Special Issue "Bacterial Toxins and Cancer".

Infection is a major contributor to the development of cancer, with more than 15% of new cancer diagnoses estimated to be caused by infection [...].

Effects of the Rho GTPase-activating toxin CNF1 on fibroblasts derived from Rett syndrome patients: A pilot study.

The bacterial product CNF1, through its action on the Rho GTPases, is emerging as a modulator of crucial signalling pathways involved in selected neurological diseases characterized by mitochondrial dysfunctions. Mitochondrial impairment has been hypothesized to have a key role in paramount mechanisms underlying Rett syndrome (RTT), a severe neurologic rare disorder. CNF1 has been already reported to have beneficial effects in mouse models of RTT. Using human RTT fibroblasts from four patients carrying different mutations, as a reliable disease-in-a-dish model, we explored the cellular and molecular mechanisms, which can underlie the CNF1-induced amelioration of RTT deficits. We found that CNF1 treatment modulates the Rho GTPases activity of RTT fibroblasts and induces a considerable re-organization of the actin cytoskeleton, mainly in stress fibres. Mitochondria of RTT fibroblasts show a hyperfused morphology and CNF1 decreases the mitochondrial mass leaving substantially unaltered the mitochondrial dynamic. From a functional perspective, CNF1 induces mitochondrial membrane potential depolarization and activation of AKT in RTT fibroblasts. Given that mitochondrial quality control is altered in RTT, our results are suggestive of a reactivation of the damaged mitochondria removal via mitophagy restoration. These effects can be at the basis of the beneficial effects of CNF1 in RTT.

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.

Gut Microbiota and Colon Cancer: A Role for Bacterial Protein Toxins?

Accumulating evidence indicates that the human intestinal microbiota can contribute to the etiology of colorectal cancer. Triggering factors, including inflammation and bacterial infections, may favor the shift of the gut microbiota from a mutualistic to a pro-carcinogenic configuration. In this context, certain bacterial pathogens can exert a pro-tumoral activity by producing enzymatically-active protein toxins that either directly induce host cell DNA damage or interfere with essential host cell signaling pathways involved in cell proliferation, apoptosis, and inflammation. This review is focused on those toxins that, by mimicking carcinogens and cancer promoters, could represent a paradigm for bacterially induced carcinogenesis.

The Bacterial Toxin CNF1 Protects Human Neuroblastoma SH-SY5Y Cells against 6-Hydroxydopamine-Induced Cell Damage: The Hypothesis of CNF1-Promoted Autophagy as an Antioxidant Strategy.

Several chronic neuroinflammatory diseases, including Parkinson's disease (PD), have the so-called 'redox imbalance' in common, a dynamic system modulated by various factors. Among them, alteration of the mitochondrial functionality can cause overproduction of reactive oxygen species (ROS) with the consequent induction of oxidative DNA damage and apoptosis. Considering the failure of clinical trials with drugs that eliminate ROS directly, research currently focuses on approaches that counteract redox imbalance, thus restoring normal physiology in a neuroinflammatory condition. Herein, we used SH-SY5Y cells treated with 6-hydroxydopamine (6-OHDA), a neurotoxin broadly employed to generate experimental models of PD. Cells were pre-treated with the Rho-modulating Escherichia coli cytotoxic necrotizing factor 1 (CNF1), before the addition of 6-OHDA. Then, cell viability, mitochondrial morphology and dynamics, redox profile as well as autophagic markers expression were assessed. We found that CNF1 preserves cell viability and counteracts oxidative stress induced by 6-OHDA. These effects are accompanied by modulation of the mitochondrial network and an increase in macroautophagic markers. Our results confirm the Rho GTPases as suitable pharmacological targets to counteract neuroinflammatory diseases and evidence the potentiality of CNF1, whose beneficial effects on pathological animal models have been already proven to act against oxidative stress through an autophagic strategy.

The Escherichia coli protein toxin cytotoxic necrotizing factor 1 induces epithelial mesenchymal transition.

