Deciphering the interplay between the genotoxic and probiotic activities of Escherichia coli Nissle 1917.
ABSTRACT: 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.
Project description:Probiotic Escherichia coli strain Nissle 1917 (EcN) has a long history of safe use. However, the recently discovered presence of a pks locus in its genome presumably producing colibactin has questioned its safety, as colibactin has been implicated in genotoxicity. Here, we assess the genotoxic potential of EcN. Metabolic products were tested in vitro by the Ames test, a mutagenicity assay developed to detect point mutation-inducing activity. Live EcN were tested by an adapted Ames test. Neither the standard nor the adapted Ames test resulted in increased numbers of revertant colonies, indicating that EcN metabolites or viable cells lacked mutagenic activity. The in vivo Mammalian Alkaline Comet Assay (the gold standard for detecting DNA-strand breaks) was used to determine potentially induced DNA-strand breaks in cells of the gastro-intestinal tract of rats orally administered with viable EcN. Bacteria were given at 109-1011 colony forming units (CFU) per animal by oral gavage on 2 consecutive days and daily for a period of 28 days to 5 rats per group. No significant differences compared to negative controls were found. These results demonstrate that EcN does not induce DNA-strand breaks and does not have any detectable genotoxic potential in the test animals.
Project description:Modular polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) comprise giant multidomain enzymes responsible for the "assembly line" biosynthesis of many genetically encoded small molecules. Site-directed mutagenesis, protein biochemical, and structural studies have focused on elucidating the catalytic mechanisms of individual multidomain proteins and protein domains within these megasynthases. However, probing their functions at the cellular level typically has invoked the complete deletion (or overexpression) of multidomain-encoding genes or combinations of genes and comparing those mutants with a control pathway. Here we describe a "domain-targeted" metabolomic strategy that combines genome editing with pathway analysis to probe the functions of individual PKS and NRPS catalytic domains at the cellular metabolic level. We apply the approach to the bacterial colibactin pathway, a genotoxic NRPS-PKS hybrid pathway found in certain Escherichia coli. The pathway produces precolibactins, which are converted to colibactins by a dedicated peptidase, ClbP. Domain-targeted metabolomics enabled the characterization of "multidomain signatures", or functional readouts of NRPS-PKS domain contributions to the pathway-dependent metabolome. These multidomain signatures provided experimental support for individual domain contributions to colibactin biosynthesis and delineated the assembly line timing events of colibactin heterocycle formation. The analysis also led to the structural characterization of two reactive precolibactin metabolites. We demonstrate the fate of these reactive intermediates in the presence and absence of ClbP, which dictates the formation of distinct product groups resulting from alternative cyclization cascades. In the presence of the peptidase, the reactive intermediates are converted to a known genotoxic scaffold, providing metabolic support of our mechanistic model for colibactin-induced genotoxicity. Domain-targeted metabolomics could be more widely used to characterize NRPS-PKS pathways with unprecedented genetic and metabolic precision.
Project description:Escherichia coli strains expressing the K1 capsule are a major cause of sepsis and meningitis in human neonates. The development of these diseases is dependent on the expression of a range of virulence factors, many of which remain uncharacterized. Here, we show that all but 1 of 34 E. coli K1 neonatal isolates carried clbA and clbP, genes contained within the pks pathogenicity island and required for the synthesis of colibactin, a polyketide-peptide genotoxin that causes genomic instability in eukaryotic cells by induction of double-strand breaks in DNA. Inactivation of clbA and clbP in E. coli A192PP, a virulent strain of serotype O18:K1 that colonizes the gastrointestinal tract and translocates to the blood compartment with very high frequency in experimental infection of the neonatal rat, significantly reduced the capacity of A192PP to colonize the gut, engender double-strand breaks in DNA, and cause invasive, lethal disease. Mutation of clbA, which encodes a pleiotropic enzyme also involved in siderophore synthesis, impacted virulence to a greater extent than mutation of clbP, encoding an enzyme specific to colibactin synthesis. Restoration of colibactin gene function by complementation reestablished the fully virulent phenotype. We conclude that colibactin contributes to the capacity of E. coli K1 to colonize the neonatal gastrointestinal tract and to cause invasive disease in the susceptible neonate.
