From ribosome to ribotoxins: understanding the toxicity of deoxynivalenol and Shiga toxin, two food borne toxins.

Ribosomes that synthesize proteins are among the most central and evolutionarily conserved organelles. Given the key role of proteins in cellular functions, prokaryotic and eukaryotic pathogens have evolved potent toxins to inhibit ribosomal functions and weaken their host. Many of these ribotoxin-producing pathogens are associated with food. For example, food can be contaminated with bacterial pathogens that produce the ribotoxin Shiga toxin, but also with the fungal ribotoxin deoxynivalenol. Shiga toxin cleaves ribosomal RNA, while deoxynivalenol binds to and inhibits the peptidyl transferase center. Despite their distinct modes of action, both groups of ribotoxins hinder protein translation, but also trigger other comparable toxic effects, which depend or not on the activation of the ribotoxic stress response. Ribotoxic stress response-dependent effects include inflammation and apoptosis, whereas ribotoxic stress response-independent effects include endoplasmic reticulum stress, oxidative stress, and autophagy. For other effects, such as cell cycle arrest and cytoskeleton modulation, the involvement of the ribotoxic stress response is still controversial. Ribotoxins affect one organelle yet induce multiple toxic effects with multiple consequences for the cell. The ribosome can therefore be considered as the cellular "Achilles heel" targeted by food borne ribotoxins. Considering the high toxicity of ribotoxins, they pose a substantial health risk, as humans are highly susceptible to widespread exposure to these toxins through contaminated food sources.

HlyF, an underestimated virulence factor of uropathogenic Escherichia coli.

OBJECTIVES: Urinary tract infections (UTIs) are primarily caused by uropathogenic Escherichia coli (UPEC). This study aims to elucidate the role of the virulence factor HlyF in the epidemiology and pathophysiology of UTIs and investigate the dissemination of plasmids carrying the hlyF gene. METHODS: An epidemiological analysis was conducted on a representative collection of 225 UPEC strains isolated from community-acquired infections. Selected hlyF+ strains were fully sequenced using a combination of Illumina and Nanopore technologies. To investigate the impact of HlyF, a murine model of UTI was utilized to compare clinical signs, bacterial loads in the bladder, kidney, and spleen, onset of bacteraemia, and inflammation through cytokine quantification among wild-type hlyF+ strains, isogenic mutants, and complemented mutants. RESULTS: Our findings demonstrate that 20% of UPEC encode the HlyF protein. These hlyF+ UPEC strains exhibited enhanced virulence, frequently leading to pyelonephritis accompanied by bloodstream infections. Unlike typical UPEC strains, hlyF+ UPEC strains demonstrate a broader phylogroup distribution and possess a unique array of virulence factors and antimicrobial resistance genes, primarily carried by ColV-like plasmids. In the murine UTI model, expression of HlyF was linked to the UPECs' capacity to induce urosepsis and elicit an exacerbated inflammatory response, setting them apart from typical UPEC strains. DISCUSSION: Overall, our results strongly support the notion that HlyF serves as a significant virulence factor for UPECs, and the dissemination of ColV-like plasmids encoding HlyF warrants further investigation.

Oxygen concentration modulates colibactin production.

Up to 25% of the E. coli strains isolated from the feces of healthy humans harbor the pks genomic island encoding the synthesis of colibactin, a genotoxic metabolite. Evidence is accumulating for an etiologic role of colibactin in colorectal cancer. Little is known about the conditions of expression of colibactin in the gut. The intestine is characterized by a unique oxygenation profile, with a steep gradient between the physiological hypoxic epithelial surface and the anaerobic lumen, which favors the dominance of obligate anaerobes. Here, we report that colibactin production is maximal under anoxic conditions and decreases with increased oxygen concentration. We show that the aerobic respiration control (ArcA) positively regulates colibactin production and genotoxicity of pks+ E. coli in response to oxygen availability. Thus, colibactin synthesis is inhibited by oxygen, indicating that the pks biosynthetic pathway is adapted to the anoxic intestinal lumen and to the hypoxic infected or tumor tissue.

