Unveiling the Mutational Mechanism of the Bacterial Genotoxin Colibactin in Colorectal Cancer.

In a recent issue of Science, Wilson et al. (2019) provide direct evidence that the bacterial-produced colibactin alkylates DNA in vivo, resulting in DNA adducts, which mediates its genotoxic effect. This work reinforces the role of colibactin-producing bacteria in colon cancer pathogenesis.

New Sorbicillinoids with Tea Pathogenic Fungus Inhibitory Effect from Marine-Derived Fungus Hypocrea jecorina H8.

Four new dimeric sorbicillinoids (1-3 and 5) and a new monomeric sorbicillinoid (4) as well as six known analogs (6-11) were purified from the fungal strain Hypocrea jecorina H8, which was obtained from mangrove sediment, and showed potent inhibitory activity against the tea pathogenic fungus Pestalotiopsis theae (P. theae). The planar structures of 1-5 were assigned by analyses of their UV, IR, HR-ESI-MS, and NMR spectroscopic data. All the compounds were evaluated for growth inhibition of tea pathogenic fungus P. theae. Compounds 5, 6, 8, 9, and 10 exhibited more potent inhibitory activities compared with the positive control hexaconazole with an ED50 of 24.25 ± 1.57 µg/mL. The ED50 values of compounds 5, 6, 8, 9, and 10 were 9.13 ± 1.25, 2.04 ± 1.24, 18.22 ± 1.29, 1.83 ± 1.37, and 4.68 ± 1.44 µg/mL, respectively. Additionally, the effects of these compounds on zebrafish embryo development were also evaluated. Except for compounds 5 and 8, which imparted toxic effects on zebrafish even at 0.625 µM, the other isolated compounds did not exhibit significant toxicity to zebrafish eggs, embryos, or larvae. Taken together, sorbicillinoid derivatives (6, 9, and 10) from H. jecorina H8 displayed low toxicity and high anti-tea pathogenic fungus potential.

Autophagy of Intestinal Epithelial Cells Inhibits Colorectal Carcinogenesis Induced by Colibactin-Producing Escherichia coli in ApcMin/+ Mice.

BACKGROUND & AIMS: Colibactin-producing Escherichia coli (CoPEC) colonize the colonic mucosa of a higher proportion of patients with vs without colorectal cancer (CRC) and promote colorectal carcinogenesis in susceptible mouse models of CRC. Autophagy degrades cytoplasmic contents, including intracellular pathogens, via lysosomes and regulates intestinal homeostasis. We investigated whether inhibiting autophagy affects colorectal carcinogenesis in susceptible mice infected with CoPEC. METHODS: Human intestinal epithelial cells (IECs) (HCT-116) were infected with a strain of CoPEC (11G5 strain) isolated from a patient or a mutant strain that does not produce colibactin (11G5ΔclbQ). Levels of ATG5, ATG16L1, and SQSTM1 (also called p62) were knocked down in HCT-116 cells using small interfering RNAs. ApcMin/+ mice and ApcMin/+ mice with IEC-specific disruption of Atg16l1 (ApcMin/+/Atg16l1ΔIEC) were infected with 11G5 or 11G5ΔclbQ. Colonic tissues were collected from mice and analyzed for tumor size and number and by immunohistochemical staining, immunoblot, and quantitative reverse transcription polymerase chain reaction for markers of autophagy, DNA damage, cell proliferation, and inflammation. We analyzed levels of messenger RNAs (mRNAs) encoding proteins involved in autophagy in colonic mucosal tissues from patients with sporadic CRC colonized with vs without CoPEC by quantitative reverse-transcription polymerase chain reaction. RESULTS: Patient colonic mucosa with CoPEC colonization had higher levels of mRNAs encoding proteins involved in autophagy than colonic mucosa without these bacteria. Infection of cultured IECs with 11G5 induced autophagy and DNA damage repair, whereas infection with 11G5ΔclbQ did not. Knockdown of ATG5 in HCT-116 cells increased numbers of intracellular 11G5, secretion of interleukin (IL) 6 and IL8, and markers of DNA double-strand breaks but reduced markers of DNA repair, indicating that autophagy is required for bacteria-induced DNA damage repair. Knockdown of ATG5 in HCT-116 cells increased 11G5-induced senescence, promoting proliferation of uninfected cells. Under uninfected condition, ApcMin/+/Atg16l1ΔIEC mice developed fewer and smaller colon tumors than ApcMin/+ mice. However, after infection with 11G5, ApcMin/+/Atg16l1ΔIEC mice developed more and larger tumors, with a significant increase in mean histologic score, than infected ApcMin/+ mice. Increased levels of Il6, Tnf, and Cxcl1 mRNAs, decreased level of Il10 mRNA, and increased markers of DNA double-strand breaks and proliferation were observed in the colonic mucosa of 11G5-infected ApcMin/+/Atg16l1ΔIEC mice vs 11G5-infected ApcMin/+ mice. CONCLUSION: Infection of IECs and susceptible mice with CoPEC promotes autophagy, which is required to prevent colorectal tumorigenesis. Loss of ATG16L1 from IECs increases markers of inflammation, DNA damage, and cell proliferation and increases colorectal tumorigenesis in 11G5-infected ApcMin/+ mice. These findings indicate the importance of autophagy in response to CoPEC infection, and strategies to induce autophagy might be developed for patients with CRC and CoPEC colonization.

