Delphine Payros

Toxicology of deoxynivalenol and its acetylated and modified forms

Mycotoxins are the most frequently occurring natural contaminants in human and animal diet. Among them, deoxynivalenol (DON), produced by Fusarium, is one of the most prevalent and thus represents an important health risk. Recent detection methods revealed new mycotoxins and new molecules derivated from the “native” mycotoxins. The main derivates of DON are the acetylated forms produced by the fungi (3- and 15-acetyl-DON), the biologically “modified” forms produced by the plant (deoxynivalenol-3-β-d-glucopyranoside), or after bacteria transformation (de-epoxy DON, 3-epi-DON and 3-keto-DON) as well as the chemically “modified” forms (norDON A-C and DON-sulfonates). High proportions of acetylated and modified forms of DON co-occur with DON, increasing the exposure and the health risk. DON and its acetylated and modified forms are rapidly absorbed following ingestion. At the molecular level, DON binds to the ribosome, induces a ribotoxic stress leading to the activation of MAP kinases, cellular cell-cycle arrest and apoptosis. The toxic effects of DON include emesis and anorexia, alteration of intestinal and immune functions, reduced absorption of the nutrients as well as increased susceptibility to infection and chronic diseases. In contrast to DON, very little information exists concerning the acetylated and modified forms; some can be converted back to DON, their ability to bind to the ribosome and to induce cellular effects varies according to the toxin. Except for the acetylated forms, their toxicity and impact on human and animal health are poorly documented.

Interplay between Siderophores and Colibactin Genotoxin Biosynthetic Pathways in Escherichia coli

In Escherichia coli, the biosynthetic pathways of several small iron-scavenging molecules known as siderophores (enterobactin, salmochelins and yersiniabactin) and of a genotoxin (colibactin) are known to require a 4′-phosphopantetheinyl transferase (PPTase). Only two PPTases have been clearly identified: EntD and ClbA. The gene coding for EntD is part of the core genome of E. coli, whereas ClbA is encoded on the pks pathogenicity island which codes for colibactin. Interestingly, the pks island is physically associated with the high pathogenicity island (HPI) in a subset of highly virulent E. coli strains. The HPI carries the gene cluster required for yersiniabactin synthesis except for a gene coding its cognate PPTase. Here we investigated a potential interplay between the synthesis pathways leading to the production of siderophores and colibactin, through a functional interchangeability between EntD and ClbA. We demonstrated that ClbA could contribute to siderophores synthesis. Inactivation of both entD and clbA abolished the virulence of extra-intestinal pathogenic E. coli (ExPEC) in a mouse sepsis model, and the presence of either functional EntD or ClbA was required for the survival of ExPEC in vivo. This is the first report demonstrating a connection between multiple phosphopantetheinyl-requiring pathways leading to the biosynthesis of functionally distinct secondary metabolites in a given microorganism. Therefore, we hypothesize that the strict association of the pks island with HPI has been selected in highly virulent E. coli because ClbA is a promiscuous PPTase that can contribute to the synthesis of both the genotoxin and siderophores. The data highlight the complex regulatory interaction of various virulence features with different functions. The identification of key points of these networks is not only essential to the understanding of ExPEC virulence but also an attractive and promising target for the development of anti-virulence therapy strategies.

The Genotoxin Colibactin Exacerbates Lymphopenia and Decreases Survival Rate in Mice Infected With Septicemic Escherichia coli

Sepsis is a life-threatening infection. Escherichia coli is the first known cause of bacteremia leading to sepsis. Lymphopenia was shown to predict bacteremia better than conventional markers of infection. The pks genomic island, which is harbored by extraintestinal pathogenic E. coli (ExPEC) and encodes the genotoxin colibactin, is epidemiologically associated with bacteremia. To investigate a possible relationship between colibactin and lymphopenia, we examined the effects of transient infection of lymphocytes with bacteria that were and those that were not producing the genotoxin. A mouse model of sepsis was used to compare the virulence of a clinical ExPEC isolate with its isogenic mutant impaired for the production of colibactin. We observed that colibactin induced double-strand breaks in the DNA of infected lymphocytes, leading to cell cycle arrest and to cell death by apoptosis. E. coli producing colibactin induced a more profound lymphopenia in septicemic mice, compared with the isogenic mutant unable to produce colibactin. In a sepsis model in which the mice were treated by rehydration and antibiotics, the production of colibactin by the bacteria was associated with a significantly lower survival rate. In conclusion, we demonstrate that production of colibactin by E. coli exacerbates lymphopenia associated with septicemia and could impair the chances to survive sepsis.

