Glutamine and Alanyl-Glutamine Increase RhoA Expression and ReduceClostridium difficileToxin-A-Induced Intestinal Epithelial Cell Damage

Abstract

Clostridium difficile is a major cause of antibiotic-associated colitis and is associated with significant morbidity and mortality. Glutamine (Gln) is a major fuel for the intestinal cell population. Alanyl-glutamine (Ala-Gln) is a dipeptide that is highly soluble and well tolerated. IEC-6 cells were used in the in vitro experiments. Cell morphology was evaluated by atomic force microscopy (AFM) and scanning electron microscopy (SEM). Cell proliferation was assessed by WST-1 and Ki-67 and apoptosis was assessed by TUNEL. Cytoskeleton was evaluated by immunofluorescence for RhoA and F-actin. RhoA was quantified by immunoblotting. TcdA induced cell shrinkage as observed by AFM, SEM, and fluorescent microscopy. Additionally, collapse of the F-actin cytoskeleton was demonstrated by immunofluorescence. TcdA decreased cell volume and area and increased cell height by 79%, 66.2%, and 58.9%, respectively. Following TcdA treatment, Ala-Gln and Gln supplementation, significantly increased RhoA by 65.5% and 89.7%, respectively at 24 h. Ala-Gln supplementation increased cell proliferation by 137.5% at 24 h and decreased cell apoptosis by 61.4% at 24 h following TcdA treatment. In conclusion, TcdA altered intestinal cell morphology and cytoskeleton organization, decreased cell proliferation, and increased cell apoptosis. Ala-Gln and Gln supplementation reduced intestinal epithelial cell damage and increased RhoA expression.

Small GTPases of the Ras superfamily regulate intestinal epithelial homeostasis and barrier function via common and unique mechanisms

“…By contrast, actin disruption alone, another well-known consequence of TcdA, by latrunculin A was not sufficient to mimick the observed apoptotic effects 72 . TcdA-induced apoptosis could be reversed by co-incubation with glutamine or alanyl-glutamine in IEC-6 cells that was accompanied by increased RhoA expression 73 . Additionally, other pathogens such as Escherichia coli, Mycobacterium avium or Salmonella typhimurium have been shown to exploit mechanisms involving RhoA activation to facilitate infection 74 - 77 .…”

The Role of Rho GTPases in Toxicity of Clostridium difficile Toxins

“…In respect of the role of Rho proteins in the toxicity of C. difficile toxins, transfection of Rho GTPases decreases the sensitivity of host cells to TcdB [ 73 , 126 ], while supplementation of glutamine and alanyl-glutamine to TcdA treated cells can increase RhoA expression and then reduce the intestinal epithelial cell damage [ 127 ]. Although ADP-ribosylation or phosphorylation of Rho proteins has not been observed in TcdB intoxicated intact oocytes [ 128 ], the protective role of phosphorylation of Rho proteins in TcdA and TcdB intoxicated cells has been reported [ 129 , 130 ].…”

Novel therapies and preventative strategies for primary and recurrent Clostridium difficile infections

“…Santos et al . additionally showed in IEC‐6 cells that alanyl‐glutamine treatment reduced TcdA toxin damage and increased RhoA expression, suggesting a potential explanation for the protective effects . Using intestinal loop models, Warren et al .…”

“…106 Santos et al additionally showed in IEC-6 cells that alanyl-glutamine treatment reduced TcdA toxin damage and increased RhoA expression, suggesting a potential explanation for the protective effects. 107 Using intestinal loop models, Warren et al showed that treatment with ATL 370 (an adenosine A2A receptor agonist) and alanyl-glutamine reduced ileal secretions, apoptosis, mucosal injury, and levels of KC and IL-10 after C. difficile toxin A exposure. 108 A phase II clinical trial (NCT02053350) testing the efficacy of alanyl-glutamine as a supplement (44 g orally daily for 10 days) during treatment of CDI was terminated due to low enrollment.…”