Cytolethal distending toxin induces the formation of transient messenger-rich ribonucleoprotein nuclear invaginations in surviving cells.

Humans are frequently exposed to bacterial genotoxins involved in digestive cancers, colibactin and Cytolethal Distending Toxin (CDT), the latter being secreted by many pathogenic bacteria. Our aim was to evaluate the effects induced by these genotoxins on nuclear remodeling in the context of cell survival. Helicobacter infected mice, coculture experiments with CDT- and colibactin-secreting bacteria and hepatic, intestinal and gastric cells, and xenograft mouse-derived models were used to assess the nuclear remodeling in vitro and in vivo. Our results showed that CDT and colibactin induced-nuclear remodeling can be associated with the formation of deep cytoplasmic invaginations in the nucleus of giant cells. These structures, observed both in vivo and in vitro, correspond to nucleoplasmic reticulum (NR). The core of the NR was found to concentrate ribosomes, proteins involved in mRNA translation, polyadenylated RNA and the main components of the complex mCRD involved in mRNA turnover. These structures are active sites of mRNA translation, correlated with a high degree of ploidy, and involve MAPK and calcium signaling. Additional data showed that insulation and concentration of these adaptive ribonucleoprotein particles within the nucleus are dynamic, transient and protect the cell until the genotoxic stress is relieved. Bacterial genotoxins-induced NR would be a privileged gateway for selected mRNA to be preferably transported therein for local translation. These findings offer new insights into the context of NR formation, a common feature of many cancers, which not only appears in response to therapies-induced DNA damage but also earlier in response to genotoxic bacteria.

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.

Isolation, stereochemical study, and racemization of (±)-pratenone A, the first naturally occurring 3-(1-naphthyl)-2-benzofuran-1(3H)-one polyketide from a marine-derived actinobacterium.

(±)-Pratenone A (1), the first representative of natural 3-(1-naphthyl)-2-benzofuran-1(3H)-one polyketides, was isolated from a marine-derived Streptomyces pratensis strain KCB-132 together with three other new analogues (2-4). Its structure was assigned by spectroscopic analysis, and the absolute configurations of the two enantiomers separated by high-performance liquid chromatography were determined by single-crystal X-ray diffraction and electronic circular dichroism calculations. The solvent-induced racemization of 1 and a proposed biogenetic pathway to 1-4 from the co-isolated angucyclinone precursor, as well as their biological activity, are also discussed.

