Clostridium difficile toxin B induces autophagic cell death in colonocytes

Abstract Toxin B (TcdB) is a major pathogenic factor of Clostridum difficile. However, the mechanism by which TcdB exerts its cytotoxic action in host cells is still not completely known. Herein, we report for the first time that TcdB induced autophagic cell death in cultured human colonocytes. The induction of autophagy was demonstrated by the increased levels of LC3‐II, formation of LC3+ autophagosomes, accumulation of acidic vesicular organelles and reduced levels of the autophagic substrate p62/SQSTM1. TcdB‐induced autophagy was also accompanied by the repression of phosphoinositide 3‐kinase (PI3K)/Akt/mechanistic target of rapamycin (mTOR) complex 1 activity. Functionally, pharmacological inhibition of autophagy by wortmannin or chloroquine or knockdown of autophagy‐related genes Beclin 1, Atg5 and Atg7 attenuated TcdB‐induced cell death in colonocytes. Genetic ablation of Atg5, a gene required for autophagosome formation, also mitigated the cytotoxic effect of TcdB. In conclusion, our study demonstrated that autophagy serves as a pro‐death mechanism mediating the cytotoxic action of TcdB in colonocytes. This discovery suggested that blockade of autophagy might be a novel therapeutic strategy for C. difficile infection.


| INTRODUCTION
Clostridium difficile is an anaerobic, Gram-positive, sporulating bacterium that gives rise to a spectrum of gastrointestinal diseases, including antibiotic-associated diarrhoea, pseudomembranous colitis and toxic megacolon. 1 C. difficile infection is a major cause of hospital-associated infection, accounting for about 3 million cases per year worldwide. 2 It is also the most common infectious cause of diarrhoea in Intensive Care Unit in which the prevalence is estimated to be about 4%. One-fifth of these ICU patients infected with C. difficile develop fulminant colitis with a mortality rate of nearly 60%. 3 The incidence of C. difficile is rapidly increasing, especially in developed countries. Therefore, C. difficile infection, which carries significant mortality and morbidity, remains a major health burden. Several factors have been associated with an increased risk for C. difficile infection, including (i) recent exposure to antibiotics, such as clindamycin, fluoroquinolones and cephalosporins; (ii) the use of acid-suppressive medications; (iii) recent hospitalization; (iv) advanced age and (v) comorbid conditions, such as the use of feeding tube and (vi) history of gastrointestinal surgery. 4 The pathogenicity of C. difficile has been attributed to the 2 major exotoxins, namely Toxin A (TcdA) and Toxin B (TcdB), encoded by its genome. Both exotoxins can glycosylate and thereby inhibiting small GTPases of host cells to (i) mediate cytotoxicity; (ii) disrupt actin cytoskeletons and tight junctions and thus impair the epithelial barrier function; and (iii) promote the release of inflammatory mediators, such as interleukin-8 and macrophage inflammatory protein 2.
To this end, TcdB is 10 times more potent than TcdA in mediating the pathogenicity. 5 Cessation of the inciting antibiotic and treatment with metronidazole and vancomycin are the mainstay in the management of C. difficile infection. Nevertheless, high rates of non-responsiveness (~22%) and relapse (~27%) have been associated with metronidazole. In addition, the emergence of vancomycin-resistant enterococci is a major concern for vancomycin. 3 Several non-antibiotic treatment modalities, such as toxin neutralization, probiotics and faecal microbiota transplantation, have been attempted, but their efficacies remain to be established. Development of other non-antibiotic therapeutics for C. difficile infection is an area of active investigation.
Autophagy is a regulated intracellular degradation system, participating in various human diseases and physiological processes, such as cancer, neurodegeneration, microbial infection, ageing and heart disease. 6,7 Although autophagy could be a cellular protective mechanism especially in times of nutrient deprivation and other stressful conditions, its extensive activation could mediate cell death. The precise role of autophagy in C. difficile-induced intestinal cell death and inflammation, however, remains unclear. In this study, we sought to investigate whether autophagy takes part in the cytotoxicity of TcdB in colonocytes.

