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Crystal structure of bacterial cytotoxic necrotizing factor CNF_Y reveals molecular building blocks for intoxication.

ABSTRACT: Cytotoxic necrotizing factors (CNFs) are bacterial single-chain exotoxins that modulate cytokinetic/oncogenic and inflammatory processes through activation of host cell Rho GTPases. To achieve this, they are secreted, bind surface receptors to induce endocytosis and translocate a catalytic unit into the cytosol to intoxicate host cells. A three-dimensional structure that provides insight into the underlying mechanisms is still lacking. Here, we determined the crystal structure of full-length Yersinia pseudotuberculosis CNF_Y . CNF_Y consists of five domains (D1-D5), and by integrating structural and functional data, we demonstrate that D1-3 act as export and translocation module for the catalytic unit (D4-5) and for a fused ?-lactamase reporter protein. We further found that D4, which possesses structural similarity to ADP-ribosyl transferases, but had no equivalent catalytic activity, changed its position to interact extensively with D5 in the crystal structure of the free D4-5 fragment. This liberates D5 from a semi-blocked conformation in full-length CNF_Y , leading to higher deamidation activity. Finally, we identify CNF translocation modules in several uncharacterized fusion proteins, which suggests their usability as a broad-specificity protein delivery tool.

SUBMITTER: Chaoprasid P

PROVIDER: S-EPMC7883292 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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Crystal structure of bacterial cytotoxic necrotizing factor CNF<sub>Y</sub> reveals molecular building blocks for intoxication.

Chaoprasid Paweena P Lukat Peer P Mühlen Sabrina S Heidler Thomas T Gazdag Emerich-Mihai EM Dong Shuangshuang S Bi Wenjie W Rüter Christian C Kirchenwitz Marco M Steffen Anika A Steffen Anika A Jänsch Lothar L Stradal Theresia E B TEB Dersch Petra P Blankenfeldt Wulf W

The EMBO journal 20210107 4

Cytotoxic necrotizing factors (CNFs) are bacterial single-chain exotoxins that modulate cytokinetic/oncogenic and inflammatory processes through activation of host cell Rho GTPases. To achieve this, they are secreted, bind surface receptors to induce endocytosis and translocate a catalytic unit into the cytosol to intoxicate host cells. A three-dimensional structure that provides insight into the underlying mechanisms is still lacking. Here, we determined the crystal structure of full-length Yer ...[more]

PMID: 33410511

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Project description:Background:Many Escherichia coli strains are considered to be a component of the normal flora found in the human and animal intestinal tracts. While most E. coli strains are commensal, some strains encode virulence factors that enable the bacteria to cause intestinal and extra-intestinal clinically-relevant infections. Colibactin, encoded by a genomic island (pks island), and cytotoxic necrotizing factor (CNF), encoded by the cnf gene, are genotoxic and can modulate cellular differentiation, apoptosis and proliferation. Some commensal and pathogenic pks+ and cnf+ E. coli strains have been associated with inflammation and cancer in humans and animals. Results:In the present study, E. coli strains encoding colibactin and CNF were identified in macaque samples. We performed bacterial cultures utilizing rectal swabs and extra-intestinal samples from clinically normal macaques. A total of 239 E. coli strains were isolated from 266 macaques. The strains were identified biochemically and selected isolates were serotyped as O88:H4, O25:H4, O7:H7, OM:H14, and OM:H16. Specific PCR for pks and cnf1 gene amplification, and phylogenetic group identification were performed on all E. coli strains. Among the 239 isolates, 41 (17.2%) were pks+/cnf1-, 19 (7.9%) were pks-/cnf1+, and 31 (13.0%) were pks+/cnf1+. One hundred forty-eight (61.9%) E. coli isolates were negative for both genes (pks-/cnf1-). In total, 72 (30.1%) were positive for pks genes, and 50 (20.9%) were positive for cnf1. No cnf2+ isolates were detected. Both pks+ and cnf1+ E. coli strains belonged mainly to phylogenetic group B2, including B21. Colibactin and CNF cytotoxic activities were observed using a HeLa cell cytotoxicity assay in representative isolates. Whole genome sequencing of 10 representative E. coli strains confirmed the presence of virulence factors and antibiotic resistance genes in rhesus macaque E. coli isolates. Conclusions:Our findings indicate that colibactin- and CNF-encoding E. coli colonize laboratory macaques and can potentially cause clinical and subclinical diseases that impact macaque models.

Project description:Background:The colibactin and cytotoxic necrotizing factor 1 (Cnf 1) are toxins with cell cycle modulating effects that contribute to tumorgenesis and hyperproliferation. This study aimed to investigate the prevalence and pathologic effects of Cnf 1 and colibactin among hemolytic uropathogenic Escherichia coli (UPEC). The bioinformatics approach incorporated in this study aimed to expand the domain of the in vitro study and explore the prevalence of both toxins among other bacterial species. A total of 125 E. coli isolates were recovered from UTIs patients. The isolates were tested for their hemolytic activity, subjected to tissue culture and PCR assays to detect the phenotypic and genotypic features of both toxins. A rat ascending UTI in vivo model was conducted using isolates expressing or non-expressing Cnf 1 and colibactin (ClbA and ClbQ). The bioinformatics analyses were inferred by Maximum likelihood method and the evolutionary relatedness was deduced by MEGA X. Results:Only 21 (16.8%) out of 125 isolates were hemolytic and 10 of these (47.62%) harbored the toxins encoding genes (cnf 1 +, clbA + and clbQ +). The phenotypic features of both toxins were exhibited by only 7 of the (cnf 1 + clbA + clbQ +) harboring isolates. The severest infections, hyperplastic and genotoxic changes in kidneys and bladders were observed in rats infected with the cnf 1 + clbA + clbQ + isolates. Conclusion:Only 33.3% of the hemolytic UPEC isolates exhibited the phenotypic and genotypic features of Cnf 1 and Colibactin. The in vivo animal model results gives an evidence of active Cnf 1 and Colibactin expression and indicates the risks associated with recurrent and chronic UTIs caused by UPEC. The bioinformatics analyses confirmed the predominance of colibactin pks island among Enterobacteriaceae family (92.86%), with the highest occurrence among Escherichia species (53.57%), followed by Klebsiella (28.57%), Citrobacter (7.14%), and Enterobacter species (3.57%). The Cnf 1 is predominant among Escherichia coli (94.05%) and sporadically found among Shigella species (1.08%), Salmonella enterica (0.54%), Yersinia pseudotuberculosis (1.08%), Photobacterium (1.08%), Moritella viscosa (0.54%), and Carnobacterium maltaromaticum (0.54%). A close relatedness was observed between the 54-kb pks island of Escherichia coli, the probiotic Escherichia coli Nissle 1917, Klebsiella aerogenes, Klebsiella pneumoniae and Citrobacter koseri.

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Crystal structure of bacterial cytotoxic necrotizing factor CNF_Y reveals molecular building blocks for intoxication.

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Crystal structure of bacterial cytotoxic necrotizing factor CNF<sub>Y</sub> reveals molecular building blocks for intoxication.

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Crystal structure of bacterial cytotoxic necrotizing factor CNFY reveals molecular building blocks for intoxication.

Publications

Crystal structure of bacterial cytotoxic necrotizing factor CNF<sub>Y</sub> reveals molecular building blocks for intoxication.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

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Crystal structure of bacterial cytotoxic necrotizing factor CNF_Y reveals molecular building blocks for intoxication.