Antimicrobial and antioxidant activities of two polyketides from lichen-endophytic fungus Preussia sp.

Two compounds, compounds 1 and 2, were isolated from Preussia sp. The molecular structures of both compounds were elucidated by analyzing one-dimensional (1D) and 2D nuclear magnetic resonance data along with high-resolution mass spectrometry data. Compound 1 was obtained as novel in structure, and compound 2 was recently reported elsewhere. Compound 1 did not show antioxidant and antimicrobial activities and brine shrimp toxicity, while compound 2 showed strong antioxidant activity (DPPH reduction capacity; IC50=3 µg/mL) and brine shrimp toxicity (LD50=50 µg/mL).

ClbS Is a Cyclopropane Hydrolase That Confers Colibactin Resistance.

Certain commensal Escherichia coli contain the clb biosynthetic gene cluster that codes for small molecule prodrugs known as precolibactins. Precolibactins are converted to colibactins by N-deacylation; the latter are postulated to be genotoxic and to contribute to colorectal cancer formation. Though advances toward elucidating (pre)colibactin biosynthesis have been made, the functions and mechanisms of several clb gene products remain poorly understood. Here we report the 2.1 Å X-ray structure and molecular function of ClbS, a gene product that confers resistance to colibactin toxicity in host bacteria and which has been shown to be important for bacterial viability. The structure harbors a potential colibactin binding site and shares similarity to known hydrolases. In vitro studies using a synthetic colibactin analog and ClbS or an active site residue mutant reveal cyclopropane hydrolase activity that converts the electrophilic cyclopropane of the colibactins into an innocuous hydrolysis product. As the cyclopropane has been shown to be essential for genotoxic effects in vitro, this ClbS-catalyzed ring-opening provides a means for the bacteria to circumvent self-induced genotoxicity. Our study provides a molecular-level view of the first reported cyclopropane hydrolase and support for a specific mechanistic role of this enzyme in colibactin resistance.

In-vitro assessment of antimicrobial properties and lymphocytotoxicity assay of benzoisochromanequinones polyketide from Streptomyces sp JRG-04.

The chromanequinone (BIQ) compound produced by the mangrove estuary derived strain, Streptomyces sp. JRG-04 was effective even at low MIC level concentration against Methicillin resistant S. aureus and other clinical pathogens. In this study, we have investigated the antimicrobial potential of chromanequinone compound by using various microscopy and imaging techniques. The flow cytometry (FACS) analysis suggested the BIQ aromatic polyketide compound produced by the Streptomyces sp. JRG-04 has toxic effect on MRSA cell membrane by increased up take of propidium iodide dye. The bacterial imaging analysis by high content screening experiment (HCS) revealed the increased number of dead MRSA cells than the live MRSA populations with chromanequinone treatment. Furthermore, atomic force microscopic study proved the MRSA cell surface ultra-structure changes when the cells exposed to chromanequinone compound at 3 h and 6 h. Further, in-vitro lymphocytotoxicity effect of chromanequinone compound at different concentrations with the combination of complement was performed on human lymphocytes by cell lysis assay. Interestingly, we have found that the higher concentration of BIQ chromanequinone (10 mg/mL) compound without complement induced apoptosis of human lymphocytes. The present investigation reveals that the toxic potential of chromanequinone on human lymphocytes might be associated with the complement dependent. This study strongly suggests that the chromanequinone compound produced by the Streptomyces strain with bioactive property can be developed as a therapeutic leads for various pharmaceutical applications.

Chaetochromones A - C, Three New Polyketides from Mangrove Plant Derived Endophytic Fungus Phomopsis sp. SCSIO 41006.

Three new polyketides, chaetochromones A - C (1 - 3), together with a chromone (4), were isolated from the ethyl acetate extract of mangrove-derived fungus Phomopsis sp. SCSIO 41006. Their structures were elucidated by means of spectroscopic techniques (UV, IR, MS, 1D- and 2D-NMR). The absolute configurations of the new compounds were established by CD data.

