A powerful machine learning approach to identify interactions of differentially abundant gut microbial subsets in patients with metastatic and non-metastatic pancreatic cancer.

Pancreatic cancer has a dismal prognosis, as it is often diagnosed at stage IV of the disease and is characterized by metastatic spread. Gut microbiota and its metabolites have been suggested to influence the metastatic spread by modulating the host immune system or by promoting angiogenesis. To date, the gut microbial profiles of metastatic and non-metastatic patients need to be explored. Taking advantage of the 16S metagenomic sequencing and the PEnalized LOgistic Regression Analysis (PELORA) we identified clusters of bacteria with differential abundances between metastatic and non-metastatic patients. An overall increase in Gram-negative bacteria in metastatic patients compared to non-metastatic ones was identified using this method. Furthermore, to gain more insight into how gut microbes can predict metastases, a machine learning approach (iterative Random Forest) was performed. Iterative Random Forest analysis revealed which microorganisms were characterized by a different level of relative abundance between metastatic and non-metastatic patients and established a functional relationship between the relative abundance and the probability of having metastases. At the species level, the following bacteria were found to have the highest discriminatory power: Anaerostipes hadrus, Coprobacter secundus, Clostridium sp. 619, Roseburia inulinivorans, Porphyromonas and Odoribacter at the genus level, and Rhodospirillaceae, Clostridiaceae and Peptococcaceae at the family level. Finally, these data were intertwined with those from a metabolomics analysis on fecal samples of patients with or without metastasis to better understand the role of gut microbiota in the metastatic process. Artificial intelligence has been applied in different areas of the medical field. Translating its application in the field of gut microbiota analysis may help fully exploit the potential information contained in such a large amount of data aiming to open up new supportive areas of intervention in the management of cancer.

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.

Protective Effect of Limosilactobacillus fermentum ME-3 against the Increase in Paracellular Permeability Induced by Chemotherapy or Inflammatory Conditions in Caco-2 Cell Models.

Chemotherapy- or inflammation-induced increase in intestinal permeability represents a severe element in disease evolution in patients suffering from colorectal cancer and gut inflammatory conditions. Emerging data strongly support the gut microbiota's role in preserving intestinal barrier integrity, whilst both chemotherapy and gut inflammation alter microbiota composition. Some probiotics might have a strong re-balancing effect on the gut microbiota, also positively affecting intestinal barrier integrity. In this study, we asked whether Limosilactobacillus fermentum ME-3 can prevent the intestinal paracellular permeability increase caused by the chemotherapeutic drug Irinotecan or by inflammatory stimuli, such as lipopolysaccharide (LPS). As an intestinal barrier model, we used a confluent and polarized Caco-2 cell monolayer and assessed the ME-3-induced effect on paracellular permeability by transepithelial electrical resistance (TEER) and fluorescent-dextran flux assays. The integrity of tight and adherens junctions was examined by confocal microscopy analysis. Transwell co-cultures of Caco-2 cells and U937-derived macrophages were used as models of LPS-induced intestinal inflammation to test the effect of ME-3 on release of the pro-inflammatory cytokines Tumor Necrosis Factor α, Interleukin-6, and Interleukin-8, was measured by ELISA. The results demonstrate that ME-3 prevents the IRI-induced increment in paracellular permeability, possibly by modulating the expression and localization of cell junction components. In addition, ME-3 inhibited both the increase in paracellular permeability and the release of pro-inflammatory cytokines in the co-culture model of LPS-induced inflammation. Our findings sustain the validity of L. fermentum ME-3 as a valuable therapeutic tool for preventing leaky gut syndrome, still currently without an available specific treatment.

Resources for Human Health from the Plant Kingdom: The Potential Role of the Flavonoid Apigenin in Cancer Counteraction.

Apigenin is one of the most widespread flavonoids in the plant kingdom. For centuries, apigenin-containing plant preparations have been used in traditional medicines to treat diseases that have an inflammatory and/or degenerative component. In the 1980s, apigenin was proposed to interfere with the process of carcinogenesis. Since then, more and more evidence has demonstrated its anticancer efficacy, both in vitro and in vivo. Apigenin has been shown to target signaling pathways involved in the development and progression of cancer, such as PI3K/Akt/mTOR, MAPK/ERK, JAK/STAT, NF-κB, and Wnt/ß-catenin pathways, and to modulate different hallmarks of cancer, such as cell proliferation, metastasis, apoptosis, invasion, and cell migration. Furthermore, apigenin modulates PD1/PD-L1 expression in cancer/T killer cells and regulates the percentage of T killer and T regulatory cells. Recently, apigenin has been studied for its synergic and additive effects when combined with chemotherapy, minimizing the side effects. Unfortunately, its low bioavailability and high permeability limit its therapeutic applications. Based on micro- and nanoformulations that enhance the physical stability and drug-loading capacity of apigenin and increase the bioavailability of apigenin, novel drug-delivery systems have been investigated to improve its solubility.

