Intestinal epithelial tight junctions as targets for enteric bacteria-derived toxins

doi:10.1016/j.addr.2003.10.045

Advanced Drug Delivery Reviews

Volume 56, Issue 6, 19 April 2004, Pages 795-807

https://doi.org/10.1016/j.addr.2003.10.045 Get rights and content

Abstract

The application of a multidisciplinary approach to study bacterial pathogenesis, along with the recent sequencing of entire microbial genomes have made possible discoveries that are changing the way scientists view the bacterium–host interaction. Today, research on the molecular basis of the pathogenesis of infectious diarrheal diseases of necessity transcends established boundaries between microbiology, cell biology, intestinal pathophysiology, and immunology. Novel multidisciplinary approaches led to the discovery of new bacteria–host cell interactions involving signals regulating intestinal permeability through the modulation of cell cytoskeleton and intercellular tight junctions (TJ). A century ago, TJ were conceptualized as a secreted extracellular cement forming an absolute and unregulated barrier within the paracellular space. Biological studies of the past several decades have shown that TJ are dynamic structures subjected to structural changes that dictate their functional status under a variety of developmental, physiological, and pathological circumstances. To meet the many diverse physiological challenges to which the intestinal epithelial barrier is subjected, TJ must be capable of rapid and coordinated responses. This requires the presence of a complex regulatory system that orchestrates the state of assembly of the TJ multiprotein network. Many pathogenic bacteria exploit this system to accomplish their pathogenic strategies by ultimately modulating intestinal permeability.

Introduction

Microorganisms represent the first species of living organisms that populated our planet and will probably continue to survive well beyond the extinction of the human race. Their distinguishing characteristics (small size, concise deployment of genetic information, and ability to survive in highly varied circumstances) contribute to their manifest virtuosity in adapting to a changing environment. To be a successful enteric, non-invasive pathogen, a microorganism has to be a good colonizer, compete effectively for nutrients, and to be able to interact with the target eukaryotic cell in order to induce secretion of water and electrolytes. Since the basic metabolism of enteric pathogens and commensals is largely the same, it follows that pathogens must possess highly specialized attributes, which enable them to activate one or more eukaryotic intracellular pathways leading to intestinal secretion. This cross talk between enteric pathogens and the host intestine may be affected by either invasion or elaboration of toxins. This article is focused on the growing number of discovered enterotoxins that have been described to exert their pathogenic effect by targeting the cell cytoskeleton/tight junctions (TJ) complex.

Section snippets

Tight junctions, a key barrier

A key function of the intercellular junction complex between neighboring intestinal epithelial cells (enterocytes) is the formation of selective barriers that permit the generation and maintenance of tissue compartments with distinct compositions. Individual enterocytes are joined to each other by a specialized complex consisting of TJ (zonula occludens, ZO), adherens junctions, gap junctions, and desmosomes [1]. TJ represent the major barrier within the paracellular pathway [2]. Evidence now

Structure of tight junctions

The actual structure of the TJ has been studied extensively (Fig. 1). Freeze fracture electron microscopy reveals that these contacts, which encircle the apical side of the lateral surface of each cell, are continuous strand-like transmembrane structures which interact with similar structures of adjacent cells. The interactions define the paracellular permeability characteristics. A number of proteins are associated with TJ.

Toxins affecting the enterocyte TJ/cytoskeleton complex

Given the key function of intestinal TJ in regulating trafficking of water and molecules between environment and host, it is not surprising that some bacterial toxins have evolved to exploit this function as part of their pathogenic arsenal. What is remarkable, however, is the breadth and complexity of strategies developed by enteric bacteria to affect intestinal permeability. The following section outlines the better-characterized examples of enteric toxins affecting TJ competency.

Concluding remarks and future directions

The paracellular pathway was once considered to be exclusively the route for passive, unregulated passage of water, electrolytes, and small molecules. Its contribution to transepithelial transports was, therefore, judged to be simply secondary to the active, transcellular transport processes. It is now becoming apparent that the elements that govern this pathway; i.e. the TJ, are extremely dynamic structures involved in developmental, physiological, and pathological circumstances. An increased

References (89)

