Interplay between Siderophores and Colibactin Genotoxin Biosynthetic Pathways in Escherichia coli

Martín, Patricia; Marcq, Ingrid; Magistro, Giuseppe; Pénary, Marie; Garcie, Christophe; Payros, Delphine; Boury, Michèle; Olier, Maı̈wenn; Nougayrède, Jean-Philippe; Audebert, Marc; Chalut, Christian; Schubert, Sören; Oswald, Éric

doi:10.1371/journal.ppat.1003437

Cited by 112 publications

(130 citation statements)

References 53 publications

(81 reference statements)

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“…This suggests that clbA could be expressed from a second promoter specifically activated under specific conditions of iron availability. This irondependent regulation of clbA transcription could constitute a fitness advantage for the bacteria, as we previously demonstrated that ClbA can also sustain siderophore biosynthesis (20).…”

Section: Discussionmentioning

confidence: 89%

“…S3). To yield pBAD33-T7-clbA, the 648-bp DNA sequence upstream of clbA in E. coli strain M1/5 (20), comprising the putative clbA promoter, the clbR gene, as well as the whole clbBR intergenic region (clbBR in. reg.…”

Section: Construction Of Venus Fusionsmentioning

confidence: 99%

“…Colibactin is a genotoxin that induces DNA double-strand breaks, senescence, and chromosomal abnormalities in eukaryotic cells (15)(16)(17) and was demonstrated to be a bona fide virulence factor in a mouse model of sepsis (18) and in a rat model of neonatal meningitis (19). We have recently highlighted the existence of cross talk between colibactin and siderophore biosynthesis (20). Indeed, ClbA can replace EntD and contribute to siderophore biosynthesis.…”

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confidence: 99%

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Iron Homeostasis Regulates the Genotoxicity of Escherichia coli That Produces Colibactin

et al. 2016

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eThe genotoxin colibactin is a secondary metabolite produced by a variety of pathogenic enterobacteria. Its biosynthesis requires the enzymatic activity of the phosphopantetheinyl transferase (PPTase) ClbA. We previously showed that ClbA can also contribute to the production of siderophores. Because the biosynthesis of siderophores is regulated by iron availability, we hypothesized that iron could also modulate the production of colibactin through the transcriptional regulation of clbA. This study revealed an increased transcription of clbA under iron-limiting conditions and a decrease of clbA expression in iron-rich media. We demonstrate that clbA transcription is regulated by both the ferric uptake regulator (Fur) and the small regulatory noncoding RNA RyhB. We evidenced that the regulation of the transcription of clbA by Fur and RyhB leads to the regulation of colibactin production. This work highlights the complex mechanism of regulation of an important virulence factor by the two major regulators of bacterial iron homeostasis, making iron a key environmental factor contributing to bacterial virulence and carcinogenesis.

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Section: Discussionmentioning

confidence: 89%

Section: Construction Of Venus Fusionsmentioning

confidence: 99%

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confidence: 99%

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Iron Homeostasis Regulates the Genotoxicity of Escherichia coli That Produces Colibactin

et al. 2016

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“…Colibactin induces senescence and consequent megalocytosis in cultured eukaryotic cells, which manifest as a progressive enlargement of the cell body and nucleus and the absence of mitosis. We quantified the extent of colibactin-induced megalocytosis using a methylene blue binding assay (12). E. coli strains were added to HeLa cells cultivated as described above at multiplicities of infection (MOIs) of between 12 and 400, cocultured for 4 h, and washed.…”

Section: Methodsmentioning

confidence: 99%

“…The 54-kb pks island encodes nonribosomal peptide synthases, polyketide synthases, and hybrid synthases, in addition to accessory, tailoring, and editing enzymes; the clbA gene encodes a phosphopantetheinyl transferase required for colibactin synthesis, and clbP specifies a D-amino peptidase involved in colibactin maturation (10,11). The pks island also contributes to the synthesis of siderophores by virtue of the broad substrate specificity of ClbA, impacting iron acquisition and the ability to survive in the blood compartment (12).…”

