Department of Pediatrics and Physiology, and Gastrointestinal Pathophysiology Section, Center for Vaccine Development, University of Maryland, School of Medicine, Baltimore, Maryland 21201
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ABSTRACT |
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Cellular microbiology is a new discipline that is emerging at the interface between cell biology and microbiology. The application of molecular techniques to the study of bacterial pathogenesis has made possible discoveries that are changing the way scientists view the bacterium-host interaction. Today, research on the molecular basis of the pathogenesis of infective diarrheal diseases of necessity transcends established boundaries between cell biology, bacteriology, intestinal pathophysiology, and immunology. The use of microbial pathogens to address questions in cell physiology is just now yielding promising applications and striking results.
bacterial pathogenesis; enterotoxins; intracellular signaling; diarrhea; intestine
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INTRODUCTION |
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INFECTIOUS DISEASES HAVE BEEN traditionally perceived as a human plague that needs to be aggressively fought in order to get rid of the harmful microorganisms. However, to bacteria, illness is often inadvertent, the results of exquisite tricks learned long ago and played on human cells to achieve the microorganisms' paramount goal: the conservation of their species. Now a new research field named cellular microbiology has emerged that is more interested in the silent, biochemical conversation between the microbe and its host. By decoding this biochemical exchange, we hope now to find new ways to chemically communicate with our own cells and to learn more about cell physiology.
The idea to learn from whom we have always considered mindless, inferior creatures should not come as a surprise. Microorganisms are among the most ancient forms of biological "intelligence" on Earth. They 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 acclaimed virtuosity to adapt and learn fast in order to survive.
Eukaryotic cells communicate with the external milieu and react to a series of physicochemical stimuli via intracellular signaling pathways. Hormones and neurotransmitters are examples of substances that can regulate cellular metabolism by binding to specific cell surface receptors, thereby triggering a signal that is relayed by guanine nucleotide binding (or G) proteins to intracellular targets or effector enzymes (4, 8, 57, 66, 91).
To be a successful enteric pathogen, a microorganism has to be a good colonizer, compete for nutrients, and be able to communicate with the target eukaryotic cell in order to induce secretion of water and electrolytes. The key question is to understand the signaling involved in this cross talk between enteric pathogens and intestinal host, leading to intestinal secretion.
Intestinal cells operate through three main intracellular signal
transduction pathways to regulate ion transport vectorially: cAMP-,
cGMP-, and Ca-dependent pathways. A fourth pathway involving cytoskeleton rearrangement has been recently described. Because the
basic metabolism of enteric pathogens and commensals is the same, it
follows that pathogens must possess highly specialized attributes that
enable them to use at least one of these four signal pathways to cause
diarrhea (Fig. 1).
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Because of the magnitude and dynamism of this new field of research, it is impossible to cover all the areas of current research in a single review. Therefore, this review is mainly focused on a few representative examples of what is presently known about the interaction between enteric pathogens and the host cell, with most of the emphasis placed on the author's personal research experience.
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THE CAMP PATHWAY |
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This is the first-described and better-studied pathway of eukaryotic
cells leading to intestinal secretion (34, 54, 81). Many physiological
modulators, including neurotrasmitters, hormones, and paracrine
substances (10, 11, 14, 33, 71, 92) are implicated in the regulation of
this signaling (see Table 1). Several
enteropathogens activate the adenylate cyclase/cAMP signaling through a
family of heat-labile enterotoxins, so-called A-B toxins (Table
2). Cholera toxin (CT) elaborated by
Vibrio cholerae represents the
archetype of this family of toxins and is certainly the most
extensively investigated. CT is a protein with a relative molecular
mass (Mr) of 84 kDa and is made up of five B subunits with
Mr of 10.5 kDa
each and an A subunit with an
Mr of 27.2 kDa.