A novel toxic effect of foodborne trichothecenes: The exacerbation of genotoxicity.

Trichothecenes (TCT) are very common mycotoxins. While the effects of DON, the most prevalent TCT, have been extensively studied, less is known about the effect of other trichothecenes. DON has ribotoxic, pro-inflammatory, and cytotoxic potential and induces multiple toxic effects in humans and animals. Although DON is not genotoxic by itself, it has recently been shown that this toxin exacerbates the genotoxicity induced by model or bacterial genotoxins. Here, we show that five TCT, namely T-2 toxin (T-2), diacetoxyscirpenol (DAS), nivalenol (NIV), fusarenon-X (FX), and the newly discovered NX toxin, also exacerbate the DNA damage inflicted by various genotoxins. The exacerbation was dose dependent and observed with phleomycin, a model genotoxin, captan, a pesticide with genotoxic potential, and colibactin, a bacterial genotoxin produced by the intestinal microbiota. For this newly described effect, the trichothecenes ranked in the following order: T-2>DAS > FX > NIV ≥ DON ≥ NX. The genotoxic exacerbating effect of TCT correlated with their ribotoxic potential, as measured by the inhibition of protein synthesis. In conclusion, our data demonstrate that TCT, which are not genotoxic by themselves, exacerbate DNA damage induced by various genotoxins. Therefore, foodborne TCT could enhance the carcinogenic potential of genotoxins present in the diet or produced by intestinal bacteria.

Uncoupling the Hsp90 and DnaK chaperone activities revealed the in vivo relevance of their collaboration in bacteria.

Chaperone proteins are essential in all living cells to ensure protein homeostasis. Hsp90 is a major adenosine triphosphate (ATP)-dependent chaperone highly conserved from bacteria to eukaryotes. Recent studies have shown that bacterial Hsp90 is essential in some bacteria in stress conditions and that it participates in the virulence of pathogenic bacteria. In vitro, bacterial Hsp90 directly interacts and collaborates with the Hsp70 chaperone DnaK to reactivate model substrate proteins; however, it is still unknown whether this collaboration is relevant in vivo with physiological substrates. Here, we used site-directed mutagenesis on Hsp90 to impair DnaK binding, thereby uncoupling the chaperone activities. We tested the mutants in vivo in two bacterial models in which Hsp90 has known physiological functions. We found that the Hsp90 point mutants were defective to support (1) growth under heat stress and activation of an essential Hsp90 client in the aquatic bacterium Shewanella oneidensis and (2) biosynthesis of the colibactin toxin involved in the virulence of pathogenic Escherichia coli. Our study therefore demonstrates the essentiality of the direct collaboration between Hsp90 and DnaK in vivo in bacteria to support client folding. It also suggests that this collaboration already functional in bacteria has served as an evolutionary basis for a more complex Hsp70-Hsp90 collaboration found in eukaryotes.

The pks island: a bacterial Swiss army knife? Colibactin: beyond DNA damage and cancer.

The structure and mode of action of colibactin with its potential involvement in cancer have been extensively studied but little is known about the intrinsic function of the biosynthetic gene cluster, coding for colibactin, as a bacterial genotoxin. Paradoxically, this pathogenicity island is also found in commensal and probiotic strains of Escherichia coli and in bacterial species colonizing olive trees and the digestive tract of bees. In this review, we summarize the available literature to address the following key questions. What does this genomic island really encode? What explains the extensive dissemination of this genetically mobile element? What do we really know about the biosynthetic and secretory pathways of colibactin? What is its inherent target/function?

The foodborne contaminant deoxynivalenol exacerbates DNA damage caused by a broad spectrum of genotoxic agents.

