Effects of the Escherichia coli Bacterial Toxin Cytotoxic Necrotizing Factor 1 on Different Human and Animal Cells: A Systematic Review

Cytotoxic necrotizing factor 1 (CNF1) is a bacterial virulence factor, the target of which is represented by Rho GTPases, small proteins involved in a huge number of crucial cellular processes. CNF1, due to its ability to modulate the activity of Rho GTPases, represents a widely used tool to unravel the role played by these regulatory proteins in different biological processes. In this review, we summarized the data available in the scientific literature concerning the observed in vitro effects induced by CNF1. An article search was performed on electronic bibliographic resources. Screenings were performed of titles, abstracts, and full-texts according to PRISMA guidelines, whereas eligibility criteria were defined for in vitro studies. We identified a total of 299 records by electronic article search and included 76 original peer-reviewed scientific articles reporting morphological or biochemical modifications induced in vitro by soluble CNF1, either recombinant or from pathogenic Escherichia coli extracts highly purified with chromatographic methods. Most of the described CNF1-induced effects on cultured cells are ascribable to the modulating activity of the toxin on Rho GTPases and the consequent effects on actin cytoskeleton organization. All in all, the present review could be a prospectus about the CNF1-induced effects on cultured cells reported so far.

The cnf1 gene is associated to an expanding Escherichia coli ST131 H30Rx/C2 sublineage and confers a competitive advantage for host colonization

Epidemiological projections point to acquisition of ever-expanding multidrug resistance (MDR) by Escherichia coli, a commensal of the digestive tract acting as a source of urinary tract pathogens. We performed a high-throughput genetic screening of predominantly clinical E. coli isolates from wide geographical origins. This revealed a preferential distribution of the Cytotoxic Necrotizing Factor 1 (CNF1)-toxin encoding gene, cnf1, in four sequence types encompassing the pandemic E. coli MDR lineage ST131. This lineage is responsible for a majority of extraintestinal infections that escape first-line antibiotic treatment and has known enhanced capacities to colonize the gastrointestinal tract (GIT). Statistical modeling uncovered a dominant global expansion of cnf1-positive strains within multidrug-resistant ST131 subclade H30Rx/C2. Despite the absence of phylogeographical signals, cnf1-positive isolates adopted a clonal distribution into clusters on the ST131-H30Rx/C2 phylogeny, sharing a similar profile of virulence factors and the same cnf1 allele. Functional analysis of the cnf1-positive clinical strain EC131GY ST131-H30Rx/C2, established that a cnf1-deleted EC131GY is outcompeted by the wildtype strain in a mouse model of competitive infection of the bladder while both strains behave similarly during monoinfections. This points for positive selection of cnf1 during UTI rather than urovirulence. Wildtype EC131GY also outcompeted the mutant when concurrently inoculated into the gastrointestinal tract, arguing for selection within the gut. Whatever the site of selection, these findings support that the benefit of cnf1 enhancing host colonization by ST131-H30Rx/C2 in turn drives a worldwide dissemination of the cnf1 gene together with extended spectrum of antibiotic resistance genes.

Molecular Characteristics Ecology Zoonotic Potential of Escherichia coli Strains that Cause Hemorrhagic Pneumonia in Animals

Hemorrhagic pneumonia (HP) is a rare but highly lethal disease, mainly of dogs and cats, caused by hemolytic Escherichia coli strains that contain cnf1 (encoding cytotoxic necrotizing factor 1). After encountering fatal HP in two dogs, we used contemporary molecular methods, including multi-locus sequence typing and whole genome sequencing, to compare the corresponding case isolates with published HP clinical isolates and newly-obtained fecal E. coli isolates from 20 humans and animals in the index HP case household. We also compared the aggregated HP clinical isolates, which represented 13 discrete strains, by pulsotype with a large, private pulsotype library of diverse-source E. coli . The HP clinical isolates represented a narrow range of phylogenetic group B2 lineages (mainly sequence types 12 and 127), O types (mainly O4 and O6), and H types (mainly H5 and H31), but diverse fimH alleles (type-1 fimbriae adhesin). Their extensive, highly conserved virulence genotypes, which qualified as extraintestinal pathogenic E. coli (ExPEC), encoded diverse adhesins, toxins, iron uptake systems, and protectins. Household surveillance identified multiple HP-like fecal strains, plus abundant between-host strain sharing, including of the household's index HP strain. The pulsotype library search identified, for five HP clinical strains, same-pulsotype human and animal fecal and clinical (predominantly urine) isolates, from diverse locales and time periods. Thus, E. coli strains that cause HP derive from a narrow range of ExPEC lineages within phylogroup B2, contain multiple virulence genes other than cnf1 , are shared extensively between hosts, and likely function in nature mainly as intestinal colonizers and uropathogens. Importance This study clarifies the clonal background and extensive virulence genotypes of the E. coli strains that cause hemorrhagic pneumonia in domestic animals (mainly dogs and cats), shows that such strains circulate among animals and humans, identifies a substantial intestinal colonization component to their lifestyle, and extends their known clinical manifestations to include bacteremia and urinary tract infection. The findings place these strains better into context vis-a-vis current understandings of E. coli phylogeny, ecology, and pathogenesis; identify questions for future research; and may prove relevant for surveillance and prevention efforts.