C. Rahner et al.
Heterogeneity in expression and subcellular localization of claudins 2, 3, 4, and 5 in the rat liver, pancreas, and gut
Gastroenterology
(2001)
J. Katahira et al.
Clostridium perfringens enterotoxin utilizes two structurally related membrane proteins as functional receptors in vivo
J. Biol. Chem.
(1997)
T. Tsukamoto et al.
Tight junction proteins form large complexes and associate with the cytoskeleton in an ATP depletion model for reversible junction assembly
J. Biol. Chem.
(1997)
G. Schmidt et al.
Rho GTPase-activating toxins: cytotoxic necrotizing factors and dermonecrotic toxin
Methods Enzymol.
(2000)
I. Just et al.
Bacterial protein toxins inhibiting low-molecular-mass GTP-binding proteins
Int. J. Med. Microbiol.
(2001)
D. He et al.
Clostridium difficile toxin A causes early damage to mitochondria in cultured cells
Gastroenterology
(2000)
C. Wilde et al.
The Rho-ADP-ribosylating C3 exoenzyme from Clostridium botulinum and related C3-like transferases
Toxicon
(2001)
P. Boquet
The cytotoxic necrotizing factor 1 (CNF-1) from Escherichia coli
Toxicon
(2001)
C.L. Sears
The toxins of Bacteroides fragilis
Toxicon
(2001)
C.L. Sears
The toxins of Bacteroides fragilis
Toxicon
(2001)

I.R. Henderson et al.

The great escape: structure and function of the autotransporter proteins

Trends Microbiol.

(1998)

M.A. De Matteis et al.

The role of ankyrin and fodrin in membrane transport and domain formation

Curr. Opin. Cell Biol.

(1998)

Z. Wu et al.

Vibrio cholerae hemagglutinin/protease (HA/protease) causes morphological changes in cultured epithelial cells and perturbs their paracellular barrier function

Microb. Pathog.

(1996)

S. Uzzau et al.

Expression of Vibrio cholerae zonula occludens toxin and analysis of its subcellular localization

Microb. Pathog.

(1999)

M. Di Pierro et al.

Zonula occludens toxin structure–function analysis: identification of the fragment biologically active on tight junctions and of the zonulin receptor binding domain

J. Biol. Chem.

(2001)

A. Fasano et al.

The enterotoxic effect of zonula occludens toxin (Zot) on rabbit small intestine involves the paracellular pathway

Gastroenterology

(1997)

S. Uzzau et al.

Purification and preliminary characterization of the zonula occludens toxin receptor from human (CaCo2) and murine (IEC6) intestinal cell lines

FEMS Microbiol. Lett.

(2001)

B.A. McClane

Clostridium perfringens enterotoxin and intestinal tight junctions

Trends Microbiol.

(2000)

B.A. McClane

The complex interactions between Clostridium perfringens enterotoxin and epithelial tight junctions

Toxicon

(2001)

A.S. Fanning et al.

Transmembrane proteins in the tight junction barrier

J. Am. Soc. Nephrol.

(1999)

J.L. Madara et al.

Occluding junction structure–function relationships in a cultured epithelial monolayer

J. Cell Biol.

(1985)

M. Furuse et al.

Occludin: a novel integral membrane protein localizing at tight junctions

J. Cell Biol.

(1993)

M. Saitou et al.

Mammalian occludin in epithelial cells: its expression and subcellular distribution

Eur. J. Cell Biol.

(1997)

M. Furuse et al.

Claudin-1 and -2: novel integral membrane proteins localizing at tight junctions with no sequence similarity to occludin

J. Cell Biol.

(1998)

M. Furuse et al.

Manner of interaction of heterogeneous claudin species within and between tight junction strands

J. Cell Biol.

(1999)

J. Katahira et al.

Molecular cloning and functional characterization of the receptor for Clostridium perfringens enterotoxin

J. Cell Biol.

(1997)

I. Martin-Padura et al.

Junctional adhesion molecule, a novel member of the immunoglobulin superfamily that distributes at intercellular junctions and modulates monocyte transmigration

J. Cell Biol.

(1998)

B.R. Stevenson et al.

Identification of ZO-1: a high molecular weight polypeptide associated with the tight junction (zonula occludens) in a variety of epithelia

J. Cell Biol.

(1986)

B. Gumbiner et al.

Identification of a 160-kDa polypeptide that binds to the tight junction protein ZO-1

Proc. Natl. Acad. Sci. USA

(1991)

J. Haskins et al.

ZO-3, a novel member of the MAGUK protein family found at the tight junction, interacts with ZO-1 and occludin

J. Cell Biol.

(1998)

M.S. Balda et al.

Tight junctions

J. Cell Sci.

(1998)

B.H. Keon et al.

Symplekin, a novel type of tight junction plaque protein

J. Cell Biol.

(1996)

C.J. Gottardi et al.

The junction-associated protein, zonula occludens-1, localizes to the nucleus before the maturation and during the remodeling of cell–cell contacts

Proc. Natl. Acad. Sci. USA

(1996)

B. Gumbiner

Structure, biochemistry, and assembly of epithelial tight junctions

Am. J. Physiol.

(1987)

J.L. Madara et al.

Effects of cytochalasin D on occluding junctions of intestinal absorptive cells: further evidence that the cytoskeleton may influence paracellular permeability and junctional charge selectivity

J. Cell Biol.

(1986)

D. Drenchahn et al.

Organization of the actin filament cytoskeleton in the intestinal brush border: a quantitative and qualitative immunoelectron microscope study

J. Cell Biol.

(1988)

F. Hecht

Expression of the catalytic domain of myosin light chain kinase increases paracellular permeability

Am. J. Physiol.

