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Clostridium difficile Hijacks Cellular Production of Reactive Oxygen Species to Kill Its Host

By: Matt Windsor, Marnett Research Laboratory
Published: November 13, 2013


A newly discovered mechanism of TcdB-mediated cell death points to potential treatment options.

Clostridium difficile (Figure 1) is the most common cause of infection acquired during antibiotic treatment at a hospital or nursing home.  The number and severity of the diarrhea-inducing infections have been growing in recent years, leading to significant effort to identify new methods of treatment.  Unfortunately, the exact mechanism of action used by the bacteria to produce the diarrhea-related symptoms of fluid secretion, inflammation, and tissue necrosis has not been identified.  Now, in a recent paper published in the Proceedings of the National Academy of Sciences, VICB member Borden Lacy, her collaborator James Goldenring, and their laboratories provide some answers to this important question. They report that C. difficile utilizes a host cell’s NADPH oxidase complex to generate an overwhelming amount of reactive oxygen species and that treatment with antioxidants can protect colonic tissue from necrosis [M.A. Farrow, et al., (2013) Proc. Natl. Acad. Sci. U.S.A., published online October 28, doi: 10.1073/pnas.1313658110].

Figure 1Photomicrograph of C. difficile isolated from the stool sample of a patient.  Image from Lois S. Wiggs, Center for Disease Control, 2004, pubic domain.

TcdA and TcdB are two exotoxins that act as the bacteria’s principal virulence factors.  These homologs act as glucosyltransferases that modify Rho and Ras family GTPases in the cell, leading to inactivation of the GTPases.  Yet, earlier work by the Lacy group had shown that TcdB causes cell death by a mechanism independent of its glucosyltransferase activity.  This initial observation motivated the group to search for a TcdB-mediated process that directly led to necrosis. 

Starting with the known interaction between TcdB and GTPases, siRNA was used to individually knock down GTPases RhoA, Rac1, and Cdc42 in HeLa and Caco-2 epithelial cells.  After introduction of TcdB, only Rac1-silenced cells were protected from cytotoxicity (Figure 2).  These data suggested that a Rac1-dependent pathway mediates TcdB’s virulence, seemingly contradicting the fact that TcdB is known to inactivate Rac1 via glucosylation.

Figure 2.   TcdB toxicity depends on Rac1. HeLa (black) and Caco-2 (white) cells transfected with siRNA were subjected to TcdB. Relative survival is normalized to the Luc control. CLTC knockdown acted as a positive control. Reproduced with permission from M.A. Farrow, et al., (2013) Proc. Natl. Acad. Sci. U.S.A., published online October 28, doi: 10.1073/pnas.1313658110 . Copyright 2013, M.A. Farrow, et al.

To resolve these opposing results, the investigators measured the kinetics of Rac1 activation and inactivation by TcdB. Rac1 reached a maximum activation level five minutes after introduction of TcdB, followed by deactivation (Figure 3). In a separate series of experiments, Rac1 glucosylation began 10 minutes after intoxication with TcdB, with complete glucosylation achieved at 25 minutes (Figure 3). These experiments highlight that Rac1 activation (required for TcdB-mediated necrosis) happens before Rac1 is deactivated by glucosylation.

Figure 3. Bottom left: Rac1 is activated in HeLa cells five minutes post introduction of TcdB, followed by deactivation. Top right: Rac1 is completely glucosylated 25 minutes after introduction of TcdB. Reproduced with permission from M.A. Farrow, et al., (2013) Proc. Natl. Acad. Sci. U.S.A., published online October 28, doi: 10.1073/pnas.1313658110 . Copyright 2013, M.A. Farrow, et al.


Rac1 is known to participate in the assembly of NADPH oxidase (NOX) complexes on the cell membrane. Upon endocytosis, NOX complexes lead to the generation of reactive oxygen species (ROS) in the endosome. As unregulated ROS can lead to cell necrosis, the investigators probed whether NOX-dependent ROS production plays a role in the TcdB-mediated necrosis pathway.

Two series of experiments were used to test the hypothesis that NOX-generated ROS were the chemical agents induced by TcdB to cause cell necrosis. First, TcdB was found to induce ROS production in cells using an intracellular, fluorescent ROS reporter. Second, siRNA was employed to knock down components of the NOX complex, resulting in cellular protection from toxin exposure. Both of these results implicate ROS in TcdB-mediated cell death and suggest that NOX complexes generate the ROS.

With a better understanding of the mechanism of action utilized by TcdB to induce cell death, the researchers evaluated several small molecules as potential therapeutic agents. Diphenyleneiodonium (DPI) and NSC 23766 prevent ROS generation by inhibiting the flavocytochrome enzymatic core of the NOX complex and a Rac1 guanine nucleotide exchange factor, respectively. N-acetylcysteine (NAC) and Tempol minimize oxidative stress by respectively acting as an antioxidant and superoxide scavenger. HeLa cells were treated with one of the compounds and then exposed to TcdB. Each of the small molecules, whether by inhibiting ROS generation or scavenging existing ROS, protected the HeLa cells from toxin-induced necrosis (Figure 4).

Figure 4.   HeLa cells pretreated with ROS inhibitors show increased viability after being challenged with TcdB. Reproduced with permission from M.A. Farrow, et al., (2013) Proc. Natl. Acad. Sci. U.S.A., published online October 28, doi: 10.1073/pnas.1313658110. Copyright 2013, M.A. Farrow, et al.


Finally, the authors evaluated the effect of NAC on human colonic explants exposed to TcdB. Tissue preincubated with NAC showed much less damage to the surface epithelial layer (after introduction of toxin) than tissue without NAC present (Figure 5). These results are particularly exciting, because NAC is an antioxidant already approved for clinical use by the Food and Drug Administration.

Figure 5.   Human colonic explant tissue exposed to TcdB. Top: Mock treatment shows significant damage. Bottom: Pretreatment of tissue with NAC is largely protected from TcdB-induced damage. Reproduced with permission from M.A. Farrow, et al., (2013) Proc. Natl. Acad. Sci. U.S.A., published online October 28, doi: 10.1073/pnas.1313658110. Copyright 2013, M.A. Farrow, et al.

By following up on their earlier work, Lacy, Goldenring, and coworkers have identified a key pathway in the mechanism of action for TcdB-induced necrosis. In addition, by determining that ROS are the active chemical agents that lead to cell death, the authors have provided a new method to treat the damage caused by C. difficile infection. Future studies identifying the TcdB receptors on the cell membrane will not only shed light on additional unknown steps in TcdB-mediate necrosis, but may offer specific targets for drug development.

 

 

 

 

 


 

 
     

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