The A subunit is proteolitically cleaved to yield two polypeptide
chains, a 195-residue A1 peptide
of 21.8 kDa and a 45-residue A2
peptide of 5.4 kDa (45). As with other toxins in this group, the
functions of the two subunits are specific: the B subunit serves to
bind the holotoxin to the eukaryotic cell receptor, and the A subunit
possesses a specific enzymatic function that acts intracellularly. The
single A subunit is presumably located on the axis of the pentameric B
subunit ring, with the fragment A2
extending some distance into the central hole (Fig. 2) (59, 75, 89, 90). The CT receptor on the
surface of the enterocyte is a GM1
ganglioside that is ubiquitous in the body, being present on such
diverse cell types as ovarian and neural cells as well as intestinal
cells (89). The neuraminidase produced by V. cholerae can increase the number of receptors by acting
on higher-order gangliosides to convert them to
GM1 (40). Binding of the toxin
appears to require that at least two of the five B subunits interact
with GM1, but it is not clear that
all five subunits need to bind (17, 90). Reduction of the disulfide bond between A1 and
A2 on the external surface of the
membrane is necessary for penetration of the
A1 into the cell (93). The fate of
the A2 peptide is not known, but
there is little evidence that it actually enters the cell. Once within
the cell, the A1 subunit activates
adenylate cyclase at the basolateral membrane, where the enzyme is
localized in intestinal epithelial cells (Fig. 2A). The
A1 subunit is thought to migrate
to the basolateral membrane through the cytosol, although there is no
convincing evidence that this actually occurs. An alternative model
proposes that generation of the A1
peptide and activation of adenylate cyclase are functionally linked to
toxin endocytosis (Fig. 2B).
A1 acts as an enzyme to
ADP-ribosylate the -subunit of
Gs at an arginine residue. Once
activated, the
-subunit of Gs
dissociates from the membrane-bound subunit of
Gs, leaving it free to transverse the cell and to attach to the catalytic subunit of adenylate cyclase in
the basolateral membrane (Fig. 2) (35). The adenylate cyclase so
activated induces the formation of cAMP, which then activates the
catalytic unit of cAMP-dependent protein kinase (A kinase). Finally,
the phosphorylation of membrane proteins is responsible for the
transepithelial ion transport changes induced by CT. These changes
consist of the inhibition of the linked Na and Cl absorptive process in
the villous cells and the stimulation of electrogenic Cl secretion in
the crypt cells (Fig. 2C) (22, 36).
The cystic fibrosis transmembrane conductance regulator (CFTR) seems to
be one of the target proteins, if not the only one, phosphorylated by
protein kinase A. CFTR is a Cl channel (2) and has multiple potential
substrate sequences for kinase A. Unlike healthy intestinal tissue,
tissues obtained from patients with cystic fibrosis do not respond to
either cAMP- or Ca-mediated secretagogues (3). It has been hypothesized
that heterozygotes have only one-half of the normal number of Cl
channels responsive to kinase. After infection with
V. cholerae, the cystic
fibrosis heterozygote may have less intestinal Cl secretion and
therefore diarrhea, suggesting a selective advantage over
"normal" homozygotes in surviving cholera.
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Enterotoxigenic Escherichia coli (ETEC) elaborate a heat-labile toxin (LT; Table 2) that closely resembles CT in structure and biochemical mode of action. Unlike CT, LT can also bind to GM2 and asialo-GM1 in addition to GM1 ganglioside (39). Toxin binding is followed by activation of the adenylate cyclase/cAMP system, resulting in water and electrolyte secretion into the lumen of the intestine, with a mechanism similar to that of CT (67). Unlike CT, however, LT is not secreted in the supernatant during bacterial growth but remains cell associated, being localized in the periplasm of the microorganism (49). Hunt and Hardy (51) have recently proved that the intestinal milieu, with bile salts, proteolytic enzymes, and very low iron concentration, can provide the ideal environment to cause the release of free LT in the intestine, thus allowing the toxin to reach its receptors.
In addition to its invasiveness (see discussion in THE CYTOSKELETON PATHWAY), Salmonella typhimurium elaborates an enterotoxin, whose role in inducing diarrhea remains controversial (Table 2) (65). A cell-free lysate of Salmonella can cause intestinal secretion and activate intestinal epithelial cell adenylate cyclase independent of any change in inflammation (65). How the Salmonella toxin activates adenylate cyclase has not been determined.
Campylobacter jejuni also produces an A-B toxin (Table 2), whose B subunit immunologically cross-reacts with CT and ETEC LT B subunits (56, 69, 76).
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THE CGMP PATHWAY |
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Besides LT, ETEC elaborate a family of heat-stable enterotoxins (STs).