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The Genotoxin Colibactin Is a Determinant of Virulence in Escherichia coli K1 Experimental Neonatal Systemic Infection

et al. 2015

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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.E scherichia coli strains belonging to phylogenetic group B2 are found with increasing frequency in the feces of healthy individuals from high-income countries (1) and are responsible for a range of extraintestinal diseases that include urinary tract infections, sepsis, pneumonia, and neonatal meningitis (2). These extraintestinal pathogenic E. coli (ExPEC) strains carry genes encoding a diverse range of virulence determinants that promote colonization, invasion, survival in the blood compartment, and the capacity to evade host defenses and damage the host (3). These virulence-enhancing genes tend to cluster on pathogenicity islands, genomic elements that facilitate dissemination by horizontal gene transfer (4). The pks genomic island is found in 30% to 40% of E. coli B2 strains and codes for the production of colibactin, a polyketide-peptide genotoxin of as-yet-unknown chemical structure that causes doublestrand (ds) breaks in DNA, leading to cell cycle arrest, chromosome instability, and increased lymphopenia in septic rodents (5-8). Carriage of the pks island is linked to long-term persistence in the gastrointestinal (GI) tract (9), and pks-bearing strains represent a high-virulence subset within the B2 group (6). The 54-kb pks island encodes nonribosomal peptide synthases, polyketide synthases, and hybrid synthases, in addition to accessory, tailoring, and editing enzymes; the clbA gene encodes a phosphopantetheinyl transferase required for colibactin synthesis, and clbP specifies a D-amino peptidase involved in colibactin maturation (10, 11). The pks island also contributes to the synthesis of siderophore...

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Validation of high‐throughput genotoxicity assay screening using γH2AX in‐cell western assay on HepG2 cells

Khoury

Zalko

Audebert

2013

Environ and Mol Mutagen

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In vitro genotoxicity tests used in regulatory toxicology studies are sensitive, but the occurrence of irrelevant positive results is high compared with carcinogenicity studies in rodents. Current in vitro genotoxicity tests are also often limited by relatively low throughput. The aim of this study was to validate an in vitro genotoxic assay in a 96-well plate format that allows the simultaneous examination of cytotoxicity and genotoxicity. The test is based on the quantification of the phosphorylation of the histone H2AX (γH2AX), which reflects a global genotoxic insult, using the In-Cell Western technique. The assay was evaluated on HepG2 cells by testing a list of 61 compounds recommended by the European Center for the Validation of Alternative Methods (ECVAM), whose genotoxic potential has already been characterized. The γH2AX assay on HepG2 cell line was highly sensitive: 75% of the genotoxic compounds gave a positive result, and specific: 90-100% of nongenotoxic compounds gave negative results. Compared with the micronucleus genotoxicity assay using the same cell line and test compounds, the γH2AX assay was more sensitive and specific. In sum, the high-throughput γH2AX assay described here can accurately detect simultaneously the genotoxic and the cytotoxic potential of compounds with different modes of mutagenic action, notably those who required metabolic activation. The use of this assay in the early discovery phase of drug development may prove to be a valuable way to assess the genotoxic potential of xenobiotics.

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Interplay between Siderophores and Colibactin Genotoxin Biosynthetic Pathways in Escherichia coli

Cited by 112 publications

References 53 publications

Iron Homeostasis Regulates the Genotoxicity of Escherichia coli That Produces Colibactin

Iron Homeostasis Regulates the Genotoxicity of Escherichia coli That Produces Colibactin

The Genotoxin Colibactin Is a Determinant of Virulence in Escherichia coli K1 Experimental Neonatal Systemic Infection

Validation of high‐throughput genotoxicity assay screening using γH2AX in‐cell western assay on HepG2 cells

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