A Mechanistic Model for Colibactin-Induced Genotoxicity.

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 ΔclbP 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.

Gut symbionts from distinct hosts exhibit genotoxic activity via divergent colibactin biosynthesis pathways.

Secondary metabolites produced by nonribosomal peptide synthetase (NRPS) or polyketide synthase (PKS) pathways are chemical mediators of microbial interactions in diverse environments. However, little is known about their distribution, evolution, and functional roles in bacterial symbionts associated with animals. A prominent example is colibactin, a largely unknown family of secondary metabolites produced by Escherichia coli via a hybrid NRPS-PKS biosynthetic pathway that inflicts DNA damage upon eukaryotic cells and contributes to colorectal cancer and tumor formation in the mammalian gut. Thus far, homologs of this pathway have only been found in closely related Enterobacteriaceae, while a divergent variant of this gene cluster was recently discovered in a marine alphaproteobacterial Pseudovibrio strain. Herein, we sequenced the genome of Frischella perrara PEB0191, a bacterial gut symbiont of honey bees and identified a homologous colibactin biosynthetic pathway related to those found in Enterobacteriaceae. We show that the colibactin genomic island (GI) has conserved gene synteny and biosynthetic module architecture across F. perrara, Enterobacteriaceae, and the Pseudovibrio strain. Comparative metabolomics analyses of F. perrara and E. coli further reveal that these two bacteria produce related colibactin pathway-dependent metabolites. Finally, we demonstrate that F. perrara, like E. coli, causes DNA damage in eukaryotic cells in vitro in a colibactin pathway-dependent manner. Together, these results support that divergent variants of the colibactin biosynthetic pathway are widely distributed among bacterial symbionts, producing related secondary metabolites and likely endowing its producer with functional capabilities important for diverse symbiotic associations.

Amphirionin-2, a novel linear polyketide with potent cytotoxic activity from a marine dinoflagellate Amphidinium species.

A novel linear polyketide, amphirionin-2 (1), with two unique hexahydrofuro[3,2-b]furan moieties has been isolated from the cultivated algal cells of a benthic dinoflagellate Amphidinium sp. (strain KCA09051). The structure was elucidated on the basis of detailed analyses of 2D NMR data, and the absolute configuration of C-5 was determined by using modified Mosher's method. Amphirionin-2 (1) exhibited potent cytotoxic activity against human colon carcinoma Caco-2 cells and human lung adenocarcinoma A549 cells.

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.

Penicillium verruculosum SG: a source of polyketide and bioactive compounds with varying cytotoxic activities against normal and cancer lines.

A newly isolated fungus Penicillium verruculosum SG was evaluated for the production and characterization of bioactive colored secondary metabolites using solid-state fermentation along with their cytotoxic activities against normal and cancer cell lines. Logical fragmentation pattern following column chromatography, thin layer chromatography and liquid chromatography and mass spectrometry of crude culture filtrate of fungus revealed the presence of different polyketide pigments and other bioactive compounds. Cytotoxicity of the selected colored fractions of fungal filtrate containing different compounds revealed IC50 (µg/ml) values ranging from 5 to 100. It was significantly higher in case of orevactaene (5 + 0.44) and monascorubrine followed by pyripyropene (8 + 0.63) against cancer cell line KA3IT. Overall, these compounds considerably showed less toxicity toward normal cell lines NIH3T3, HSCT6, HEK293 and MDCK. XRD of a yellow crystalline compound (224.21 m/z) confirmed its 3-dimensional structure as phenazine 1 carboxylic acid (C13H8N2O2) (broad spectrum antibiotic), and it is first time reported in fungi.

Klebsiella pneumoniae producing bacterial toxin colibactin as a risk of colorectal cancer development - A systematic review.

