Lathyrus sativus diamine oxidase reduces Clostridium difficile toxin A‐induced toxicity in Caco‐2 cells by rescuing RhoA‐GTPase and inhibiting pp38‐MAPK/NF‐κB/HIF‐1α activation

Abstract

Clostridium difficile toxin A (TcdA) impairs the intestinal epithelial barrier, increasing the mucosa permeability and triggering a robust inflammatory response. Lathyrus sativus diamino oxidase (LSAO) is a nutraceutical compound successfully used in various gastrointestinal dysfunctions. Here, we evaluated the LSAO (0.004–0.4 μM) ability to counter TcdA‐induced (30 ng/mL) toxicity and damage in Caco‐2 cells, investigating its possible mechanism of action. LSAO has improved the transepithelial electrical resistance (TEER) score and increased cell viability in TcdA‐treated cells, significantly rescuing the protein expression of Ras homolog family members, A‐GTPase (RhoA‐GTPase), occludin, and zonula occludens‐1 (ZO‐1). LSAO has also exhibited an anti‐apoptotic effect by inhibiting the TcdA‐induced expression of Bcl‐2‐associated X protein (Bax), p50 nuclear factor‐kappa‐B (p50), p65nuclear factor‐kappa‐B (p65), and hypoxia‐inducible transcription factor‐1 alpha (HIF‐1α), and the release of tumor necrosis factor‐alpha (TNF‐α), interleukin‐6 (IL‐6), and vascular endothelial growth factor (VEGF) in the cell milieu. Our data showed that LSAO exerts a protective effect on TcdA‐induced toxicity in Caco‐2 cells, placing itself as an interesting nutraceutical to supplement the current treatment of the Clostridium difficile infections.

Protective Effects of Alginate and Chitosan Oligosaccharides against Clostridioides difficile Bacteria and Toxin

“…This is per previous studies demonstrating the impotence of TcdA in this concentration range on causing IL-8 production by Caco-2 monolayers [55,56]. By contrast, in other studies using HT-29 and T84 monolayers, TcdA mediated the release of IL-8 [56,57], while in a study on Caco-2 cells, TcdA managed to induce TNF-α and IL-6 release [11]. Even though TcdA is a well-recognized inflammatory enterotoxin, little is known regarding the underlying mechanisms of innate immune system activation and stimulation of pro-inflammatory cytokine release [58].…”

“…Strikingly, despite the evident TcdA-mediated increased paracellular permeability, no alterations at the protein expression of the crucial TJ proteins, ZO-1 and occludin, were observed according to the Western blot assay. In previous studies using Caco-2 cells, TcdA challenge altered ZO-1 and occludin protein expression and localization; though in all of these studies, the TcdA concentration applied was cytotoxic [11,50,63]. TcdA has a time-and concentration-dependent toxicity, thus a plausible explanation for the absence of TJ protein level changes is that the concentration of 1 ng/mL was not capable of suppressing TJ protein expression within this time frame.…”

“…Transport studies using nonelectrolyte tracers indicate the TJ pore size and the paracellular water flow [60]. Numerous studies have demonstrated the TcdA-induced cytopathic effects, principally involving disruption of intercellular cell-cell junctions, thus increasing epithelial permeability in vitro [7,11,15,50]. Exposure of the Caco-2 monolayers to TcdA 100 pg/mL, 1 ng/mL (non-toxic concentrations 24 h post-exposure), and 10 ng/mL (toxic concentration 24 h post-exposure) impaired the monolayer's integrity in a time-and concentration-dependent manner.…”

“…TcdA possesses glucosyltransferase activity, with a primary target of A-GTPase, a Ras homolog family member (RhoA-GTPases), which is a crucial regulator of actin cytoskeleton dynamics and tight junction assembly. TcdA inactivates RhoA-GTPase by inducing its transition from the guanosine triphosphate (GTP)-bound form (active) to the guanosine diphosphate (GDP)-bound form (inactive), ultimately leading to cytoskeletal breakdown and tight junction (TJ) disassembly [11]. Each TJ is formed by the assembly of various proteins (mainly ZO-1, -2, -3, occludin, claudins, and JAMs) and is located near the apical side of the lateral membrane of the epithelium.…”

“…At present, available therapeutic strategies for CDI include the use of specific antibiotics against C. difficile, fecal transplantation, and surgery. However, severe and recurrent CDI treatment is still exigent, with limited treatment options available [11]. Both C. difficile and TcdA constitute appealing pharmacological targets; thus, identifying agents with antibacterial capacity against C. difficile or with barrier-protective properties against the TcdA-induced toxicity, or even better with both abilities, are very promising strategies.…”