(1996)

K. Tsuneo et al.

Extracellular ATP, intracellular calcium and canalicular contraction in rat hepatocye doublets

Hepatology

(1991)

A. Fasano et al.

Zonula occludens toxin modulates tight junctions through protein kinase C-dependent actin reorganization, in vitro

J. Clin. Invest.

(1995)

L.L. Mitic et al.

Occludin and claudins: transmembrane proteins of the tight junction

M. Itoh et al.

Direct binding if three tight junction-associated MAGUKs, ZO-1, ZO-2, and ZO-3, with the COOH termini of claudins

J. Cell Biol.

(1999)

G. Schmidt et al.

Bacterial cytotoxins target Rho GTPases

Naturwissenschaften

(1998)

H.J. Schnittler et al.

Role of actin filaments in endothelial cell–cell adhesion and membrane stability under fluid shear stress

Pflug. Arch.

(2001)

I. Just et al.

Molecular mode of action of the large clostridial cytotoxins

Curr. Top. Microbiol. Immunol.

(2000)

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Intestinal epithelial tight junctions as targets for enteric bacteria-derived toxins

Abstract

Introduction

Section snippets

Tight junctions, a key barrier

Structure of tight junctions

Toxins affecting the enterocyte TJ/cytoskeleton complex

Concluding remarks and future directions

Gastroenterology

J. Biol. Chem.

J. Biol. Chem.

Methods Enzymol.

Int. J. Med. Microbiol.

Gastroenterology

Toxicon

Toxicon

Toxicon

Toxicon

Trends Microbiol.

Curr. Opin. Cell Biol.

Microb. Pathog.

Microb. Pathog.

J. Biol. Chem.

Gastroenterology

FEMS Microbiol. Lett.

Trends Microbiol.

Toxicon

Transmembrane proteins in the tight junction barrier

J. Am. Soc. Nephrol.

Occluding junction structure–function relationships in a cultured epithelial monolayer

J. Cell Biol.

Occludin: a novel integral membrane protein localizing at tight junctions

J. Cell Biol.

Mammalian occludin in epithelial cells: its expression and subcellular distribution

Eur. J. Cell Biol.

Claudin-1 and -2: novel integral membrane proteins localizing at tight junctions with no sequence similarity to occludin

J. Cell Biol.

Manner of interaction of heterogeneous claudin species within and between tight junction strands

J. Cell Biol.

Molecular cloning and functional characterization of the receptor for Clostridium perfringens enterotoxin

J. Cell Biol.

Junctional adhesion molecule, a novel member of the immunoglobulin superfamily that distributes at intercellular junctions and modulates monocyte transmigration

J. Cell Biol.

Identification of ZO-1: a high molecular weight polypeptide associated with the tight junction (zonula occludens) in a variety of epithelia

J. Cell Biol.

Identification of a 160-kDa polypeptide that binds to the tight junction protein ZO-1

Proc. Natl. Acad. Sci. USA

ZO-3, a novel member of the MAGUK protein family found at the tight junction, interacts with ZO-1 and occludin

J. Cell Biol.

Tight junctions

J. Cell Sci.

Symplekin, a novel type of tight junction plaque protein

J. Cell Biol.

The junction-associated protein, zonula occludens-1, localizes to the nucleus before the maturation and during the remodeling of cell–cell contacts

Proc. Natl. Acad. Sci. USA

Structure, biochemistry, and assembly of epithelial tight junctions

Am. J. Physiol.

Effects of cytochalasin D on occluding junctions of intestinal absorptive cells: further evidence that the cytoskeleton may influence paracellular permeability and junctional charge selectivity

J. Cell Biol.

Organization of the actin filament cytoskeleton in the intestinal brush border: a quantitative and qualitative immunoelectron microscope study

J. Cell Biol.

Expression of the catalytic domain of myosin light chain kinase increases paracellular permeability

Am. J. Physiol.

Extracellular ATP, intracellular calcium and canalicular contraction in rat hepatocye doublets

Hepatology

Zonula occludens toxin modulates tight junctions through protein kinase C-dependent actin reorganization, in vitro

J. Clin. Invest.

Occludin and claudins: transmembrane proteins of the tight junction

Direct binding if three tight junction-associated MAGUKs, ZO-1, ZO-2, and ZO-3, with the COOH termini of claudins

J. Cell Biol.

Bacterial cytotoxins target Rho GTPases

Naturwissenschaften

Role of actin filaments in endothelial cell–cell adhesion and membrane stability under fluid shear stress

Pflug. Arch.

Molecular mode of action of the large clostridial cytotoxins

Curr. Top. Microbiol. Immunol.