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

Abstract

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.

Keywords: actin cytoskeleton; adenosine triphosphate; cytotoxic necrotizing factor 1; mitochondrial diseases; myoclonic epilepsy with ragged red fibers syndrome.

Figure 1

CNF1 effects ATP content and mitochondrial morphology and mass in fibroblasts from a patient carrying a mitochondrial disease. (A) Muscle pathology in a control patient (WT): the muscle looks normal without mitochondrial proliferation or COX-deficient fibers. Muscle pathology in the MA19 patient: SDH histochemistry shows mitochondrial proliferation with classic RBF, which appeared pale with the COX staining (magnification 20×); (B) Graph showing the relative increase of ATP content in WT and MA19 fibroblasts after 48 h of exposure to CNF1, either in the presence or absence of oligomycin added during the last 4 h of incubation. Data are expressed as percentages and represent mean ± standard error of the mean (SEM) from at least three independent experiments. *** p < 0.001 for WT versus MA19; aa p < 0.01 for WT versus WT + CNF1; b p < 0.05 for WT + oligo versus WT + CNF1 + oligo; cc p < 0.01 for MA19 versus MA19 + CNF1; d p < 0.05 for MA19 + oligo versus MA19 + CNF1 + oligo; (C) Fluorescence micrographs of WT and MA19 fibroblasts treated with CNF1 for 48 h and then stained with the mitochondrial dye Mitotracker (red) and with Hoechst 33258 (blue). Note that CNF1 treatment modifies the mitochondrial network in MA19 fibroblasts, rescuing the mitochondrial morphology of untreated WT fibroblasts. Bar = 10 µm; (D) Immunoblots showing representative Western blot experiments in whole cells lysates from control WT and MA19 fibroblasts. The amount of Tom20 is normalized as a function of GAPDH (bottom histograms). The histogram on the right shows, in percentage, the relative increase in Tom20 expression for both cell types (untreated WT and MA19 at time 0 = 100) following 48 h of toxin challenge, both normalized to their controls (treatment at time 0 = 100%). Data are represented as the means ± SEM from three independent experiments. * p < 0.05 for MA19 versus MA19 + CNF1.

Figure 2

In the MA19 patient’s fibroblasts, CNF1 restores the actin stress fibers’ organization typical of WT cells. Fluorescence micrographs of WT fibroblasts (left) column and MA19 fibroblasts (right) column treated with CNF1 at the indicated time points. Note that the actin stress fibers’ organization appeared improved in WT fibroblasts with an apparent reinforcement of the actin fibers. The reorganization promoted by the toxin was more evident in pathological fibroblasts that showed an impressive restoration of the cytoskeletal phenotype typical of fibroblasts. Arrows show the stress fibers. Bar = 10 µm.