Some toxigenic bacteria produce protein toxins with carcinogenic signatures, which either directly damage DNA or stimulate signalling pathways related to cancer. So far, however, only a few of them have been proved to favour the induction or progression of cancer. In this work, we report that the Rho-activating Escherichia coli protein toxin, cytotoxic necrotising factor 1 (CNF1), induces epithelial to mesenchymal transition (EMT) in intestinal epithelial cells. EMT is a crucial step in malignant tumour conversion and invasiveness. In the case of CNF1, it occurs by up-regulation of the transcription factors ZEB1 and Snail1, delocalisation of E-cadherin and ß-catenin, activation of the serine/threonine kinase mTOR, accelerated wound healing, and invasion. However, our results highlight that nontransformed epithelial cells entail the presence of inflammatory factors, in addition to CNF1, to acquire a mesenchymal-like behaviour. All this suggests that the surrounding microenvironment, as well as the cell type, dramatically influences the CNF1 ability to promote carcinogenic traits.

Eye Drop Instillation of the Rac1 Modulator CNF1 Attenuates Retinal Gliosis and Ameliorates Visual Performance in a Rat Model of Hypertensive Retinopathy.

In hypertensive retinopathy, the retinal damage due to high blood pressure is accompanied by increased expression of Glial Fibrillary Acidic Protein (GFAP), which indicates a role of neuroinflammatory processes in such a retinopathy. Proteins belonging to the Rho GTPase family, particularly Rac1, are involved in the activation of Müller glia and in the progression of photoreceptor degeneration, and may thus represent a novel candidate for therapeutic intervention following central nervous system inflammation. In this paper, we have observed that topical administration as eye drops of Cytotoxic Necrotizing Factor 1 (CNF1), a Rho GTPase modulator, surprisingly improves electrophysiological and behavioral visual performances in aged spontaneously hypertensive rats. Furthermore, such functional improvement is accompanied by a reduction of Rac1 activity and retinal GFAP expression. Our results suggest that Rac1 inhibition through CNF1 topical administration may represent a new strategy to target retinal gliosis.

The bacterial protein CNF1 as a new strategy against Plasmodium falciparum cytoadherence.

Plasmodium falciparum severe malaria causes more than 400,000 deaths every year. One feature of P. falciparum-parasitized erythrocytes (pRBC) leading to cerebral malaria (CM), the most dangerous form of severe malaria, is cytoadherence to endothelium and blockage of the brain microvasculature. Preventing ligand-receptor interactions involved in this process could inhibit pRBC sequestration and insurgence of severe disease whilst reversing existing cytoadherence could be a saving life adjunct therapy. Increasing evidence indicate the endothelial Rho signaling as a crucial player in malaria parasite cytoadherence. Therefore, we have used the cytotoxic necrotizing factor 1 (CNF1), an Escherichia coli protein able to modulate the activity of Cdc42, Rac, and Rho, three subfamilies of the Rho GTPases family, to study interactions between infected erythrocytes and cerebral endothelium in co-culture models. The main results are that CNF1 not only prevents cytoadherence but, more importantly, induces the detachment of pRBCs from endothelia monolayers. We first observed that CNF1 does affect neither parasite growth, nor the morphology and concentration of knobs that characterize the parasitized erythrocyte surface, as viewed by scanning electron microscopy. On the other hand, flow cytometry experiments show that cytoadherence reversion induced by CNF1 occurs in parallel with a decreased ICAM-1 receptor expression on the cell surface, suggesting the involvement of a toxin-promoted endocytic activity in such a response. Furthermore, since the endothelial barrier functionality is compromised by P. falciparum, we conducted a permeability assay on endothelial cells, revealing the CNF1 capacity to restore the brain endothelial barrier integrity. Then, using pull-down assays and inhibitory studies, we demonstrated, for the first time, that CNF1 is able not only to prevent but also to cause the parasite detachment by simultaneously activating Rho, Rac and Cdc42 in endothelial cells. All in all our findings indicate that CNF1 may represent a potential novel therapeutic strategy for preventing neurological complications of CM.

The Bacterial Protein CNF1 as a Potential Therapeutic Strategy against Mitochondrial Diseases: A Pilot Study.

The Escherichia coli protein toxin cytotoxic necrotizing factor 1 (CNF1), which acts on the Rho GTPases that are key regulators of the actin cytoskeleton, is emerging as a potential therapeutic tool against certain neurological diseases characterized by cellular energy homeostasis impairment. In this brief communication, we show explorative results on the toxin's effect on fibroblasts derived from a patient affected by myoclonic epilepsy with ragged-red fibers (MERRF) that carries a mutation in the m.8344A>G gene of mitochondrial DNA. We found that, in the patient's cells, besides rescuing the wild-type-like mitochondrial morphology, CNF1 administration is able to trigger a significant increase in cellular content of ATP and of the mitochondrial outer membrane marker Tom20. These results were accompanied by a profound F-actin reorganization in MERRF fibroblasts, which is a typical CNF1-induced effect on cell cytoskeleton. These results point at a possible role of the actin organization in preventing or limiting the cell damage due to mitochondrial impairment and at CNF1 treatment as a possible novel strategy against mitochondrial diseases still without cure.