| Cytotoxicity assay
Cells were incubated in the absence or presence of different concen-

| RNA interference
Cells were seeded at a density of 8000 cells per well in 96-well plates prior to the transfection. During the transfection, the cells were transfected with small interfering RNA (Thermo Fisher) against autophagy-related genes (ie Beclin 1, Atg5 and Atg7) using the jet-PRIME transfection reagent (Polyplus) according to the manufacturer's instructions.

| Detection of LC3 + autophagosomes
NCM460 cells were grown on glass chamber slides overnight and then transfected with mCherry-GFP-LC3 for 24 hours. After transfection, cells were exposed to TcdB (10 ng/mL) for different periods of time. Cells were then rinsed twice with 19 PBS and fixed in 4% paraformaldehyde for 15 minutes at room temperature. After rinsing twice with 1 9 PBS, the slides were mounted in prolong gold anti-fade reagent (Invitrogen, Carlsbad, CA, USA) and then analysed on a confocal microscope (Leica).

| Statistical analysis
Statistical analysis was performed with one-way analysis of variance (ANOVA) followed by the Tukey's post hoc test. P values <.05 will be considered statistically significant.

| TcdB reduces viability of colonocytes without inducing apoptosis
Colon is the primary anatomic site of C. difficile infection. We therefore determined the effect of TcdB on the viability of cultured human colonocytes. NCM460 cells were exposed to increasing concentrations of TcdB from 0.1 to 100 ng/mL for 24 or 48 hours followed by the CCK-8 assay. As shown in Figure 1A, TcdB reduced the viability of NCM460 cells in a dose-and time-dependent manner. The significant cytotoxic effect of TcdB could be detected at the concentration as low as 10 ng/mL at the 24-hour time-point.
Dose-response analysis for assessing the half maximal inhibitory concentration (IC 50 ) of TcdB was also conducted. It was found that the IC 50 of TcdB in NCM460 cells was 235.6 ng/mL at the 48-hour time-point. To assess whether TcdB reduced viability of NCM460 cells, TUNEL staining was performed. As shown in Figure Figure 2B), suggesting that apoptosis was not the primary cell death mechanism in TcdB-exposed colonocytes.

| TcdB induces autophagy in colonocytes
To further investigate the cell death mechanism underlying the cytotoxicity of TcdB in NCM460 cells, autophagy was assayed. First, the level of LC3B-II as a biomarker of autophagosome formation was determined in TcdB-exposed cells. As shown in Figure

