information for EWAS. Information included serves as a guideline – snapshot in time without any claim to comprehensiveness – to interpret HBM data and offers opportunities to collect information about the internal exposure of stressors if no specific BoE is available

Regulation of gene expression in Vibrio cholerae by ToxT involves both antirepression and RNA polymerase stimulation

Co-ordinate expression of many virulence genes in the human pathogen Vibrio cholerae is under the direct control of the ToxT protein, including genes whose products are required for the biogenesis of the toxin-co-regulated pilus (TCP) and cholera toxin (CTX). This work examined interactions between ToxT and the promoters of ctx and tcpA genes. We found that a minimum of three direct repeats of the sequence TTTTGAT is required for ToxT-dependent activation of the ctx promoter, and that the region from –85 to –41 of the tcpA promoter contains elements that are responsive to ToxT-dependent activation. The role of H-NS in transcription of ctx and tcpA was also analysed. The level of activation of ctx–lacZ in an E. coli hns – strain was greatly increased even in the absence of ToxT, and was further enhanced in the presence of ToxT. In contrast, H-NS plays a lesser role in the regulation of the tcpA promoter. Electrophoretic mobility shift assays demonstrated that 6 × His-tagged ToxT directly, and specifically, interacts with both the ctx and tcpA promoters. DNase I footprinting analysis suggests that there may be two ToxT binding sites with different affinity in the ctx promoter and that ToxT binds to –84 to –41 of the tcpA promoter. In vitro transcription experiments demonstrated that ToxT alone is able to activate transcription from both promoters. We hypothesize that under conditions appropriate for ToxT-dependent gene expression, ToxT binds to AT-rich promoters that may have a specific secondary conformation, displaces H-NS and stimulates RNA polymerase resulting in transcription activation.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/71500/1/j.1365-2958.2002.02721.x.pd