Humans are exposed to different contaminants including mycotoxins. Deoxynivalenol (DON), a potent ribosome inhibitor, is a highly prevalent mycotoxin in the food chain worldwide. Although DON is not genotoxic, we previously showed that it exacerbates the genotoxicity of colibactin, a DNA-crosslinking toxin produced by bacteria in the gut. In the present study, we investigated whether this phenotype can be extended to other genotoxic compounds with different modes of action. Our data showed that, at a dose that can be found in food, DON exacerbated the DNA damage caused by etoposide, cisplatin and phleomycin. In contrast, de-epoxy-deoxynivalenol (DOM-1), a modified form of DON that does not induce ribotoxic stress, did not exacerbate DNA damage. The effect of DON was mimicked with other ribosome inhibitors such as anisomycin and cycloheximide, suggesting that ribotoxicity plays a key role in exacerbating DNA damage. In conclusion, a new effect of DON was identified, this toxin aggravates the DNA damage induced by a broad spectrum of genotoxic agents with different modes of action. These results are of utmost importance as our food can be co-contaminated with DON and DNA-damaging agents.

Tackling the Threat of Cancer Due to Pathobionts Producing Colibactin: Is Mesalamine the Magic Bullet?

Colibactin is a genotoxin produced primarily by Escherichia coli harboring the genomic pks island (pks+ E. coli). Pks+ E. coli cause host cell DNA damage, leading to chromosomal instability and gene mutations. The signature of colibactin-induced mutations has been described and found in human colorectal cancer (CRC) genomes. An inflamed intestinal environment drives the expansion of pks+ E. coli and promotes tumorigenesis. Mesalamine (i.e., 5-aminosalycilic acid), an effective anti-inflammatory drug, is an inhibitor of the bacterial polyphosphate kinase (PPK). This drug not only inhibits the production of intestinal inflammatory mediators and the proliferation of CRC cells, but also limits the abundance of E. coli in the gut microbiota and diminishes the production of colibactin. Here, we describe the link between intestinal inflammation and colorectal cancer induced by pks+ E. coli. We discuss the potential mechanisms of the pleiotropic role of mesalamine in treating both inflammatory bowel diseases and reducing the risk of CRC due to pks+ E. coli.

A Toxic Friend: Genotoxic and Mutagenic Activity of the Probiotic Strain Escherichia coli Nissle 1917.

The probiotic Escherichia coli strain Nissle 1917 (DSM 6601, Mutaflor), generally considered beneficial and safe, has been used for a century to treat various intestinal diseases. However, Nissle 1917 hosts in its genome the pks pathogenicity island that codes for the biosynthesis of the genotoxin colibactin. Colibactin is a potent DNA alkylator, suspected to play a role in colorectal cancer development. We show in this study that Nissle 1917 is functionally capable of producing colibactin and inducing interstrand cross-links in the genomic DNA of epithelial cells exposed to the probiotic. This toxicity was even exacerbated with lower doses of the probiotic, when the exposed cells started to divide again but exhibited aberrant anaphases and increased gene mutation frequency. DNA damage was confirmed in vivo in mouse models of intestinal colonization, demonstrating that Nissle 1917 produces the genotoxin in the gut lumen. Although it is possible that daily treatment of adult humans with their microbiota does not produce the same effects, administration of Nissle 1917 as a probiotic or as a chassis to deliver therapeutics might exert long-term adverse effects and thus should be considered in a risk-versus-benefit evaluation. IMPORTANCE Nissle 1917 is sold as a probiotic and considered safe even though it has been known since 2006 that it harbors the genes for colibactin synthesis. Colibactin is a potent genotoxin that is now linked to causative mutations found in human colorectal cancer. Many papers concerning the use of this strain in clinical applications ignore or elude this fact or misleadingly suggest that Nissle 1917 does not induce DNA damage. Here, we demonstrate that Nissle 1917 produces colibactin in vitro and in vivo and induces mutagenic DNA damage. This is a serious safety concern that must not be ignored in the interests of patients, the general public, health care professionals, and ethical probiotic manufacturers.

Insights into the acquisition of the pks island and production of colibactin in the Escherichia coli population.