STa is a small peptide that
stimulates guanylate cyclase (GC), causing the increased intracellular
concentration of cGMP (Table 2), which, like cAMP, evokes Cl secretion
and diarrhea. A variety of other enteric pathogens elaborate
STa or
STa-like toxins and, therefore,
activate the cGMP pathway (Table 2). Enterocytes isolated from rat
jejunal or ileal villi specifically bind
STa and do so with identical
kinetics, that is, an affinity constant of ~4 × 108 l/mol (43). This binding
constant is two log orders less than that of CT for its receptor and
accounts for the rapid reversibility of
STa-induced secretion and the
relative "permanency" of CT-induced secretion. The
STa epithelial surface receptor is
distinct from the CT and LT toxin receptors and coincides with GC
activity (38, 44). Ileal villous epithelial cells have approximately
twice as many receptors as crypt cells for the enterotoxin (47). GC exists in two major forms: soluble and particulate. These are distinct
proteins encoded by separate genes. Soluble GC is a dimeric cytosolic
protein that is activated by nitric oxide (43). Particulate GC is a
family of brush-border membrane glycoproteins that are activated by
only two classes of substances: atrial natriuretic peptides (ANPs;
Table 1) and STa. In the
intestine, ~80% of total GC is particulate (41, 85). So far, three
different members of the particulate GC family have been cloned (12,
41, 83-85, 88). GC-A and GC-B are ANP receptor cyclases, whereas
GC-C is the specific receptor for
STa. All three members of the GC
family share the property of being proteins that span the cell membrane and that contain an extracellular domain, a transmembrane domain, an
intracytoplasmic domain made up of a protein kinase-like enzyme, and a
catalytic domain. These proteins show minimal similarities in their
extracellular domain, whereas there is a higher degree of similarity of
their intracellular domains. This finding suggests that the
extracellular domain represents the ligand-binding domain (41, 85). The
physiological role of STa
receptors in the mammalian intestine was unknown until six years ago,
when Currie et al. (15) extracted and purified from the rat small
intestine a peptide that is homologous to
STa. This endogenous peptide,
named guanylin, has been shown to be 50% homologous to
STa and to bind competitively to
the STa binding site on T84 cells,
thereby stimulating cGMP production (Table 1) (15, 37, 55, 95). Our
group has described a heat-stable enterotoxin (named EAST1) elaborated
by enteroaggragative E. coli (EaggEC)
(78) that proved to be structurally and functionally similar to
guanylin (79) (Fig. 3). Studies on T84
cells and COS cells tranfected with GC-C suggest that EAST1 interacts
with GC-C to elicit an increase in cGMP (D. Robertson, A. Fasano, and
S. Savarino, personal communication). The relation of guanylin with
STa and EAST1 entreats close
scrutiny. Given that these three moieties all act by means of the
GC/cGMP system and that all share significant amino acid sequence
homology (Fig. 3), a comparison of their primary structure may provide
insight as to the absolute requirements of the receptor-ligand
interaction involved in the enterocyte cGMP-mediated signal
transduction. Certain structural elements within the COOH-terminal
tridecapeptide of STa are
functionally critical, one of which is the occurrence of six cysteine
residues participating in three disulfide bridges (Fig. 3). Listed in
descending order of importance in terms of their respective
contribution to enterotoxic activity, the predicted disulfide linkages
between Cys-7 and Cys-15, Cys-6 and Cys-11, and Cys-10 and Cys-18
(referring to STa) are all
necessary for full biological activity (42). In contrast, EAST1 and
guanylin possess only four cysteine residues. Their four cysteine
residues align with Cys-7, Cys-10, Cys-15, and Cys-18. Based on this
evidence, it is attractive to predict that the cysteine residues of
EAST1 and guanylin form the same bridge pairs as in
STa. Site-directed mutagenesis
experiments, in which the Cys-7 (referring to
STa) of EAST1 was
replaced with an alanine residue, confirmed the key role of the
disulfide bridge between Cys-7 and Cys-15 in GC/cGMP activation and
subsequent enterotoxic activity of EAST1 (D. Robertson, A. Fasano, and
S. Savarino, personal communication).
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The EAST1 genotype is not restricted to EaggEC, being detected with notable frequency in enterohemorragic E. coli (EHEC), ETEC, and enteropathogenic E. coli (EPEC) (80). The high prevalence of the EAST1 genotype in EHEC, particularly in serogroups O157:H7 (100%) and O26 (89%), is striking. Genetic analysis demonstrated that the EAST1 gene present in EHEC is chromosomally encoded, whereas this gene is plasmid encoded in ETEC (41%), EPEC (21.5%), and EaggEC (41.4%) (80). These data suggest that EAST1 was acquired by O157 EHEC early in its clonal lineage and that some selective edge is maintained by its conservation. Based on this finding, it is also tantalizing to postulate that the transfection of EHEC by a bacteriophage was responsible for the translocation of the EAST1 gene on plasmid(s) subsequently acquired by other pathogenic E. coli.