New therapeutics from Nature: The odd case of the bacterial cytotoxic necrotizing factor 1.

Natural products may represent a rich source of new drugs. The enthusiasm toward this topic has recently been fueled by the 2015 Nobel Prize in Physiology or Medicine, awarded for the discovery of avermectin and artemisinin, natural products from Bacteria and Plantae, respectively, which have targeted one of the major global health issues, the parasitic diseases. Specifically, bacteria either living in the environment or colonizing our body may produce compounds of unexpected biomedical value with the potentiality to be employed as therapeutic drugs. In this review, the fascinating history of CNF1, a protein toxin produced by pathogenic strains of Escherichia coli, is divulged. Even if produced by bacteria responsible for a variety of diseases, CNF1 can behave as a promising benefactor to mankind. By modulating the Rho GTPases, this bacterial product plays a key role in organizing the actin cytoskeleton, enhancing synaptic plasticity and brain energy level, rescuing cognitive deficits, reducing glioma growth in experimental animals. These abilities strongly suggest the need to proceed with the studies on this odd drug in order to pave the way toward clinical trials.

CSF tau is associated with impaired cortical plasticity, cognitive decline and astrocyte survival only in APOE4-positive Alzheimer's disease.

In Alzheimer's disease (AD) patients, apopoliprotein (APOE) polymorphism is the main genetic factor associated with more aggressive clinical course. However, the interaction between cerebrospinal fluid (CSF) tau protein levels and APOE genotype has been scarcely investigated. A possible key mechanism invokes the dysfunction of synaptic plasticity. We investigated how CSF tau interacts with APOE genotype in AD patients. We firstly explored whether CSF tau levels and APOE genotype influence disease progression and long-term potentiation (LTP)-like cortical plasticity as measured by transcranial magnetic stimulation (TMS) in AD patients. Then, we incubated normal human astrocytes (NHAs) with CSF collected from sub-groups of AD patients to determine whether APOE genotype and CSF biomarkers influence astrocytes survival. LTP-like cortical plasticity differed between AD patients with apolipoprotein E4 (APOE4) and apolipoprotein E3 (APOE3) genotype. Higher CSF tau levels were associated with more impaired LTP-like cortical plasticity and faster disease progression in AD patients with APOE4 but not APOE3 genotype. Apoptotic activity was higher when cells were incubated with CSF from AD patients with APOE4 and high tau levels. CSF tau is detrimental on cortical plasticity, disease progression and astrocyte survival only when associated with APOE4 genotype. This is relevant for new therapeutic approaches targeting tau.

CNF1 Enhances Brain Energy Content and Counteracts Spontaneous Epileptiform Phenomena in Aged DBA/2J Mice.

Epilepsy, one of the most common conditions affecting the brain, is characterized by neuroplasticity and brain cell energy defects. In this work, we demonstrate the ability of the Escherichia coli protein toxin cytotoxic necrotizing factor 1 (CNF1) to counteract epileptiform phenomena in inbred DBA/2J mice, an animal model displaying genetic background with an high susceptibility to induced- and spontaneous seizures. Via modulation of the Rho GTPases, CNF1 regulates actin dynamics with a consequent increase in spine density and length in pyramidal neurons of rat visual cortex, and influences the mitochondrial homeostasis with remarkable changes in the mitochondrial network architecture. In addition, CNF1 improves cognitive performances and increases ATP brain content in mouse models of Rett syndrome and Alzheimer's disease. The results herein reported show that a single dose of CNF1 induces a remarkable amelioration of the seizure phenotype, with a significant augmentation in neuroplasticity markers and in cortex mitochondrial ATP content. This latter effect is accompanied by a decrease in the expression of mitochondrial fission proteins, suggesting a role of mitochondrial dynamics in the CNF1-induced beneficial effects on this epileptiform phenotype. Our results strongly support the crucial role of brain energy homeostasis in the pathogenesis of certain neurological diseases, and suggest that CNF1 could represent a putative new therapeutic tool for epilepsy.