| Toxin glucosyltransferase activity is required for the pro-autophagic effect of TcdB
TcdB is known to exert its cytotoxic action through its glucosyltransferase activity, 9 which subsequently leads to cell rounding and cell death. To examine the critical role of glucosyltransferase activity in TcdB-induced autophagy, a non-competitive glucosyltransferase inhibitor phloretin 10 was used. As shown in Figure 5D, phloretin inhibited the TcdB-mediated induction of LC3B-II level. ( Figure 6B). Similar results were also found in murine embryonic fibroblasts (MEFs) derived from wild-type and Atg5 knockout mice.
Atg5 encodes an E3 ubiquitin ligase necessary for autophagy due to its role in autophagosome elongation. As compared with the wild-type MEFs, the loss of cell viability induced by TcdB (10 and 100 ng/mL at the 48-hour time-point) was mitigated by Atg5 knockout (Figure 6C), confirming the pro-death nature of TcdBinduced autophagy.
F I G U R E 4 Enhanced formation of acidic vesicular organelles and autophagic flux in TcdB-exposed colonocytes. A, NCM460 cells were transfected with mCherry-GFP-labelled LC3 plasmid for 24 h followed by exposure to TcdB (10 ng/mL) or rapamycin (1.1 lmol L À1 ). Acidified and non-acidified LC3-positive autophagosomes were visualized and counted under a confocal microscope. Quantitative data represent means AE SEM of 3 independent experiments. **P < .01; ***P < .001; ****P < .0001 significantly different between groups. B, Acridine orange staining was performed to visualize TcdB-induced formation of acidic vesicular organelles (orange colour) in NCM460 under fluorescence microscopy. Quantitation of the red-to-green ratio represents means AE SEM of 3 independent experiments. *P < .05 significantly different between groups Another opinion, based on results from a hamster model, is that TcdB is vital for C. difficile virulence while TcdA was avirulent. 12 As the problems of unclear infection mechanism and emerging antibiotic resistance are serious, C. difficile infection has become more difficult to treat. Investigating the pathogenic mechanism would therefore help to devise new therapeutic approaches.
Previous studies have shown that TcdB is a potent cytotoxin capable of inducing enzyme-independent necrosis in both cells and tissue. 13 In this study, we focus on the role of autophagy. As autophagy is a complicated and dynamics process, various methods are required for more accurate analysis in monitoring autophagy.
Acridine orange is one of the methods to examine the formation of acidic compartments during autophagy. However, staining with acridine orange by itself is not a sufficient method for monitoring autop- Although autophagy has been generally reported as an essential mechanism for the maintenance of cell survival, especially in times F I G U R E 6 Attenuation of the cytotoxic effect of TcdB by autophagy inhibition. A, NCM460 cells were exposed to TcdB (10 ng/mL) in the absence or presence of wortmannin (Wort; 200 nmol L À1 ) or chloroquine (CQ; 20 lmol L À1 ) for 48 h. B, NCM460 cells were transfected with control siRNA or mixture of siRNAs targeting Beclin 1, Atg5 and Atg7 before the exposure to TcdB at the indicated concentrations for 48 h. Cell viability was assessed by CCK-8 assay. C, Wild-type and Atg5 knockout mouse embryonic fibroblasts (MEFs) were exposed to TcdB at the indicated concentrations for 48 h. Cell viability was assessed by CCK-8 assay. Results are expressed as means of percentage of control AE SEM of 3 independent experiments. *P < .05; **P < .01; ***P < .001; ****P < .0001 significantly different between groups of nutrient deprivation and other stressful conditions, which could protect host cells against various forms of cellular insults, extensive activation of autophagy could mediate cell death.
mTOR complex 1 signalling pathway serves as a major player in transmitting the autophagic signals. In Saccharomyces cerevisiae, inhibition of TOR is required to control the Atg1-dependent organization of pre-autophagosomal elements and the expansion of the pre-autophagosomal membrane. 14,15 In humans, inhibition of mTORC1 has been reported to induce autophagy in different biological context. For instances, mTOR inhibitors induce autophagy in lung cancer, glioma, mantle cell lymphoma and disseminated gastric cancer cells. [16][17][18][19] In this study, we demonstrated that TcdB suppressed mTORC1 activity as demonstrated by reduced phosphorylation of mTOR and its substrate p70-S6K. We further demonstrated that TcdB suppressed mTOR complex 1 activity through the PTEN-PI3K-AKT-mTOR pathway as indicated by increased phosphorylation of PTEN and the reduced phosphorylation of Akt at Serine 473 and Threonine 308. However, the precise upstream mechanism by which TcdB regulates the PTEN-PI3K-AKT-mTOR pathway is still unclear as TcdB could mediate its signal through targeting multiple small GTPases, such as RhoA, Rac1 and Cdc42. Nevertheless, in this study, the glucosyltransferase activity of TcdB was found to be required for its pro-autophagic effect.
The current findings implicate that the use of autophagy inhibitor might attenuate the virulence of TcdB. Further animal studies would be required to assess the in vivo therapeutic effect of autophagy inhibition during C. difficile infection.