The pks island codes for the enzymes necessary for synthesis of the genotoxin colibactin, which contributes to the virulence of Escherichia coli strains and is suspected of promoting colorectal cancer. From a collection of 785 human and bovine E. coli isolates, we identified 109 strains carrying a highly conserved pks island, mostly from phylogroup B2, but also from phylogroups A, B1 and D. Different scenarios of pks acquisition were deduced from whole genome sequence and phylogenetic analysis. In the main scenario, pks was introduced and stabilized into certain sequence types (STs) of the B2 phylogroup, such as ST73 and ST95, at the asnW tRNA locus located in the vicinity of the yersiniabactin-encoding High Pathogenicity Island (HPI). In a few B2 strains, pks inserted at the asnU or asnV tRNA loci close to the HPI and occasionally was located next to the remnant of an integrative and conjugative element. In a last scenario specific to B1/A strains, pks was acquired, independently of the HPI, at a non-tRNA locus. All the pks-positive strains except 18 produced colibactin. Sixteen strains contained mutations in clbB or clbD, or a fusion of clbJ and clbK and were no longer genotoxic but most of them still produced low amounts of potentially active metabolites associated with the pks island. One strain was fully metabolically inactive without pks alteration, but colibactin production was restored by overexpressing the ClbR regulator. In conclusion, the pks island is not restricted to human pathogenic B2 strains and is more widely distributed in the E. coli population, while preserving its functionality.

Uropathogenic E. coli induces DNA damage in the bladder.

Urinary tract infections (UTIs) are among the most common outpatient infections, with a lifetime incidence of around 60% in women. We analysed urine samples from 223 patients with community-acquired UTIs and report the presence of the cleavage product released during the synthesis of colibactin, a bacterial genotoxin, in 55 of the samples examined. Uropathogenic Escherichia coli strains isolated from these patients, as well as the archetypal E. coli strain UTI89, were found to produce colibactin. In a murine model of UTI, the machinery producing colibactin was expressed during the early hours of the infection, when intracellular bacterial communities form. We observed extensive DNA damage both in umbrella and bladder progenitor cells. To the best of our knowledge this is the first report of colibactin production in UTIs in humans and its genotoxicity in bladder cells.

Two Polyketides Intertwined in Complex Regulation: Posttranscriptional CsrA-Mediated Control of Colibactin and Yersiniabactin Synthesis in Escherichia coli.

Bacteria have to process several levels of gene regulation and coordination of interconnected regulatory networks to ensure the most adequate cellular response to specific growth conditions. Especially, expression of complex and costly fitness and pathogenicity-associated traits is coordinated and tightly regulated at multiple levels. We studied the interconnected regulation of the expression of the colibactin and yersiniabactin polyketide biosynthesis machineries, which are encoded by two pathogenicity islands found in many phylogroup B2 Escherichia coli isolates. Comparative phenotypic and genotypic analyses identified the BarA-UvrY two-component system as an important regulatory element involved in colibactin and yersiniabactin expression. The carbon storage regulator (Csr) system controls the expression of a wide range of central metabolic and virulence-associated traits. The availability of CsrA, the key translational regulator of the Csr system, depends on BarA-UvrY activity. We employed reporter gene fusions to demonstrate UvrY- and CsrA-dependent expression of the colibactin and yersiniabactin determinants and confirmed a direct interaction of CsrA with the 5' untranslated leader transcripts of representative genes of the colibactin and yersiniabactin operons by RNA electrophoretic mobility shift assays. This posttranscriptional regulation adds an additional level of complexity to control mechanisms of polyketide expression, which is also orchestrated at the level of ferric uptake regulator (Fur)-dependent regulation of transcription and phosphopantetheinyl transferase-dependent activation of polyketide biosynthesis. Our results emphasize the interconnection of iron- and primary metabolism-responsive regulation of colibactin and yersiniabactin expression by the fine-tuned action of different regulatory mechanisms in response to variable environmental signals as a prerequisite for bacterial adaptability, fitness, and pathogenicity in different habitats. IMPORTANCE Secondary metabolite expression is a widespread strategy among bacteria to improve their fitness in habitats where they constantly compete for resources with other bacteria. The production of secondary metabolites is associated with a metabolic and energetic burden. Colibactin and yersiniabactin are two polyketides, which are expressed in concert and promote the virulence of different enterobacterial pathogens. To maximize fitness, they should be expressed only in microenvironments in which they are required. Accordingly, precise regulation of colibactin and yersiniabactin expression is crucial. We show that the expression of these two polyketides is also interconnected via primary metabolism-responsive regulation at the posttranscriptional level by the CsrA RNA-binding protein. Our findings may help to optimize (over-)expression and further functional characterization of the polyketide colibactin. Additionally, this new aspect of concerted colibactin and yersiniabactin expression extends our knowledge of conditions that favor the expression of these virulence- and fitness-associated factors in different Enterobacterales members.