A screening pipeline identifies a broad-spectrum inhibitor of bacterial AB toxins with cross protection against influenza A virus H1N1 and SARS-CoV-2

A challenge for the development of host-targeted anti-infectives against a large spectrum of AB-like toxin-producing bacteria encompasses the identification of chemical compounds corrupting toxin transport through both endolysosomal and retrograde pathways. Here, we performed a high-throughput screening of small chemical compounds blocking active Rac1 proteasomal degradation triggered by the Cytotoxic Necrotizing Factor-1 (CNF1) toxin, followed by orthogonal screens against two AB toxins hijacking defined endolysosomal (Diphtheria toxin) or retrograde (Shiga-like toxin 1) pathways to intoxicate cells. This led to the identification of the molecule N-(3,3-diphenylpropyl)-1-propyl-4-piperidinamine, referred to as C910. This compound induces the swelling of EEA1-positive early endosomes, in absence of PIKfyve kinase inhibition, and disturbs the trafficking of CNF1 and the B-subunit of Shiga toxin along the endolysosomal or retrograde pathways, respectively. Together, we show that C910 protects cells against 8 bacterial AB toxins including large clostridial glucosylating toxins from Clostridium difficile. Of interest, C910 also reduced viral infection in vitro including influenza A virus subtype H1N1 and SARS-CoV-2. Moreover, parenteral administration of C910 to the mice resulted in its accumulation in lung tissues and reduced lethal influenza infection.

CTX-CNF1 Recombinant Protein Selectively Targets Glioma Cells In Vivo

Current strategies for glioma treatment are only partly effective because of the poor selectivity for tumoral cells. Hence, the necessity to identify novel approaches is urgent. Recent studies highlighted the effectiveness of the bacterial protein cytotoxic necrotizing factor 1 (CNF1) in reducing tumoral mass, increasing survival of glioma-bearing mice and protecting peritumoral neural tissue from dysfunction. However, native CNF1 needs to be delivered into the brain, because of its incapacity to cross the blood–brain barrier (BBB) per se, thus hampering its clinical translation. To allow a non-invasive administration of CNF1, we here developed a chimeric protein (CTX-CNF1) conjugating CNF1 with chlorotoxin (CTX), a peptide already employed in clinics due to its ability of passing the BBB and selectively binding glioma cells. After systemic administration, we found that CTX-CNF1 is able to target glioma cells and significantly prolong survival of glioma-bearing mice. Our data point out the potentiality of CTX-CNF1 as a novel effective tool to treat gliomas.

Cnf1 Variants Endowed with the Ability to Cross the Blood–Brain Barrier: A New Potential Therapeutic Strategy for Glioblastoma

Among gliomas, primary tumors originating from glial cells, glioblastoma (GBM) identified as WHO grade IV glioma, is the most common and aggressive malignant brain tumor. We have previously shown that the Escherichia coli protein toxin cytotoxic necrotizing factor 1 (CNF1) is remarkably effective as an anti-neoplastic agent in a mouse model of glioma, reducing the tumor volume, increasing survival, and maintaining the functional properties of peritumoral neurons. However, being unable to cross the blood–brain barrier (BBB), CNF1 requires injection directly into the brain, which is a very invasive administration route. Thus, to overcome this pitfall, we designed a CNF1 variant characterized by the presence of an N-terminal BBB-crossing tag. The variant was produced and we verified whether its activity was comparable to that of wild-type CNF1 in GBM cells. We investigated the signaling pathways engaged in the cell response to CNF1 variants to provide preliminary data to the subsequent studies in experimental animals. CNF1 may represent a novel avenue for GBM therapy, particularly because, besides blocking tumor growth, it also preserves the healthy surrounding tissue, maintaining its architecture and functionality. This renders CNF1 the most interesting candidate for the treatment of brain tumors, among other potentially effective bacterial toxins.

Revisiting Old Ionophore Lasalocid as a Novel Inhibitor of Multiple Toxins

The ionophore lasalocid is widely used as a veterinary drug against coccidiosis. We found recently that lasalocid protects cells from two unrelated bacterial toxins, the cytotoxic necrotizing factor-1 (CNF1) from Escherichia. coli and diphtheria toxin. We evaluated lasalocid’s capacity to protect cells against other toxins of medical interest comprising toxin B from Clostridium difficile, Shiga-like toxin 1 from enterohemorrhagic E. coli and exotoxin A from Pseudomonas aeruginosa. We further characterized the impact of lasalocid on the endolysosomal and the retrograde pathways and organelle integrity, especially the Golgi apparatus. We found that lasalocid protects cells from all toxins tested and impairs the drop of vesicular pH along the trafficking pathways that are required for toxin sorting and translocation to the cytoplasm. Lasalocid also has an impact on the cellular distribution of GOLPH4 and GOLPH2 Golgi markers. Other intracellular trafficking compartments positive for EEA1 and Rab9A display a modified cellular pattern. In conclusion, lasalocid protects cells from multiple deadly bacterial toxins by corrupting vesicular trafficking and Golgi stack homeostasis.