Project description:The pks genomic island of Escherichia coli encodes polyketide (PK) and nonribosomal peptide (NRP) synthases that allow assembly of a putative hybrid PK-NRP compound named colibactin that induces DNA double-strand breaks in eukaryotic cells. The pks-encoded machinery harbors an atypical essential protein, ClbP. ClbP crystal structure and mutagenesis experiments revealed a serine-active site and original structural features compatible with peptidase activity, which was detected by biochemical assays. Ten ClbP homologs were identified in silico in NRP genomic islands of closely and distantly related bacterial species. All tested ClbP homologs were able to complement a clbP-deficient E. coli mutant. ClbP is therefore a prototype of a new subfamily of extracytoplasmic peptidases probably involved in the maturation of NRP compounds. Such peptidases will be powerful tools for the manipulation of NRP biosynthetic pathways.
Project description: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.
Project description:Colibactin represents a structurally undefined class of bacterial genotoxin inducing DNA damage and genomic instability in mammalian cells, thus promoting tumour development and exacerbating lymphopenia in animal models. The colibactin biosynthetic gene cluster (clb) has been known for ten years and it encodes a hybrid nonribosomal peptide synthetase (NRPS)/polyketide synthase (PKS) assembly line. Nevertheless, the final chemical product(s) remain unknown. Previously, we and others reported several colibactin pathway-related metabolites including N-myristoyl-d-asparagine (1) as part of a prodrug precursor that is cleaved from the putative precolibactin to form active colibactin by the peptidase ClbP. Herein, we report two new colibactin pathway-related metabolites (2 and 3) isolated from a clbP mutant of the probiotic E. coli Nissle 1917 strain. Their structures were established by HRMS and NMR. Compound 2 shows an additional 4-aminopenatanoic acid moiety with respect to 1, while 3 is characterized by the presence of an unusual 7-methyl-4-azaspiro[2.4]hept-6-en-5-one residue. Moreover, we propose the biosynthetic pathway towards both intermediates on the basis of extensive gene inactivation and feeding experiments. The identification of 2 and 3 provides further insight into colibactin biosynthesis including the involvement and formation of a rare 1-aminocyclopropanecarboxylic acid unit. Thus, our work establishes additional steps of the pathway forming the bacterial genotoxin colibactin.
Project description:Cellular senescence is an irreversible state of proliferation arrest evoked by a myriad of stresses including oncogene activation, telomere shortening/dysfunction and genotoxic insults. It has been associated with tumor activation, immune suppression and aging, owing to the secretion of proinflammatory mediators. The bacterial genotoxin colibactin, encoded by the pks genomic island is frequently harboured by Escherichia coli strains of the B2 phylogenetic group. Mammalian cells exposed to live pks+ bacteria exhibit DNA-double strand breaks (DSB) and undergo cell-cycle arrest and death. Here we show that cells that survive the acute bacterial infection with pks+ E. coli display hallmarks of cellular senescence: chronic DSB, prolonged cell-cycle arrest, enhanced senescence-associated ?-galactosidase (SA-?-Gal) activity, expansion of promyelocytic leukemia nuclear foci and senescence-associated heterochromatin foci. This was accompanied by reactive oxygen species production and pro-inflammatory cytokines, chemokines and proteases secretion. These mediators were able to trigger DSB and enhanced SA-?-Gal activity in bystander recipient cells treated with conditioned medium from senescent cells. Furthermore, these senescent cells promoted the growth of human tumor cells. In conclusion, the present data demonstrated that the E. coli genotoxin colibactin induces cellular senescence and subsequently propel bystander genotoxic and oncogenic effects.
