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.


Introduction
Cytotoxic necrotizing factor 1 (CNF1) is a bacterial virulence factor associated with some pathogenic Escherichia coli strains causing urinary tract infection and meningitis [1]. It belongs to the cytotoxic necrotizing factors family that includes proteins from E. coli (CNF1, CNF2, and CNF3) and Yersinia pseudotuberculosis (CNFY). CNF1 is an AB-type toxin, composed of a cell-binding domain and the C-terminal catalytic domain, bearing deamidase activity. The cell-binding domain encompasses two interaction sites in CNF1: an N-terminus domain, which interacts with the 37-kDa laminin receptor precursor (LRP), and a domain directly adjacent to the catalytic domain, which is a high affinity interaction site for the Lutheran (Lu) adhesion glycoprotein/basal cell adhesion molecule (BCAM) Lu/BCAM [2,3]. Following endocytosis, the catalytic domain of CNF1 is cleaved off and released into the cytosol [4]. The CNF1 target is represented by small GTPases belonging to the Rho family, Rho, Rac, and Cdc42. CNF1 deamidates a specific glutamine residue, located in the switch 2 domain and involved in GTP hydrolysis (glutamine 63 in RhoA [3,5,6] or 61 in Cdc42 and Rac1 [7]) and this modification results in the constitutive association of the Rho GTPase with GTP, namely, its constitutive activation.
Nonetheless, some of the Rho-regulated signaling pathways have been found to be only transiently activated. That is because, once constitutively activated by CNF1,  Tables 1-3, the observed effects induced by CNF1 toxin and the related references have been grouped together for each cell type origin i.e., cancer, immortalized/transformed, and finite/primary.  Tables 1-3, the observed effects induced by CNF1 toxin and the related references have been grouped together for each cell type origin i.e., cancer, immortalized/transformed, and finite/primary. SH-SY5Y hu brain, neuroblastoma, epithelial [29] • counteraction of the 6-OHDA-induced: − cell toxicity − phospho-Drp1 decrease − oxidative stress − mitochondrial fragmentation • enrichment of the mitochondrial network • autophagy induction: − increase in LC3-II expression − colocalization of LC3-II and LAMP1 recombinant, purified by chromatography SK-N-SH hu brain, neuroblastoma, epithelial [30] • Rac1/AKT/NF-κB-dependent MORs protein upregulation • redistribution of MORs protein at the cell surface recombinant, purified by chromatography U87 hu brain, glioblastoma like, epithelial [31] • increase in SA β-gal activity • p21 upregulation (mRNA) recombinant, purified by chromatography GBM (Glioblastoma) hu brain, glioblastoma multiforme [31,32]

Effects on Rho GTPases and on Actin Cytoskeleton
Not all selected papers report the description of the CNF1 effects on its direct targets, Rho, Rac, and Cdc42 (i.e., analyzed by pulldown, band shift, or glutamine 63 deamidation experiments), probably depending on the specific purpose of the single experimental work.
In most studies, the induction of at least some of the morphological effects characteristic of Rho GTPase activation is described, that is, changes in the actin cytoskeleton organization, demonstrated by actin stress fibers or the formation of actin cables, membrane ruffles, filopodia and lamellipodia assembly (Figure 2A)

Effects on Rho GTPases and on Actin Cytoskeleton
Not all selected papers report the description of the CNF1 effects on its direct targets, Rho, Rac, and Cdc42 (i.e., analyzed by pulldown, band shift, or glutamine 63 deamidation experiments), probably depending on the specific purpose of the single experimental work.
Of interest, in T-lymphocytes and IEC-6 cells, CNF1 was able to raise cell motility, but only in the presence of SDF-1α [44] or inflammation mediators [22].
Effects of CNF1 treatment on phagocytic activity has been observed. In particular, two publications [60,95] report that, in different monocyte/macrophagic cell lines, of both primary and cancer origin (BMDM, mouse peritoneal macrophages, human monocytes, THP-1, Raw264.7), CNF1 reduced the phagocytosis of nonopsonized beads and of nonopsonized bacteria. Conversely, in HEp-2 and in 804G epithelial cancer cell lines, CNF1 confers the ability to ingest latex beads as well as bacteria [8,46,47,54].
These results suggest that CNF1 might contribute to bacterial infection by favoring epithelium colonization and/or affecting the host innate immune defense, thus reducing the pathogenic E. coli clearance ability of macrophages (by decreasing scavenger receptor CD36 expression).
On cell lines of endothelial origin, activation of Rho GTPases by CNF1 seems to have different effects on the regulation of cell permeability depending on the background of the endothelial cell lines. Baumer and co-workers [70] show, in fact, that CNF1-induced activation of Rho GTPases reduces permeability in microvascular endothelial cell types; whereas, in macrovascular endothelial cells CNF1 stabilizes barrier functions.

