Adopting Mechanistic Molecular Biology Approaches in Exposome Research for Causal Understanding.

Through investigating the combined impact of the environmental exposures experienced by an individual throughout their lifetime, exposome research provides opportunities to understand and mitigate negative health outcomes. While current exposome research is driven by epidemiological studies that identify associations between exposures and effects, new frameworks integrating more substantial population-level metadata, including electronic health and administrative records, will shed further light on characterizing environmental exposure risks. Molecular biology offers methods and concepts to study the biological and health impacts of exposomes in experimental and computational systems. Of particular importance is the growing use of omics readouts in epidemiological and clinical studies. This paper calls for the adoption of mechanistic molecular biology approaches in exposome research as an essential step in understanding the genotype and exposure interactions underlying human phenotypes. A series of recommendations are presented to make the necessary and appropriate steps to move from exposure association to causation, with a huge potential to inform precision medicine and population health. This includes establishing hypothesis-driven laboratory testing within the exposome field, supported by appropriate methods to read across from model systems research to human.

Current EU research activities on combined exposure to multiple chemicals.

Humans and wildlife are exposed to an intractably large number of different combinations of chemicals via food, water, air, consumer products, and other media and sources. This raises concerns about their impact on public and environmental health. The risk assessment of chemicals for regulatory purposes mainly relies on the assessment of individual chemicals. If exposure to multiple chemicals is considered in a legislative framework, it is usually limited to chemicals falling within this framework and co-exposure to chemicals that are covered by a different regulatory framework is often neglected. Methodologies and guidance for assessing risks from combined exposure to multiple chemicals have been developed for different regulatory sectors, however, a harmonised, consistent approach for performing mixture risk assessments and management across different regulatory sectors is lacking. At the time of this publication, several EU research projects are running, funded by the current European Research and Innovation Programme Horizon 2020 or the Seventh Framework Programme. They aim at addressing knowledge gaps and developing methodologies to better assess chemical mixtures, by generating and making available internal and external exposure data, developing models for exposure assessment, developing tools for in silico and in vitro effect assessment to be applied in a tiered framework and for grouping of chemicals, as well as developing joint epidemiological-toxicological approaches for mixture risk assessment and for prioritising mixtures of concern. The projects EDC-MixRisk, EuroMix, EUToxRisk, HBM4EU and SOLUTIONS have started an exchange between the consortia, European Commission Services and EU Agencies, in order to identify where new methodologies have become available and where remaining gaps need to be further addressed. This paper maps how the different projects contribute to the data needs and assessment methodologies and identifies remaining challenges to be further addressed for the assessment of chemical mixtures.

European Union research in support of environment and health: Building scientific evidence base for policy.

Opinion polls show that the European Union citizens are increasingly concerned about the impact of environmental factors on their health. In order to respond and provide solid scientific evidence for the numerous policies related to the protection of human health and the environment managed at the Union level, the European Union made a substantial investment in research and innovation in the past two decades through its Framework Programmes for Research and Technological Development, including the current programme, Horizon 2020, which started in 2014. This policy review paper analysed the portfolio of forty collaborative projects relevant to environment and health, which received a total amount of around 228 million euros from the EU. It gives details on their contents and general scientific trends observed, the profiles of the participating countries and institutions, and the potential policy implications of the results obtained. The increasing knowledge base is needed to make informed policy decisions in Europe and beyond, and should be useful to many stakeholders including the scientific community and regulatory authorities.

Development of an integrative vector for the expression of antisense RNA in Clostridium difficile.

A method was developed to use the conjugative transposon Tn916 as a vector for introducing recombinant DNA into Clostridium difficile. This was used to introduce antisense RNA for the adhesin encoding gene cwp66 into C. difficile 79-685. RT-PCR demonstrated that cwp66 specific antisense RNA was produced. However, there was no statistically significant difference in the protein expression or in the adherence of recombinant C. difficile strains. This may be due to the amount of transcripts of the wild-type (sense) cwp66 outnumbering the antisense transcripts or secondary structures present within the cwp66 mRNA. Unlike in other strains of C. difficile, where Tn916 inserts into the genome at highly preferred sites, in C. difficile 79-685, it integrates into multiple sites opening up the possibility of using Tn916 as a mutagen in this strain.

Identification and characterization of a fibronectin-binding protein from Clostridium difficile.

A 68 kDa fibronectin-binding protein (Fbp68) from Clostridium difficile displaying significant homology to several established or putative Fbps from other bacteria was identified. The one-copy gene is highly conserved in C. difficile isolates. Fbp68 was expressed in Escherichia coli in fusion with glutathione S-transferase; the fusion protein and the native Fbp68 were purified. Immunoblot analysis and cell fractionation experiments revealed that Fbp68 is present on the surface of the bacteria. Far-immuno dot-blotting demonstrated that Fbp68 was capable of fixing fibronectin. Indirect immunofluorescence and ELISA were employed to demonstrate that C. difficile could bind both soluble and immobilized fibronectin. With competitive adherence inhibition assays it was shown that antibodies raised against Fbp68 partially inhibited attachment of C. difficile to fibronectin and Vero cells. Furthermore, Vero cells could fix purified membrane-immobilized Fbp68. Thus Fbp68 appears to be one of the several adhesins identified to date in C. difficile.