Mode of action of fibrous amphiboles: the case of Biancavilla (Sicily, Italy).

BACKGROUND: The inhalation of fibrous amphiboles can result in pulmonary fibrosis, lung cancer and mesothelioma. Although these fibres have the same disease-causing potential, their different morphologies and chemical composition can determine different biological activities. An unusual cluster of mesothelioma was evidenced in Biancavilla (Sicily) where no inhabitant had been significantly exposed to asbestos. OBJECTIVE: We herein discuss the mechanism of action of amphiboles, focusing on the fibres identified in the study area. RESULTS: Human lung carcinoma cells have been exposed to two different materials: prismatic fluoro-edenite and fibres with fluoro-edenitic composition. Only in the second case, they exhibit features typical of transformed cells, such as multinucleation, prosurvival activity and pro-inflammatory cytokine release. Accordingly, in vivo studies demonstrated that the fibrous sample only could induce a mesotheliomatogenic effect. CONCLUSIONS: Fibres with fluoro-edenitic composition behave similarly to the asbestos crocidolite, whose connection with inflammation and lung cancer is well established.

Enhancement of mitochondrial ATP production by the Escherichia coli cytotoxic necrotizing factor 1.

Mitochondria are dynamic organelles that constantly change shape and structure in response to different stimuli and metabolic demands of the cell. The Escherichia coli protein toxin cytotoxic necrotizing factor 1 (CNF1) has recently been reported to influence mitochondrial activity in a mouse model of Rett syndrome and to increase ATP content in the brain tissue of an Alzheimer's disease mouse model. In the present work, the ability of CNF1 to influence mitochondrial activity was investigated in IEC-6 normal intestinal crypt cells. In these cells, the toxin was able to induce an increase in cellular ATP content, probably due to an increment of the mitochondrial electron transport chain. In addition, the CNF1-induced Rho GTPase activity also caused changes in the mitochondrial architecture that mainly consisted in the formation of a complex network of elongated mitochondria. The involvement of the cAMP-dependent protein kinase A signaling pathway was postulated. Our results demonstrate that CNF1 positively affects mitochondria by bursting their energetic function and modifying their morphology.

The E. coli CNF1 as a pioneering therapy for the central nervous system diseases.

The Cytotoxic Necrotizing Factor 1 (CNF1), a protein toxin from pathogenic E. coli, modulates the Rho GTPases, thus, directing the organization of the actin cytoskeleton. In the nervous system, the Rho GTPases play a key role in several processes, controlling the morphogenesis of dendritic spines and synaptic plasticity in brain tissues. This review is focused on the peculiar property of CNF1 to enhance brain plasticity in in vivo animal models of central nervous system (CNS) diseases, and on its possible application in therapy.

The cytotoxic necrotizing factor 1 from E. coli: a janus toxin playing with cancer regulators.

Certain strains of Escherichia coli have been indicated as a risk factor for colon cancer. E. coli is a normal inhabitant of the human intestine that becomes pathogenic, especially in extraintestinal sites, following the acquisition of virulence factors, including the protein toxin CNF1. This Rho GTPases-activating toxin induces dysfunctions in transformed epithelial cells, such as apoptosis counteraction, pro-inflammatory cytokines' release, COX2 expression, NF-kB activation and boosted cellular motility. As cancer may arise when the same regulatory pathways are affected, it is conceivable to hypothesize that CNF1-producing E. coli infections can contribute to cancer development. This review focuses on those aspects of CNF1 related to transformation, with the aim of contributing to the identification of a new possible carcinogenic agent from the microbial world.

CNF1 increases brain energy level, counteracts neuroinflammatory markers and rescues cognitive deficits in a murine model of Alzheimer's disease.

Overexpression of pro-inflammatory cytokines and cellular energy failure are associated with neuroinflammatory disorders, such as Alzheimer's disease. Transgenic mice homozygous for human ApoE4 gene, a well known AD and atherosclerosis animal model, show decreased levels of ATP, increased inflammatory cytokines level and accumulation of beta amyloid in the brain. All these findings are considered responsible for triggering cognitive decline. We have demonstrated that a single administration of the bacterial E. coli protein toxin CNF1 to aged apoE4 mice, beside inducing a strong amelioration of both spatial and emotional memory deficits, favored the cell energy restore through an increment of ATP content. This was accompanied by a modulation of cerebral Rho and Rac1 activity. Furthermore, CNF1 decreased the levels of beta amyloid accumulation and interleukin-1ß expression in the hippocampus. Altogether, these data suggest that the pharmacological modulation of Rho GTPases by CNF1 can improve memory performances in an animal model of Alzheimer's disease via a control of neuroinflammation and a rescue of systemic energy homeostasis.