Project description:Klebsiella pneumoniae is the most common pathogen of community-acquired meningitis in Taiwan. However, the lack of a physiologically relevant meningitis model for K. pneumoniae has impeded research into its pathogenesis mechanism. Based on the core genome MLST analyses, the hypervirulent K1 K. pneumoniae strains, which are etiologically implicated in adult meningitis, mostly belong to a single clonal complex, CC23. Some K1 CC23 K. pneumoniae strains carry a gene cluster responsible for colibactin production. Colibactin is a small genotoxic molecule biosynthesized by an NRPS-PKS complex, which is encoded by genes located on the pks island. Compared to other hypervirulent K. pneumoniae which primarily infect the liver, the colibactin-producing (pks+) K1 CC23 strains had significant tropism toward the brain of BALB/c mice. We aimed in this study to develop a physiologically relevant meningitis model with the use of pks+ K1 CC23 K. pneumoniae. Acute meningitis was successfully induced in adult BALB/c male mice through orogastric, intranasal, and intravenous inoculation of pks+ K1 CC23 K. pneumoniae. Besides the typical symptoms of bacterial meningitis, severe DNA damages, and caspase 3-independent cell death were elicited by the colibactin-producing K1 CC23 K. pneumoniae strain. The deletion of clbA, which abolished the production of colibactin, substantially hindered K. pneumoniae hypervirulence in the key pathogenic steps toward the development of meningitis. Our findings collectively demonstrated that colibactin was necessary but not sufficient for the meningeal tropism of pks+ K1 CC23 K. pneumoniae, and the mouse model established in this study can be applied to identify other virulence factors participating in the development of this life-threatening disease.
Project description:The gut bacterial genotoxin colibactin is linked to the development of colorectal cancer. In the final stages of colibactin's biosynthesis, an inactive precursor (precolibactin) undergoes proteolytic cleavage by ClbP, an unusual inner-membrane-bound periplasmic peptidase, to generate the active genotoxin. This enzyme presents an opportunity to monitor and modulate colibactin biosynthesis, but its active form has not been studied in vitro and limited tools exist to measure its activity. Here, we describe the in vitro biochemical characterization of catalytically active, full-length ClbP. We elucidate its substrate preferences and use this information to develop a fluorogenic activity probe. This tool will enable the discovery of ClbP inhibitors and streamline identification of colibactin-producing bacteria.
Project description:Precolibactins and colibactins represent a family of natural products that are encoded by the clb gene cluster and are produced by certain commensal, extraintestinal, and probiotic E. coli. clb+ E. coli induce megalocytosis and DNA double-strand breaks in eukaryotic cells, but paradoxically, this gene cluster is found in the probiotic Nissle 1917. Evidence suggests precolibactins are converted to genotoxic colibactins by colibactin peptidase (ClbP)-mediated cleavage of an N-acyl-d-Asn side chain, and all isolation efforts have employed ?clbP strains to facilitate accumulation of precolibactins. It was hypothesized that colibactins form unsaturated imines that alkylate DNA by cyclopropane ring opening (2 ? 3). However, as no colibactins have been isolated, this hypothesis has not been tested experimentally. Additionally, precolibactins A-C (7-9) contain a pyridone that cannot generate the unsaturated imines that form the basis of this hypothesis. To resolve this, we prepared 13 synthetic colibactin derivatives and evaluated their DNA binding and alkylation activity. We show that unsaturated imines, but not the corresponding pyridone derivatives, potently alkylate DNA. The imine, unsaturated lactam, and cyclopropane are essential for efficient DNA alkylation. A cationic residue enhances activity. These studies suggest that precolibactins containing a pyridone are not responsible for the genotoxicity of the clb cluster. Instead, we propose that these are off-pathway fermentation products produced by a facile double cyclodehydration route that manifests in the absence of viable ClbP. The results presented herein provide a foundation to begin to connect metabolite structure with the disparate phenotypes associated with clb+ E. coli.