Role of the Microbiota in Lung Cancer: Insights on Prevention and Treatment.

The microbiota is increasingly recognized as a critical player in cancer onset and progression and response to cancer chemotherapy treatment. In recent years, several preclinical and clinical studies have evidenced the involvement of microbiota in lung cancer, one of the world's deadliest cancers. However, the mechanisms by which the microbiota can impact this type of cancer and patient survival and response to treatments remain poorly investigated. In this review, the peculiarities of the gut and lung microbial ecosystems have been highlighted, and recent findings illustrating the possible mechanisms underlying the microbiota-lung cancer interaction and the host immune response have been discussed. In addition, the mucosal immune system has been identified as a crucial communication frame to ease interactive dynamics between the immune system and the microbiota. Finally, the use of specific next-generation intestinal probiotic strains in counteracting airway diseases has been evaluated. We believe that restoring homeostasis and the balance of bacterial microflora should become part of the routine of integrated cancer interventions, using probiotics, prebiotics, and postbiotics, and promoting a healthy diet and lifestyle.

The Combination of AHCC and ETAS Decreases Migration of Colorectal Cancer Cells, and Reduces the Expression of LGR5 and Notch1 Genes in Cancer Stem Cells: A Novel Potential Approach for Integrative Medicine.

The AHCC standardized extract of cultured Lentinula edodes mycelia, and the standardized extract of Asparagus officinalis stem, trademarked as ETAS, are well known supplements with immunomodulatory and anticancer potential. Several reports have described their therapeutic effects, including antioxidant and anticancer activity and improvement of immune response. In this study we aimed at investigating the effects of a combination of AHCC and ETAS on colorectal cancer cells and biopsies from healthy donors to assess the possible use in patients with colorectal cancer. Our results showed that the combination of AHCC and ETAS was synergistic in inducing a significant decrease in cancer cell growth, compared with single agents. Moreover, the combined treatment induced a significant increase in apoptosis, sparing colonocytes from healthy donors, and was able to induce a strong reduction in migration potential, accompanied by a significant modulation of proteins involved in invasiveness. Finally, combined treatment was able to significantly downregulate LGR5 and Notch1 in SW620 cancer stem cell (CSC) colonospheres. Overall, these findings support the potential therapeutic benefits of the AHCC and ETAS combinatorial treatment for patients with colorectal cancer.

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.

Association of Polygenic Risk Score and Bacterial Toxins at Screening Colonoscopy with Colorectal Cancer Progression: A Multicenter Case-Control Study.

Colorectal cancer (CRC) is a leading cause of cancer death worldwide, and its incidence is correlated with infections, chronic inflammation, diet, and genetic factors. An emerging aspect is that microbial dysbiosis and chronic infections triggered by certain bacteria can be risk factors for tumor progression. Recent data suggest that certain bacterial toxins implicated in DNA attack or in proliferation, replication, and death can be risk factors for insurgence and progression of CRC. In this study, we recruited more than 300 biopsy specimens from people undergoing colonoscopy, and we analyzed to determine whether a correlation exists between the presence of bacterial genes coding for toxins possibly involved in CRC onset and progression and the different stages of CRC. We also analyzed to determine whether CRC-predisposing genetic factors could contribute to bacterial toxins response. Our results showed that CIF toxin is associated with polyps or adenomas, whereas pks+ seems to be a predisposing factor for CRC. Toxins from Escherichia coli as a whole have a higher incidence rate in adenocarcinoma patients compared to controls, whereas Bacteroides fragilis toxin does not seem to be associated with pre-cancerous nor with cancerous lesions. These results have been obtained irrespectively of the presence of CRC-risk loci.

Treatment with the Bacterial Toxin CNF1 Selectively Rescues Cognitive and Brain Mitochondrial Deficits in a Female Mouse Model of Rett Syndrome Carrying a MeCP2-Null Mutation.

Rett syndrome (RTT) is a rare neurological disorder caused by mutations in the X-linked MECP2 gene and a major cause of intellectual disability in females. No cure exists for RTT. We previously reported that the behavioural phenotype and brain mitochondria dysfunction are widely rescued by a single intracerebroventricular injection of the bacterial toxin CNF1 in a RTT mouse model carrying a truncating mutation of the MeCP2 gene (MeCP2-308 mice). Given the heterogeneity of MECP2 mutations in RTT patients, we tested the CNF1 therapeutic efficacy in a mouse model carrying a null mutation (MeCP2-Bird mice). CNF1 selectively rescued cognitive defects, without improving other RTT-related behavioural alterations, and restored brain mitochondrial respiratory chain complex activity in MeCP2-Bird mice. To shed light on the molecular mechanisms underlying the differential CNF1 effects on the behavioural phenotype, we compared treatment effects on relevant signalling cascades in the brain of the two RTT models. CNF1 provided a significant boost of the mTOR activation in MeCP2-308 hippocampus, which was not observed in the MeCP2-Bird model, possibly explaining the differential effects of CNF1. These results demonstrate that CNF1 efficacy depends on the mutation beared by MeCP2-mutated mice, stressing the need of testing potential therapeutic approaches across RTT models.