Microbiota can significantly contribute to colorectal cancer initiation and development. It was described that E. coli harbouring polyketide synthase (pks) genes can synthetize bacterial toxin colibactin, which was first described by Nougayrede's group in 2006. E. coli positive for pks genes were overrepresented in colorectal cancer biopsies and, therefore, prevalence and the effect of pks positive bacteria as a risk factor in colorectal cancer development is in our interest. Interestingly, pks gene cluster in E. coli shares a striking 100% sequence identity with K. pneumoniae, suggesting that their function and regulation are conserved. Moreover, K. pneumoniae can express a variety of virulence factors, including capsules, siderophores, iron-scavenging systems, adhesins and endotoxins. It was reported that pks cluster and thereby colibactin is also related to the hypervirulence of K. pneumoniae. Acquisition of the pks locus is associated with K. pneumoniae gut colonisation and mucosal invasion. Colibactin also increases the likelihood of serious complications of bacterial infections, such as development of meningitis and potentially tumorigenesis. Even though K. pneumoniae is undoubtedly a gut colonizer, the role of pks positive K. pneumoniae in GIT has not yet been investigated. It seems that CRC-distinctive microbiota is already present in the early stages of cancer development and, therefore, microbiome analysis could help to discover the early stages of cancer, which are crucial for effectiveness of anticancer therapy. We hypothesize, that pks positive K. pneumoniae can be a potential biomarker of tumour prevalence and anticancer therapy response.

Mechanistic dissection unmasks colibactin as a prevalent mutagenic driver of cancer.

Gut colonization by colibactin-producing bacteria is associated with colorectal cancer. A mutational signature of this genotoxin in human cancer indicates causality but only partially accounts for cell transformation. Instead, the failure of adequately resolving DNA damage causes genomic aberrations and chromosomal instability, constituting the main starting point for colibactin-driven cancer.

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.

Shining a Light on Colibactin Biology.

Colibactin is a secondary metabolite encoded by the pks gene island identified in several Enterobacteriaceae, including some pathogenic Escherichia coli (E. coli) commonly enriched in mucosal tissue collected from patients with inflammatory bowel disease and colorectal cancer. E. coli harboring this biosynthetic gene cluster cause DNA damage and tumorigenesis in cell lines and pre-clinical models, yet fundamental knowledge regarding colibactin function is lacking. To accurately assess the role of pks+ E. coli in cancer etiology, the biological mechanisms governing production and delivery of colibactin by these bacteria must be elucidated. In this review, we will focus on recent advances in our understanding of colibactin's structural mode-of-action and mutagenic potential with consideration for how this activity may be regulated by physiologic conditions within the intestine.

ClbG in Avian Pathogenic Escherichia coli Contributes to Meningitis Development in a Mouse Model.

Colibactin is a complex secondary metabolite that leads to genotoxicity that interferes with the eukaryotic cell cycle. It plays an important role in many diseases, including neonatal mouse sepsis and meningitis. Avian pathogenic Escherichia coli (APEC) is responsible for several diseases in the poultry industry and may threaten human health due to its potential zoonosis. In this study, we confirmed that clbG was necessary for the APEC XM strain to produce colibactin. The deletion of clbG on APEC XM contributed to lowered γH2AX expression, no megalocytosis, and no cell cycle arrest in vitro. None of the 4-week Institute of Cancer Research mice infected with the APEC XM ΔclbG contracted meningitis or displayed weakened clinical symptoms. Fewer histopathological lesions were observed in the APEC XM ΔclbG group. The bacterial colonization of tissues and the relative expression of cytokines (IL-1ß, IL-6, and TNF-α) in the brains decreased significantly in the APEC XM ΔclbG group compared to those in the APEC XM group. The tight junction proteins (claudin-5, occludin, and ZO-1) were not significantly destroyed in APEC XM ΔclbG group in vivo and in vitro. In conclusion, clbG is necessary for the synthesis of the genotoxin colibactin and affects the development of APEC meningitis in mice.