CNF1 improves astrocytic ability to support neuronal growth and differentiation in vitro.

Modulation of cerebral Rho GTPases activity in mice brain by intracerebral administration of Cytotoxic Necrotizing Factor 1 (CNF1) leads to enhanced neurotransmission and synaptic plasticity and improves learning and memory. To gain more insight into the interactions between CNF1 and neuronal cells, we used primary neuronal and astrocytic cultures from rat embryonic brain to study CNF1 effects on neuronal differentiation, focusing on dendritic tree growth and synapse formation, which are strictly modulated by Rho GTPases. CNF1 profoundly remodeled the cytoskeleton of hippocampal and cortical neurons, which showed philopodia-like, actin-positive projections, thickened and poorly branched dendrites, and a decrease in synapse number. CNF1 removal, however, restored dendritic tree development and synapse formation, suggesting that the toxin can reversibly block neuronal differentiation. On differentiated neurons, CNF1 had a similar effacing effect on synapses. Therefore, a direct interaction with CNF1 is apparently deleterious for neurons. Since astrocytes play a pivotal role in neuronal differentiation and synaptic regulation, we wondered if the beneficial in vivo effect could be mediated by astrocytes. Primary astrocytes from embryonic cortex were treated with CNF1 for 48 hours and used as a substrate for growing hippocampal neurons. Such neurons showed an increased development of neurites, in respect to age-matched controls, with a wider dendritic tree and a richer content in synapses. In CNF1-exposed astrocytes, the production of interleukin 1ß, known to reduce dendrite development and complexity in neuronal cultures, was decreased. These results demonstrate that astrocytes, under the influence of CNF1, increase their supporting activity on neuronal growth and differentiation, possibly related to the diminished levels of interleukin 1ß. These observations suggest that the enhanced synaptic plasticity and improved learning and memory described in CNF1-injected mice are probably mediated by astrocytes.

Human melanoma cells express FGFR/Src/Rho signaling that entails an adhesion-independent caveolin-1 membrane association.

Caveolae have been indicated as a center of cytoskeleton regulation for Src kinase/Rho GTPase signaling. In addition, Src recruitment on intact cortical actin cytoskeleton appears to be required for bFGF/FGFR signal activation. Recently, we established a relationship between caveolin-1 (Cav-1) expression and cell migration in human malignant melanoma, constitutively activated by a bFGF autoregulatory loop. This work intends to investigate whether caveolae's asset, through bFGF/FGFR/c-Src/Rho signaling, could be related to melanoma cell anchorage. Accordingly, we revealed the existence of a FGFR/Src kinase pathway in Cav-1 enriched detergent-resistant membranes (DRMs) of Me665/1 metastatic melanoma cells, as confirmed by FGFR silencing. Moreover, we determined the expression and phosphorylation levels of Cav-1/Src/Erk signal pathway as a function of FGFR activation and cell density. A sucrose density gradient ultracentrifugation was employed to monitor Cav-1 membrane association and buoyancy in Me665/1 cells treated for actin fragmentation or for altered phosphorylation signals. As a result, melanoma cells show remarkable resistance to Cav-1 disassembly, together with persisting cell signal activity, being Src and Cav-1 crucial modulators of Rho GTPases. In conclusion, our study primarily highlights, in a metastatic melanoma cell line expressing caveolin, the circumstances whereby caveola structural and functional endurance enables the FGFR/Src/Rho GTPases pathway to keep on cell progression.

Escherichia coli cytotoxic necrotizing factor 1 (CNF1): toxin biology, in vivo applications and therapeutic potential.

CNF1 is a protein toxin produced by certain pathogenic strains of Escherichia coli. It permanently activates the regulatory Rho, Rac, and Cdc42 GTPases in eukaryotic cells, by deamidation of a glutamine residue. This modification promotes new activities in cells, such as gene transcription, cell proliferation and survival. Since the Rho GTPases play a pivotal role also in several processes in vivo, the potentiality of CNF1 to act as a new pharmacological tool has been explored in experimental animals and in diverse pathological contexts. In this review, we give an update overview on the potential in vivo applications of CNF1.