Multinucleation, Cell Cycle, Cell Death, Apoptosis, and Senescence
It is well known that Rho signaling pathways are involved in cell proliferation and cell cycle regulation, also through actin cytoskeleton regulation.
In four cell lines, the inhibition of cell cycle progression leads cells to a senescence state (U87 GL261, human GBM [31]; HCT-116 [45]). Of interest, Zhang and co-workers [45] showed that in HCT-116 human colon cancer cells, CNF1 elicited endoreplication and polyploidization driving cells into a reversible senescence state, which provided a survival route to the cells via depolyploidization. Indeed, authors showed that when CNF1-induced polyploid cells were cultured in fresh medium, in the absence of the toxin, a population of depolyploidized cells able to re-enter the mitotic cycle was selected [45]. Importantly, progeny derived from the CNF1 treatment exhibited genomic instability exemplified by an increased aneuploidy.
In three cell lines, after prolonged treatment, the block of proliferation resulted in cell death (5637 [27]; 3T3-L1 [75]; GL261 [32]), which in one case occurred by an apoptotic mechanism (5637 [27]). In other cell lines, CNF1 treatment seems to protect cells from apoptosis induced by exposure to UV [57,96] or in simulated microgravity conditions [64,65]. Although the molecular mechanisms are still unknown, it seems reasonable, that the fate (senescence, cell death, or survival) of CNF1-treated cells largely depends on the cell type and on the transformation degree of the cells exposed.
On the other hand, one paper shows that CNF1 triggers the activation and phenotypic maturation of cultured monocyte-derived DCs, with an increased level of IL-6 and TNFα secretion and the proliferation of allogenic naïve CD4+ T cells (DC monocytes [83]). In bone-derived macrophages, CNF1 toxin activates the NLRP3 inflammasome via a signaling cascade that involves PAK1/Rac2, thus inducing caspase-1 activation and IL1-β secretion [85].
In cells of lymphoid origin, both primary (T-lymphocytes, BMDM) and leukemic (Jurkat), CNF1 treatment enhances cell migration ability across acellular filters and their adherence to colonic epithelial cell monolayers. In particular, treated T-lymphocytes are able to adhere more tightly to monolayers of human intestinal epithelial cell lines resulting in cytotoxicty for the epithelial cells. In these cells, CNF1 also stimulates the production of high levels of TGF-β1, TGF-β2, TGF-β3, and TNF-α proteins (Jurkat, T-lymphocytes [44]).
In NK cells, CNF1 causes a strong increase in the binding efficiency and killing capacity of effector cells. An augmented expression of cell adhesion and activation-associated molecules, as well as reshaping of the actin and microtubule networks, are also described and probably represent the basis of the enhanced binding ability and cytotoxicity of NKtreated cells [82].
Overall, the in vitro described effects of CNF1 toxin on cells of immune origin suggest its ability to affect innate immune defenses, facilitating bacterial infection and increasing the virulence of E. coli pathogenic strains (in the intestinal epithelia). On the other hand, CNF1 also seems to elicit a protective immune mechanism, which is consistent with in vivo studies indicating CNF1 as promoter of antibacterial immunity [101,102]. This apparent discrepancy between pro-and antibacterial activity induced by CNF1 probably depends on the experimental settings and on the specific purpose of the study.

CNF1 Effects on Different Cellular Pathways
Rho proteins have over 60 known downstream effectors, which determine the outcome of activation for a given Rho GTPase protein. The activation of CNF1-induced Rho GTPases affects different cellular pathways that, in turn, drive different new cell states. Actually, in the reviewed papers, CNF1 cell intoxication resulted in a number of proteins being modulated. The described effects on actin organization and cytoskeletal rearrangement (see Section 3.1.1.) are often accompanied by modifications in the distribution and/or expression of proteins involved in specific signal transduction pathways regulating cytoskeleton organization, as well as cell adhesion, motility, and migration.
It is evident that the heterogeneity of the reported results reflects the great variability of the experimental models, cell lines, experimental conditions, and authors' purpose between the different reviewed articles.

Discussion and Conclusions
CNF1 is a bacterial protein toxin mainly produced by E. coli, associated with extraintestinal disease, but occasionally detected in intestinal infections [105]. For this reason, many studies have been carried out on epithelial cells that represent the actual target of the toxin, in an attempt to analyze its role in E. coli pathogenesis. However, due to its specific activity on Rho GTPases, CNF1 has also been used as a tool for studies aimed at deciphering the involvement of Rho GTPases in certain pathways. In the present review, we aimed at giving a comprehensive examination of the CNF1 effects described in different human and animal cell lines, in an attempt to provide an easy-to-use guide of the results obtained so far. A schematic summary of the overall CNF1-induced effects reported in the literature is shown in Figure 3.