The Polyphosphate Kinase of Escherichia coli Is Required for Full Production of the Genotoxin Colibactin.

Colibactin induces DNA damage in mammalian cells and has been linked to the virulence of Escherichia coli and the promotion of colorectal cancer (CRC). By looking for mutants attenuated in the promoter activity of clbB encoding one of the key enzymes for the production of colibactin, we found that a mutant of the gene coding for the polyphosphate kinase (PPK) produced less colibactin than the parental strain. We observed this phenotype in different strains ranging from pathogens responsible for meningitis, urinary tract infection, or mouse colon carcinogenesis to the probiotic Nissle 1917. We confirmed the role of PPK by using an inhibitor of PPK enzymatic activity, mesalamine (also known as 5-aminosalicylic acid). Interestingly, mesalamine has a local anti-inflammatory effect on the epithelial cells of the colon and is used to treat inflammatory bowel disease (IBD). Upon treatment with mesalamine, a decreased genotoxicity of colibactin-producing E. coli was observed both on epithelial cells and directly on purified DNA. This demonstrates the direct effect of mesalamine on bacteria independently from its anti-inflammatory effect on eukaryotic cells. Our results suggest that the mechanisms of action of mesalamine in treating IBD and preventing CRC could also lie in the inhibition of colibactin production. All in all, we demonstrate that PPK is required for the promoter activity of clbB and the production of colibactin, which suggests that PPK is a promising target for the development of anticolibactin and antivirulence strategies.IMPORTANCE Colibactin-producing E. coli induces DNA damage in eukaryotic cells and promotes tumor formation in mouse models of intestinal inflammation. Recent studies have provided strong evidence supporting the causative role of colibactin in human colorectal cancer (CRC) progression. Therefore, it is important to understand the regulation of the production of this genotoxin. Here, we demonstrate that polyphosphate kinase (PPK) is required for the promoter activity of clbB and the production of colibactin. Interestingly, PPK is a multifunctional player in bacterial virulence and stress responses and has been proposed as a new target for developing antimicrobial medicine. We observed inhibition of colibactin production by using a previously identified PPK inhibitor (i.e., mesalamine, an anti-inflammatory drug commonly prescribed for inflammatory bowel diseases). These data brought us a new perspective on the regulatory network of colibactin production and provided us a clue for the development of anticolibactin strategies for CRC treatment/prophylaxis.

ClbR Is the Key Transcriptional Activator of Colibactin Gene Expression in Escherichia coli.

