Clostridium difficile toxin A perturbs cytoskeletal structure and tight junction permeability of cultured human intestinal epithelial monolayers.

Abstract

Toxin A of Clostridium difficile causes severe inflammatory enterocolitis in man and animals that appears to be mediated in part by acute inflammatory cells that migrate into the toxin A-exposed mucosa. To determine the direct effects of toxin A on intestinal epithelial permeability and structure in the absence of other modulating factors, we used cultured monolayers of a human intestinal epithelial cell line (T84). A toxin A concentration of 7 X 10-1 ,ug/ml (3 X i0-' M) nearly abolished monolayer transepithelial resistance within 6-8 h. This marked permeability defect occurred while the monolayers were still confluent. Dual sodium-mannitol flux studies localized the permeability defect to the intercellular tight junction. Cytotoxicity assays and morphological evaluation using Nomarski optics and electron microscopy failed to demonstrate any evidence of cell damage at the time the maximum resistance response was observed. Fluorescent staining for F actin, however, revealed a marked decrease in fluorescent intensity in toxin-treated monolayers versus controls. These data show that toxin A can directly affect the barrier function of this model intestinal epithelium and initially does so by selectively enhancing tight junction permeability. Furthermore, cytoskeletal structure is markedly altered over the same time course, although the integrity of individual cells is maintained. Because the cytoskeleton of intestinal epithelial cells is known to be capable of regulating tight junction permeability, we speculate that the above effects of toxin A on epithelial barrier function result from alterations of the cytoskeleton.

Protein Kinase C Signaling Regulates ZO-1 Translocation and Increased Paracellular Flux of T84 Colonocytes Exposed toClostridium difficile Toxin A

“…However, we previously observed a rapid drop in TER in the absence of any changes in cell morphology or TJ ultrastructure in T84 cells exposed to C. difficile toxin A (24). Further-more, TJ permeability changes in response to toxin A preceded the disassembly of perijunctional actin filaments, suggesting that the early increase in TJ permeability may be mediated by factor(s) other than actin disassembly (24). In this study, we focused on the early effects of toxin A on TJ permeability and ZO-1 redistribution.…”

“…The increase in TJ permeability and redistribution of TJ proteins in T84 cells exposed to C. difficile toxins has been correlated with depolymerization of the actin cytoskeleton (23,24). However, we previously observed a rapid drop in TER in the absence of any changes in cell morphology or TJ ultrastructure in T84 cells exposed to C. difficile toxin A (24).…”

“…Freeze fracture electron micrographs reveal abnormal TJ morphology in transfectants overexpressing mutant forms of the Rho family of GTPases, suggesting involvement of Rho proteins in TJ assembly (21). In polarized intestinal epithelial cells (T84 cells) infected with a chimeric protein containing Clostridium botulinum C3 exoenzyme that specifically inactivates RhoA, perijunctional actin rings are disrupted, leading to a decrease in TER and redistribution of .The increase in TJ permeability and redistribution of TJ proteins in T84 cells exposed to C. difficile toxins has been correlated with depolymerization of the actin cytoskeleton (23,24). However, we previously observed a rapid drop in TER in the absence of any changes in cell morphology or TJ ultrastructure in T84 cells exposed to C. difficile toxin A (24).…”

Neutrophil migration across cultured intestinal epithelial monolayers is modulated by epithelial exposure to IFN-gamma in a highly polarized fashion.

“…Monolayers exposed to even 1,000 U/ml IFN-'y for 72 h maintain resistances generally exceeding 250-300 ohm.cm 2 (Madara and Stafford, 1989), and we have previously shown that modulation of epithelial resistance in the range of 200-2,000 ohm.cm 2 has little effect on the efficiency of PMN transmigration (Nash et al, 1987). This is likely due to the fact that the relationship between resistance and the flux of solutes, such as those in the size-range of FMLP is asymptotic (Madara and Dharmsathaphorn, 1985;Hecht et al, 1988); that is, at higher resistance values (>200-250 ohm.cm:), large changes in resistance are accompanied by only modest changes in solute flux. The reasons for this are clear when one considers the behavior of a circuit composed of parallel resistances as we have discussed in the past (Madara and Dharmsathaphorn, 1985).…”

Modulatory Effects of Gut Microbiota on the Central Nervous System: How Gut Could Play a Role in Neuropsychiatric Health and Diseases

“…28 For example, Clostridium difficile that can colonize the gut in the absence of normal gut flora produces an enterotoxin that increase the gut permeability by impairing epithelial tight junctions through damaging aggregation of actin filaments. 29 Another way that gut microbiota can enhance the function of the intestinal barrier is through protecting and improving epithelial tight junctions. Most of the evidence that supports this role of microbiota in the normal function of the intestinal barrier comes from studies that have shown that probiotic treatment can reduced gut permeability in models of GI tract disorders.…”