Impact of mycotoxins on the intestine: are mucus and microbiota new targets?

ABSTRACT There is an increasing awareness of the deleterious effects attributed to mycotoxins during their fate within the gut, particularly for deoxynivalenol (DON), zearalenone (ZEN), ochratoxin A (OTA), fumonisin B1 (FB1), aflatoxin B1 (AFB1), and patulin (PAT). Evidence indicates that disruption of the epithelial barrier is well established. However, intestinal barrier function on its luminal side involves two other partners, mucus and microbiota, which have rarely been considered in the context of mycotoxin exposure. The current review aimed at providing a summary of DON, ZEN, OTA, FB1, AFB1, and PAT effects on intestinal barrier function, with special focus on mucus and microbiota. DON, ZEN, OTA, FB1, AFB1, and PAT are known to markedly affect epithelial cell integrity and functions. Regarding mucus, DON is the most documentated mycotoxin. In vivo, toxicological impact of DON generally has only been assessed through goblet cell number. Evaluation of the mycotoxins/mucus interplay considering other indicators such as composition, thickness, and penetrability of mucus, mucin O-glycosylation thus warrants further attention. With respect to microbiota, few short-term studies to date have been reported indicating deleterious effects. However, long-term exposure to mycotoxins may also produce significant changes in microbiota composition and metabolic activity, which requires further experimentation. In conclusion, mucus and microbiota are key targets for dietary mycotoxins although assessment of induced effects is preliminary. A significant research effort is now underway to determine the adverse consequences of mycotoxins on mucus and microbiota considered as individual but also as tightly connected gut players.

Maternally acquired genotoxic Escherichia coli alters offspring's intestinal homeostasis.

The neonatal gut is rapidly colonized by a newly dominant group of commensal Escherichia coli strains among which a large proportion produces a genotoxin called colibactin. In order to analyze the short- and long-term effects resulting from such evolution, we developed a rat model mimicking the natural transmission of E. coli from mothers to neonates. Genotoxic and non-genotoxic E. coli strains were equally transmitted to the offspring and stably colonized the gut across generations. DNA damage was only detected in neonates colonized with genotoxic E. coli strains. Signs of genotoxic stress such as anaphase bridges, higher occurrence of crypt fission and accelerated renewal of the mature epithelium were detected at adulthood. In addition, we observed alterations of secretory cell populations and gut epithelial barrier. Our findings illustrate how critical is the genotype of E. coli strains acquired at birth for gut homeostasis at adulthood.

The Food Contaminant Deoxynivalenol Exacerbates the Genotoxicity of Gut Microbiota

ABSTRACT An increasing number of human beings from developed countries are colonized by Escherichia coli strains producing colibactin, a genotoxin suspected to be associated with the development of colorectal cancers. Deoxynivalenol (DON) is the most prevalent mycotoxin that contaminates staple food—especially cereal products—in Europe and North America. This study investigates the effect of the food contaminant DON on the genotoxicity of the E. coli strains producing colibactin. In vitro, intestinal epithelial cells were coexposed to DON and E. coli producing colibactin. In vivo, newborn rats colonized at birth with E. coli producing colibactin were fed a DON-contaminated diet. Intestinal DNA damage was estimated by the phosphorylation of histone H2AX. DON exacerbates the genotoxicity of the E. coli producing colibactin in a time- and dose-dependent manner in vitro. Although DON had no effect on the composition of the gut microbiota, and especially on the number of E. coli, a significant increase in DNA damage was observed in intestinal epithelial cells of animals colonized by E. coli strains producing colibactin and coexposed to DON compared to animals colonized with E. coli strains unable to produce colibactin or animals exposed only to DON. In conclusion, our data demonstrate that the genotoxicity of E. coli strains producing colibactin, increasingly present in the microbiota of asymptomatic human beings, is modulated by the presence of DON in the diet. This raises questions about the synergism between food contaminants and gut microbiota with regard to intestinal carcinogenesis. IMPORTANCE An increasing number of human beings from developed countries are colonized by Escherichia coli strains producing colibactin, a genotoxin suspected to be associated with the development of colorectal cancers. Deoxynivalenol (DON) is the most prevalent mycotoxin that contaminates staple food—especially cereal products—in Europe and North America. Our in vitro and in vivo results demonstrate that the intestinal DNA damage induced by colibactin-producing E. coli strains was exacerbated by the presence of DON in the diet. This raises questions about the synergism between food contaminants and gut microbiota with regard to intestinal carcinogenesis. IMPORTANCE An increasing number of human beings from developed countries are colonized by Escherichia coli strains producing colibactin, a genotoxin suspected to be associated with the development of colorectal cancers. Deoxynivalenol (DON) is the most prevalent mycotoxin that contaminates staple food—especially cereal products—in Europe and North America. Our in vitro and in vivo results demonstrate that the intestinal DNA damage induced by colibactin-producing E. coli strains was exacerbated by the presence of DON in the diet. This raises questions about the synergism between food contaminants and gut microbiota with regard to intestinal carcinogenesis.