cGMP signaling is a typical example of how microorganisms have been able to study the intestinal physiology of complex animals, to obtain information about guanylin, the natural ligand of GC/cGMP signaling, to genetically engineer agonists (i.e., EAST1) that activate this system, and to share this knowledge with other bacteria. Even more remarkable is the observation that some microorganisms were clever enough to synthesize a long-lived superagonist of guanylin, since STa turned out to be 40-fold more active than guanylin (7). Given the time needed by procaryotic organisms to assemble new genes, to maintain them in their limited genome, and to share this information with other microorganisms, it follows that bacteria probably learned about the cGMP pathway thousands, if not millions, of years before us.
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THE CA PATHWAY |
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Intracellular Ca ions are major regulators of mammalian electrolyte transport, being involved directly or indirectly in the regulation of active electrolyte transport, both in the small and large intestines (18, 21, 24). Nearly all living cells use this pathway as a way to transduce a variety of signals into a plethora of biological responses. The concentration of free intracellular Ca ([Ca]i) in the cytosol of most cells, including enterocytes, is in the submicromolar range, usually ~100 nM. The levels of [Ca]i involved in regulation are transient and low (less than 5- to 10-fold increase) and are tightly controlled, since higher concentrations are destructive to the cells. Higher concentrations of Ca (100-10,000-fold) are sequestered in three major compartments in living tissues: the extracellular or interstitial fluid; the mitochondria; and a third, physically ambiguous compartment, which is called the "nonmitochondrial" intracellular Ca store (73). The conduits for Ca entry into the cytosol from these stores as well as the mechanism(s) by which Ca is returned to the baseline state form a fascinating and far from complete saga. In the stimulated state, low permeabilities of the various membranes to Ca retain low [Ca]i. A series of neurohumoral substances normally present in the intestinal mucosa, including acetylcholine (6), serotonin (6, 19), substance P (20), neurotensin (20, 52), and bradykinin (Table 1) (16), can alter [Ca]i by modifying the permeability(ies) of either one or more of these compartments. This can be achieved by either voltage-gated channels or receptor-mediated mechanisms. Binding of ligands to receptors activates a membrane-associated inositol lipid-specific phospholipase C via the mediation of G proteins (73). This enzyme hydrolyzes phosphatidylinositol 4,5-bisphosphate to release inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). The soluble IP3 causes an increase in [Ca]i by either releasing Ca from the nonmitochondrial intracellular pool or stimulating entry of extracellular Ca. The membrane-bound DAG is a key activator of the phospholipid-dependent Ca kinase, protein kinase C (PKC) (73). In almost all studies of the enterocyte, regardless of the species, an increase in [Ca]i has one or more of the following effects on ion transport: it inhibits Na and Cl absorption, it stimulates anion secretion, and/or it modulates either the apical or basolateral membrane K conductance (22, 74). As in the case of the other two intracellular signaling mechanisms outlined above, these effects result in net luminal fluid accumulation, even if the accumulation is less in magnitude compared with cAMP. Ca can also activate the Ca-binding protein, calmodulin, which in turn activates calmodulin-dependent protein kinases and presumably certain plasma membrane transport proteins.
Until recently, there was no well-defined example of an enterotoxin that required Ca ions for its secretory effects. Raimondi et al. (72) have now demonstrated, using direct [Ca]i measurement, that the enterotoxic effect of the thermostable direct hemolysin elaborated by V. parahaemolyticus is mediated by Ca (Table 2). This toxin seems to interact with a polysialoganglioside GT1b surface receptor whose physiological function remains to be established (72). Other toxins probably act through Ca (Table 2); however, only indirect evidence was provided (29, 48, 50).