Colibactin is a nonribosomal peptide/polyketide hybrid natural product expressed by different members of the Enterobacteriaceae which can be correlated with induction of DNA double-strand breaks and interference with cell cycle progression in eukaryotes. Regulatory features of colibactin expression are only incompletely understood. We used Escherichia coli strain M1/5 as a model to investigate regulation of expression of the colibactin determinant at the transcriptional level and to characterize regulatory elements located within the colibactin pathogenicity island itself. We measured clbR transcription in vitro and observed that cultivation in defined minimal media led to increased colibactin expression relative to rich media. Transcription of clbR directly responds to iron availability. We also characterized structural DNA elements inside the colibactin determinant involved in ClbR-dependent regulation, i.e., ClbR binding sites and a variable number of tandem repeats located upstream of clbR We investigated the impact of clbR overexpression or deletion at the transcriptome and proteome levels. Moreover, we compared global gene regulation under these conditions with that occurring upon overexpression or deletion of clbQ, which affects the flux of colibactin production. Combining the results of the transcriptome and proteome analyses with indirect measurements of colibactin levels by cell culture assays and an approximate quantification of colibactin via the second product of colibactin cleavage from precolibactin, N-myristoyl-d-asparagine, we demonstrate that the variable number of tandem repeats plays a significant regulatory role in colibactin expression. We identify ClbR as the only transcriptional activator known so far that is specific and essential for efficient regulation of colibactin production.IMPORTANCE The nonribosomal peptide/polyketide hybrid colibactin can be considered a bacterial virulence factor involved in extraintestinal infection and also a procarcinogen. Nevertheless, and despite its genotoxic effect, colibactin expression can also inhibit bacterial or tumor growth and correlates with probiotic anti-inflammatory and analgesic properties. Although the biological function of this natural compound has been studied extensively, our understanding of the regulation of colibactin expression is still far from complete. We investigated in detail the role of regulatory elements involved in colibactin expression and in the growth conditions that promote colibactin expression. In this way, our data shed light on the regulatory mechanisms involved in colibactin expression and may support the expression and purification of this interesting nonribosomal peptide/polyketide hybrid for further molecular characterization.

The Polyamine Spermidine Modulates the Production of the Bacterial Genotoxin Colibactin.

Colibactin is a polyketide/nonribosomal peptide produced by Escherichia coli strains that harbor the pks island. This toxin induces DNA double-strand breaks and DNA interstrand cross-links in infected eukaryotic cells. Colibactin-producing strains are found associated with colorectal cancer biopsy specimens and promote intestinal tumor progression in various murine models. Polyamines are small polycationic molecules produced by both microorganisms and eukaryotic cells. Their levels are increased in malignancies, where they contribute to disease progression and metastasis. In this study, we demonstrated that the endogenous spermidine synthase SpeE is required for full genotoxic activity of colibactin-producing E. coli Supplying spermidine in a ΔspeE pks+E. coli strain restored genotoxic activity. Spermidine is involved in the autotoxicity linked to colibactin and is required for direct damaging activity on DNA. The production of the colibactin prodrug motif is impaired in ΔspeE mutants. Therefore, we demonstrated that spermidine has a direct impact on colibactin synthesis.IMPORTANCE Colibactin-producing Escherichia coli strains are associated with cancerous and precancerous colorectal tissues and are suspected of promoting colorectal carcinogenesis. In this study, we describe a new interplay between the synthesis of the genotoxin colibactin and the polyamine spermidine. Polyamines are highly abundant in cancer tissue and are associated with cell proliferation. The need for spermidine in genotoxic activity provides a new perspective on the role of these metabolites in the pathogenicity of colibactin-producing E. coli strains in colorectal cancer.

Deciphering the interplay between the genotoxic and probiotic activities of Escherichia coli Nissle 1917.