Discussion and Conclusions
CNF1 is a bacterial protein toxin mainly produced by E. coli, associated with extraintestinal disease, but occasionally detected in intestinal infections [105]. For this reason, many studies have been carried out on epithelial cells that represent the actual target of the toxin, in an attempt to analyze its role in E. coli pathogenesis. However, due to its specific activity on Rho GTPases, CNF1 has also been used as a tool for studies aimed at deciphering the involvement of Rho GTPases in certain pathways. In the present review, we aimed at giving a comprehensive examination of the CNF1 effects described in different human and animal cell lines, in an attempt to provide an easy-to-use guide of the results obtained so far. A schematic summary of the overall CNF1-induced effects reported in the literature is shown in Figure 3. From a general analysis of the published studies, a different activity of CNF1 does not emerge between primary, transformed and cancer cells. All in all, it is evident that the heterogeneity of the reported results reflects the great variability between the different reviewed articles in terms of experimental models, cell line tissue and type origin, experimental conditions and authors' purpose.
Almost all the in vitro studies report the direct CNF1 enzymatic activation of Rho proteins and/or its effects on cytoskeletal organization, irrespective of the cell type and transformation status of a cell. It is also interesting to note that the depletion of Rho GTPases, ensuing CNF1 exposure, does not seem to be related to the transformation status or cell type, but rather to a specific alteration of the ubiquitin pathway in certain cells (see introduction). Rho GTPases are important transducers in signaling pathways crucial for the maintenance of normal tissue. It is well known that the same signaling pathway regulated by a specific Rho can elicit distinct responses in different cell types, depending on From a general analysis of the published studies, a different activity of CNF1 does not emerge between primary, transformed and cancer cells. All in all, it is evident that the heterogeneity of the reported results reflects the great variability between the different reviewed articles in terms of experimental models, cell line tissue and type origin, experimental conditions and authors' purpose.
Almost all the in vitro studies report the direct CNF1 enzymatic activation of Rho proteins and/or its effects on cytoskeletal organization, irrespective of the cell type and transformation status of a cell. It is also interesting to note that the depletion of Rho GTPases, ensuing CNF1 exposure, does not seem to be related to the transformation status or cell type, but rather to a specific alteration of the ubiquitin pathway in certain cells (see introduction). Rho GTPases are important transducers in signaling pathways crucial for the maintenance of normal tissue. It is well known that the same signaling pathway regulated by a specific Rho can elicit distinct responses in different cell types, depending on the biological context, such as the extracellular stimuli and signaling pathways involved in that particular cell type [93]. This is also evident from reviewing the effects of CNF1, since various TFs are activated and different proteins are regulated in many of the experimental models analyzed.
Finally, we would like to point out that none of the published papers seem to take into account the possible further consequence elicited by CNF1 interaction with its receptors, the 37 kDa LRP (37LRP) and the Lutheran adhesion glycoprotein/basal cell adhesion molecule (Lu/BCAM) [2,3]. Actually, these two distinct laminin receptors are known to be involved in a number of cellular functions, resembling those regulated by CNF1. In particular, 37LRP, acting as a mediator of cell adhesion, cell proliferation and differentiation, hampers apoptosis, plays a major role as a cell surface receptor in prion disorders and could possibly be involved in the cell biology of neurodegenerative diseases, such as Alzheimer's disease (AD) [106,107]. In this context, it is interesting to underline that CNF1 is able to rescue cognitive deficits in a murine model of AD by increasing brain energy levels and counteracting neuroinflammatory markers [108]. Furthermore, the overexpression of 37LRP is evident in several cancer types and has been demonstrated to enhance the invasiveness of cancer cells [109].
Another aspect that could be taken into account in future studies is the possible effect due to the alteration of the balance between the F-and G-actin cellular pools, as a consequence of the CNF1-induced actin polymerization state. It has now been established that, in addition to its function in the cytoplasm, actin is actively imported into the nucleus, where it directly regulates transcription and participates in chromatin organization, mRNA transport, translation, post-translational modifications, chromosome positioning, DNA rearrangements and repair. All these functions are tightly linked to the balance between nuclear actin monomers and polymers in the nucleus and indirectly, to actin polymerization/depolymerization in the cytoplasm, which affects nuclear import/export [110].
Not least, the carcinogenic capacity of CNF1, in line with other toxins, is an emerging feature. Several modifications induced by CNF1 are, in fact, reminiscent of a procarcinogenic potential [111,112]. In recent years, studies have been carried out to corroborate this hypothesis [21,113], but the subject of study is still in its infancy.
In conclusion, although several aspects have already been addressed in studies dealing with CNF1, there are still completely new fields of investigation concerning the cellular activity of the toxin that deserve careful investigation.

Acknowledgments:
The authors thank Rossella Di Nallo for her invaluable technical contribution.

Conflicts of Interest:
The authors declare no conflict of interest.