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.

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.

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.

The Bacterial Toxin CNF1 Induces Activation and Maturation of Human Monocyte-Derived Dendritic Cells.

Cytotoxic necrotizing factor 1 (CNF1) is a bacterial protein toxin primarily expressed by pathogenic Escherichia coli strains, causing extraintestinal infections. The toxin is believed to enhance the invasiveness of E. coli by modulating the activity of Rho GTPases in host cells, but it has interestingly also been shown to promote inflammation, stimulate host immunity and function as a potent immunoadjuvant. The mechanisms underlying the immunostimulatory properties of CNF1 are, however, poorly characterized, and little is known about the direct effects of the toxin on immune cells. Here, we show that CNF1 induces expression of maturation markers on human immature monocyte-derived dendritic cells (moDCs) without compromising cell viability. Consistent with the phenotypic maturation, CNF1 further triggered secretion of proinflammatory cytokines and increased the capacity of moDCs to stimulate proliferation of allogenic naïve CD4+ T cells. A catalytically inactive form of the toxin did not induce moDC maturation, indicating that the enzymatic activity of CNF1 triggers immature moDCs to undergo phenotypic and functional maturation. As the maturation of dendritic cells plays a central role in initiating inflammation and activating the adaptive immune response, the present findings shed new light on the immunostimulatory properties of CNF1 and may explain why the toxin functions as an immunoadjuvant.

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.

Integrated Cancer Treatment in the Course of Metastatic Pancreatic Cancer: Complete Resolution in 2 Cases.

Pancreatic cancer (PC) has a very low average survival, but its prognosis is further reduced in the case of metastatic spread. Medical therapy in these cases is the only applicable methodology in the international guidelines. During anticancer treatments, common side effects are nausea, vomiting, arthralgia, neuropathy, and alopecia as well as a myelosuppressive effect. The toxicity of various drugs not only affects the quality of life of the patient, but often its severity requires a reduction in if not the termination of drug administration. Scientific studies have shown that a combined use of chemotherapy and certain natural substances, in the form of standardized extracts, can lead to an enhancement of the action of the chemotherapy. Here, we describe 2 cases of metastatic PC. The first case concerns the integrated treatment of a patient with cancer of the pancreas tail with metastatic involvement ab initio of peripancreatic lymph nodes and liver parenchyma, with numerous secondary lesions greater than 9.5 cm. The second case concerns the integrated treatment of a patient with cancer of the pancreatic body with metastatic involvement of the liver parenchyma, with a small secondary lesion. In both cases, an integrated cancer treatment approach, combining chemotherapy with natural remedies, extracts, and hyperthermia, induced a notable remission of primary and metastatic lesions.

High yield purification and first structural characterization of the full-length bacterial toxin CNF1.

The Cytotoxic Necrotizing Factor 1 (CNF1) is a bacterial toxin secreted by certain Escherichia coli strains causing severe pathologies, making it a protein of pivotal interest in toxicology. In parallel, the CNF1 capability to influence important neuronal processes, like neuronal arborization, astrocytic support, and efficient ATP production, has been efficiently used in the treatment of neurological diseases, making it a promising candidate for therapy. Nonetheless, there are still some unsolved issues about the CNF1 mechanism of action and structuration probably caused by the difficulty to achieve sufficient amounts of the full-length protein for further studies. Here, we propose an efficient strategy for the production and purification of this toxin as a his-tagged recombinant protein from E. coli extracts (CNF1-H8). CNF1-H8 was expressed at the low temperature of 15°C to diminish its characteristic degradation. Then, its purification was achieved using an immobilized metal affinity chromatography (IMAC) and a size exclusion chromatography so as to collect up to 8 mg of protein per liter of culture in a highly pure form. Routine dynamic light scattering (DLS) experiments showed that the recombinant protein preparations were homogeneous and preserved this state for a long time. Furthermore, CNF1-H8 functionality was confirmed by testing its activity on purified RhoA and on HEp-2 cultured cells. Finally, a first structural characterization of the full-length toxin in terms of secondary structure and thermal stability was performed by circular dichroism (CD). These studies demonstrate that our system can be used to produce high quantities of pure recombinant protein for a detailed structural analysis. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:150-159, 2018.