Although Escherichia coli Nissle 1917 (EcN) has been used therapeutically for over a century, the determinants of its probiotic properties remain elusive. EcN produces two siderophore-microcins (Mcc) responsible for an antagonistic activity against other Enterobacteriaceae. EcN also synthesizes the genotoxin colibactin encoded by the pks island. Colibactin is a virulence factor and a putative pro-carcinogenic compound. Therefore, we aimed to decouple the antagonistic activity of EcN from its genotoxic activity. We demonstrated that the pks-encoded ClbP, the peptidase that activates colibactin, is required for the antagonistic activity of EcN. The analysis of a series of ClbP mutants revealed that this activity is linked to the transmembrane helices of ClbP and not the periplasmic peptidase domain, indicating the transmembrane domain is involved in some aspect of Mcc biosynthesis or secretion. A single amino acid substitution in ClbP inactivates the genotoxic activity but maintains the antagonistic activity. In an in vivo salmonellosis model, this point mutant reduced the clinical signs and the fecal shedding of Salmonella similarly to the wild type strain, whereas the clbP deletion mutant could neither protect nor outcompete the pathogen. The ClbP-dependent antibacterial effect was also observed in vitro with other E. coli strains that carry both a truncated form of the Mcc gene cluster and the pks island. In such strains, siderophore-Mcc synthesis also required the glucosyltransferase IroB involved in salmochelin production. This interplay between colibactin, salmochelin, and siderophore-Mcc biosynthetic pathways suggests that these genomic islands were co-selected and played a role in the evolution of E. coli from phylogroup B2. This co-evolution observed in EcN illustrates the fine margin between pathogenicity and probiotic activity, and the need to address both the effectiveness and safety of probiotics. Decoupling the antagonistic from the genotoxic activity by specifically inactivating ClbP peptidase domain opens the way to the safe use of EcN.

The Colibactin Genotoxin Generates DNA Interstrand Cross-Links in Infected Cells.

Colibactins are hybrid polyketide-nonribosomal peptides produced by Escherichia coli, Klebsiella pneumoniae, and other Enterobacteriaceae harboring the pks genomic island. These genotoxic metabolites are produced by pks-encoded peptide-polyketide synthases as inactive prodrugs called precolibactins, which are then converted to colibactins by deacylation for DNA-damaging effects. Colibactins are bona fide virulence factors and are suspected of promoting colorectal carcinogenesis when produced by intestinal E. coli Natural active colibactins have not been isolated, and how they induce DNA damage in the eukaryotic host cell is poorly characterized. Here, we show that DNA strands are cross-linked covalently when exposed to enterobacteria producing colibactins. DNA cross-linking is abrogated in a clbP mutant unable to deacetylate precolibactins or by adding the colibactin self-resistance protein ClbS, confirming the involvement of the mature forms of colibactins. A similar DNA-damaging mechanism is observed in cellulo, where interstrand cross-links are detected in the genomic DNA of cultured human cells exposed to colibactin-producing bacteria. The intoxicated cells exhibit replication stress, activation of ataxia-telangiectasia and Rad3-related kinase (ATR), and recruitment of the DNA cross-link repair Fanconi anemia protein D2 (FANCD2) protein. In contrast, inhibition of ATR or knockdown of FANCD2 reduces the survival of cells exposed to colibactin-producing bacteria. These findings demonstrate that DNA interstrand cross-linking is the critical mechanism of colibactin-induced DNA damage in infected cells.IMPORTANCE Colorectal cancer is the third-most-common cause of cancer death. In addition to known risk factors such as high-fat diets and alcohol consumption, genotoxic intestinal Escherichia coli bacteria producing colibactin are proposed to play a role in colon cancer development. Here, by using transient infections with genotoxic E. coli, we showed that colibactins directly generate DNA cross-links in cellulo Such lesions are converted into double-strand breaks during the repair response. DNA cross-links, akin to those induced by metabolites of alcohol and high-fat diets and by widely used anticancer drugs, are both severely mutagenic and profoundly cytotoxic lesions. This finding of a direct induction of DNA cross-links by a bacterium should facilitate delineating the role of E. coli in colon cancer and engineering new anticancer agents.

Protocol for HeLa Cells Infection with Escherichia coli Strains Producing Colibactin and Quantification of the Induced DNA-damage.

Strains of Escherichia coli bearing the pks genomic island synthesize the genotoxin colibactin. Exposure of eukaryotic cells to E. coli producing colibactin induces DNA damages, ultimately leading to cell cycle arrest, senescence and death. Here we describe a simple method to demonstrate the genotoxicity of bacteria producing colibactin following a short infection of cultured mammalian cells with pks + E. coli.