Oral Tolerance Failure upon Neonatal Gut Colonization with Escherichia coli Producing the Genotoxin Colibactin

ABSTRACT The intestinal barrier controls the balance between tolerance and immunity to luminal antigens. When this finely tuned equilibrium is deregulated, inflammatory disorders can occur. There is a concomitant increase, in urban populations of developed countries, of immune-mediated diseases along with a shift in Escherichia coli population from the declining phylogenetic group A to the newly dominant group B2, including commensal strains producing a genotoxin called colibactin that massively colonized the gut of neonates. Here, we showed that mother-to-offspring early gut colonization by colibactin-producing E. coli impairs intestinal permeability and enhances the transepithelial passage of luminal antigen, leading to an increased immune activation. Functionally, this was accompanied by a dramatic increase in local and systemic immune responses against a fed antigen, decreased regulatory T cell population, tolerogenic dendritic cells, and enhanced mucosal delayed-type hypersensitivity response. Conversely, the abolition of colibactin expression by mutagenesis abrogates the alteration of oral tolerance induced by neonatal colonization by E. coli. In conclusion, the vertical colonization by E. coli producing the genotoxin colibactin enhances intestinal translocation and subsequently alters oral tolerance. Thus, early colonization by E. coli from the newly dominant phylogenetic group B2, which produces colibactin, may represent a risk factor for the development of immune-mediated diseases.

Intestinal toxicity of deoxynivalenol is limited by Lactobacillus rhamnosus RC007 in pig jejunum explants

Probiotics have been explored to stimulate gut health in weaned pigs, when they started to consume solid diet where mycotoxins could be present. The aim of this study was to evaluate the effect of Lactobacillus rhamnosus RC007 on the intestinal toxicity of deoxynivalenol (DON) in an ex vivo model. Jejunal explants, obtained from 5-week-old crossbred castrated male piglets, were kept as control, exposed for 3xa0h to 10xa0μM DON, incubated for 4xa0h with 109 CFU/mL L. rhamnosus, or pre-incubated 1xa0h with 109L. rhamnosus and exposed to DON. Histological lesions were observed, para- and transcellular intestinal permeability was measured in Ussing chambers. The expression levels of mRNA encoding six inflammatory cytokines (CCL20, IL-10, IL-1β, TNFα, IL-8 and IL-22) were determined by RT-PCR. The expressions of the phosphorylated MAP kinases p42/p44 and p38 were assessed by immunoblotting. Exposure to DON induced histological changes, significantly increased the expression of CCL20, IL-1β, TNFα, IL-8, IL-22 and IL-10, increased the intestinal paracellular permeability and activated MAP kinases. Incubation with L. rhamnosus alone did not have any significant effect. By contrast, the pre-incubation with L. rhamnosus reduced all the effects of DON: the histological alterations, the pro-inflammatory response, the paracellular permeability and the phosphorylation of MAP kinases. Of note, L. rhamnosus did not adsorb DON and only slightly degrade the toxin. In conclusion, L. rhamnosus RC007 is a promising probiotic which, included as feed additive, can decrease the intestinal toxicity of DON.

Protein O -mannosylation deficiency increases LprG-associated lipoarabinomannan release by Mycobacterium tuberculosis and enhances the TLR2-associated inflammatory response

Protein O-mannosylation is crucial for the biology of Mycobacterium tuberculosis but the key mannosylated protein(s) involved and its(their) underlying function(s) remain unknown. Here, we demonstrated that the M. tuberculosis mutant (Δpmt) deficient for protein O-mannosylation exhibits enhanced release of lipoarabinomannan (LAM) in a complex with LprG, a lipoprotein required for LAM translocation to the cell surface. We determined that LprG is O-mannosylated at a unique threonine position by mass spectrometry analyses of the purified protein. However, although replacement of this amino acid by an alanine residue completely abolished LprG O-mannosylation, the increased release of the LAM/LprG complex was preserved. We found that the increased secretion of this complex is due to enhanced LAM production in the Δpmt M. tuberculosis and M. smegmatis mutants relative to their wild-type counterparts. This abnormal release of LAM/LprG has functional consequences on the induction of inflammatory responses and provides a possible explanation for the reduced virulence of the M. tuberculosis Δpmt mutant.