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THE CYTOSKELETON PATHWAY |
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The cytoskeleton of enterocytes has always been considered important for maintaining the polarity and structure of epithelial cells. At the cytoplasmic core of each microvillus there is a longitudinal bundle of parallel microfilaments that projects into the cytoplasm and associates with a complex network of cytoskeletal proteins known as the terminal web. The terminal web appears to be attached to the lateral plasma membrane in the region of the adherence junction. Throughout the body of the enterocyte are numerous keratin and actin filaments that provide the necessary infrastructure for enterocytes to maintain their polarity and columnar configuration.
It is only recently that changes in cytoskeleton arrangement have been associated with functional modifications of the intestinal epithelium. Therefore, the cytoskeleton remains the least defined yet most fascinating signaling mechanism used by enteric pathogens to communicate with target enterocytes. The result of this interaction may lead to three main mechanisms of secretion involving the cell cytoskeleton: invasion, spreading, and modulation of intestinal permeability.
Invasion
Yersinia and Salmonella represent two typical examples of microorganisms that invade their target cells through the cytoskeletal pathway. The interaction of pathogenic Yersinia and mammalian cells is proposed to occur in two sequential steps. Initially, the bacteria communicate with the host cell cytoskeleton throughMembrane ruffling, cytoskeletal rearrangements, and
[Ca]i fluxes occur as
part of a global cellular response to mitogens, oncogene expression,
and growth factors (1, 53, 64). The induction of membrane ruffles is
critical for entry of S. typhimurium (Fig. 1), since mutants unable to induce these changes are severely impeded in their ability to enter cultured mammalian cells (5). The
appearance of membrane ruffles on the surface of infected cells is
accompanied by profound cytoskeletal rearrangements at the point of the
bacterial-host contact, and a number of cytoskeletal proteins,
including actin, -actinin, talin, tubulin, tropomyosin, and ezrin,
accumulate at these sites (5). S. typhimurium infection of cultured cells is accompanied
by a marked increase of
[Ca]i, an event that
seems to be crucial for internalization, since Ca chelators and
antagonists of Ca channels block the bacterium entry into the cell (5).
Interestingly, Shigella invasion also
involves membrane ruffling and cytoskeletal alteration; however, no
increase in [Ca]i
seems to occur. Membrane ruffling, cytoskeletal rearrangement, and
[Ca]i fluxes have been
observed as a consequence of the activation of a number of host cell
surface receptors, including the epidermal growth factor receptor
(EGFR) (53). Interestingly, infection in Henle-407 cells by
S. typhimurium is accompanied by
activation of EGFR (Fig. 1) (5). However, the microorganism can enter into a number of cultured cell lines that do not express EGFR (5),
suggesting that S. typhimurium can stimulate more than one signal
transduction pathway to promote entry into mammalian cells. Recently,
Pier and co-workers (70) have provided compelling evidence suggesting
that S. typhi (but not
S. typhimurium) uses CFTR for entry
into epithelial cells. Heterozygous mice expressing the most common
CFTR mutation, a phenylalanine deleted at residue 508 (
F508
Cftr) translocated 86% fewer
S. typhi into the
gastrointestinal submucosa than wild-type
Cftr mice, whereas no translocation
occurred in
F508 Cftr homozygous
mice (70).
Spreading
Shigella flexneri is unusual among pathogenic bacteria in the nature of its intimate interactions with host cells to complete its vital cycle. The microorganism invades the intestinal mucosa via M cells (46). Within 15 min of entry into the epithelial cell, the bacterium escapes from its phagocytic vacuole and thereby enters the cytoplasmic compartment of the host cell. Immediately, short filaments of polymerized cytoplasmic actin accumulate at one extremity of the bacterium (46). The actin is bundled to form an actin-containing tail several micrometers in length behind the microorganism as it moves forward in spurts through the cytoplasm (Fig. 1) (46). Several cytoskeletal proteins have been shown to be incorporated into the tail that trails the bacterium. In particular, it contains large amounts of polymerized actin (46). Therefore, Shigella utilize at least some of the resources of the cellular cytoskeleton apparatus, probably in conjunction with certain elements, to motor itself through and between the host cells. To spread from one cell to another, the bacterium forms a fingerlike protrusion from the surface of the infected cell (Fig. 1). Around the site of exit of the protrusion at the cell surface, major rearrangement of the cytoskeleton occurs, with the formation of numerous tiny villosities (46). Within the protrusion, the bacterium is trailed by its actin tail. The tip of the protrusion penetrates the surface membrane of an adjacent cell, and, subsequently, the bacterium lyses these membranes and is thereby released into the cytoplasm of the adjacent cell.This entire process represents a remarkable example of the ability of S. flexneri to utilize the host cytoskeleton for its own purpose to adapt to intracellular survival and, therefore, to induce diarrhea.