Transcription and analysis of polymorphism in a cluster of genes encoding surface-associated proteins of Clostridium difficile.

Recent investigations of the Clostridium difficile genome have revealed the presence of a cluster of 17 genes, 11 of which encode proteins with similar two-domain structures, likely to be surface-anchored proteins. Two of these genes have been proven to encode proteins involved in cell adherence: slpA encodes the precursor of the two proteins of the S-layer, P36 and P47, whereas cwp66 encodes the Cwp66 adhesin. To gain further insight into the function of this cluster, we further focused on slpA, cwp66, and cwp84, the latter of which encodes a putative surface-associated protein with homology to numerous cysteine proteases. It displayed nonspecific proteolytic activity when expressed as a recombinant protein in Escherichia coli. Polymorphism of cwp66 and cwp84 genes was analyzed in 28 strains, and transcriptional organization of the three genes was explored by Northern blots. The slpA gene is strongly transcribed during the entire growth phase as a bicistronic transcript; cwp66 is transcribed only in the early exponential growth phase as a polycistronic transcript encompassing the two contiguous genes upstream. The putative proteins encoded by the cotranscribed genes have no significant homology with known proteins but may have a role in adherence. No correlation could be established between sequence patterns of Cwp66 and Cwp84 and virulence of the strains. The cwp84 gene is strongly transcribed as a monocistronic message. This feature, together with the highly conserved sequence pattern of cwp84, suggests a significant role in the physiopathology of C. difficile for the Cwp84 protease, potentially in the maturation of surface-associated adhesins encoded by the gene cluster.

Inhibition of in vitro cell adherence of Clostridium difficile by Saccharomyces boulardii.

The influence on the adherence of Clostridium difficile to Vero cells of the yeast Saccharomyces boulardii, the yeast fractions (cytoplasm and cell wall) and the culture supernatant was investigated in vitro. C. difficile adherence was significantly inhibited when bacteria were pre-incubated with the whole yeast and the cell wall fraction; this adherence inhibition was dose-dependent. The cell wall fraction also acts upon the target cultured cells inasmuch as the level of adherence was significantly decreased when Vero cells were preincubated with it. The same experiments carried out in the presence of an inhibitor of serine proteases resulted in no inhibition of bacterial adherence. These results suggest that the yeast could inhibit adherence of C. difficile to cells thanks to its proteolytic activity but also through steric hindrance.

Clostridium difficile genotyping based on slpA variable region in S-layer gene sequence: an alternative to serotyping.

Recent investigations of Clostridium difficile cell wall components have revealed the presence of an S-layer encoded by the slpA gene. The aim of this study was to determine whether slpA genotyping can be used as an alternative to serotyping. The variable regions of slpA were amplified by PCR from serogroup reference strains and various clinical isolates chosen randomly. Amplified products were analyzed after restriction enzyme digestion and DNA sequencing. The sequences of the variable region of the SlpA protein were found to be strictly identical within a given serogroup but divergent between serogroups. These preliminary results suggest that PCR-restriction fragment length polymorphism, in conjunction with DNA sequencing of the slpA variable region, could constitute an alternative typing method for determining C. difficile serotypes.

A Clostridium difficile gene encoding flagellin.

Six strains of Clostridium difficile examined by electron microscopy were found to carry flagella. The flagella of these strains were extracted and the N-terminal sequences of the flagellin proteins were determined. Four of the strains carried the N-terminal sequence MRVNTNVSAL exhibiting up to 90% identity to numerous flagellins. Using degenerate primers based on the N-terminal sequence and the conserved C-terminal sequence of several flagellins, the gene encoding the flagellum subunit (fliC) was isolated and sequenced from two virulent strains. The two gene sequences exhibited 91% inter-strain identity. The gene consists of 870 nt encoding a protein of 290 amino acids with an estimated molecular mass of 31 kDa, while the extracted flagellin has an apparent molecular mass of 39 kDa on SDS-PAGE. The FliC protein displays a high degree of identity in the N- and C-terminal amino acids whereas the central region is variable. A second ORF is present downstream of fliC displaying homology to glycosyltransferases. The fliC gene was expressed in fusion with glutathione S-transferase, purified and a polyclonal monospecific antiserum was obtained. Flagella of C. difficile do not play a role in adherence, since the antiserum raised against the purified protein did not inhibit adherence to cultured cells. PCR-RFLP analysis of amplified flagellin gene products and Southern analysis revealed inter-strain heterogeneity; this could be useful for epidemiological and phylogenetic studies of this organism.