Modulation of Intestinal Permeability
The intestinal epithelium represents the largest interface (>2,000,000 cm2) between the external environment and the internal host milieu and constitutes the major barrier through which molecules can either be absorbed or secreted. There is now substantial evidence that tight junctions play a major role in regulating epithelial permeability by influencing paracellular flow of fluid and solutes. Moreover, structural features of occluding junctions such as strand number often correlate inversely with the permeability of epithelia as measured electrophysiologically (13). A century ago, tight junctions were thought to be a secreted extracellular cement forming an absolute and unregulated barrier within the paracellular space (9). There is now abundant evidence that tight junctions are dynamic structures that readily adapt to a variety of developmental (61, 82), physiological (60, 62, 77), and pathological (63, 68, 87) circumstances. In recent years much has been discovered about the structure, function, and regulation of tight junctions. However, the precise mechanism(s) through which they operate are still incompletely understood. Several microorganisms have been shown to exert a cytopathic, pathological effect on epithelial cells that involves the cytoskeletal structure and the tight junctions functioning in an irreversible manner. These bacteria alter intestinal permeability either directly (i.e., EPEC) or through the elaboration of toxins (i.e., Clostridium difficile, Bacteroides fragilis; for a more complete review see Ref. 86). A more physiological mechanism of regulation of tight junction permeability has been proposed for the zonula occludens toxin (Zot) elaborated by V. cholerae (27). As often occurs in science, the discovery of this toxin was made by accident. A few years ago, researchers at the Center for Vaccine Development at the University of Maryland in Baltimore engineered what was believed to be an ideal attenuated vaccine for cholera. At that time CT was the only described toxin elaborated by V. cholerae to induce diarrhea. Therefore, the deletion of the gene encoding the active subunit of CT appeared to be the best approach to eliminate the key pathogenic factor of the microorganism while maintaining the expression of other Vibrio antigens necessary for a protective immune response. When fed to volunteers, these vaccine candidates still caused mild diarrhea in more than one-half of the vaccinees (58). In search of other factors responsible for this residual diarrhea, our group identified Zot, a protein elaborated by V. cholerae that increases the permeability of the small intestine by affecting the structure of tight junctions (27). We have subsequently demonstrated that Zot activates a complex intracellular cascade of events that regulate intestinal permeability (Fig. 4) (28). Zot induces a dose- and time-dependent PKC
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Based on this observation, we postulated that Zot may mimic the effect of a functionally and immunologically related endogenous modulator of epithelial tight junctions. The combination of affinity-purified anti-Zot antibodies and the Ussing chamber assay allowed us to identify an intestinal Zot analog that we named zonulin (32). When zonulin was studied in a nonhuman primate model, it reversibly opened intestinal tight junctions. Because V. cholerae infections are strictly confined to the gastrointestinal tract, we anticipated an exclusively intraintestinal role for zonulin. Much to our surprise, this protein(s) was detected in a wide range of extraintestinal tissues and has now been purified from human intestine, heart, and brain. We have provided evidence that the zonulins comprise a family of tissue-specific regulators of tight junctions (32). Each family member has a molecular mass of ~47 kDa, a distinct NH2-terminal receptor binding motif that confers tissue specificity, and a COOH-terminal tau-like domain probably involved in the cytoskeleton rearrangement. Amino acid substitution within the NH2-terminal binding motif identified three amino acid residues that dictate tissue specificity, allowing local autocrine/paracrine regulation in response to local requirements (32). The physiological function of the zonulins remains to be established; however, it is likely that they are involved in tight junction regulation responsible for the movement of fluid, macromolecules, and leukocytes between body compartments. With regard to the intestine, it is conceivable to speculate that this pathway is also involved in the process of nutrient digestion. The complete digestion of complex molecules depends on an ideal solute-to-solvent ratio to enable pancreatic enzymes to intimately interact with their target nutrients. A neuroendocrine system sensitive to intestinal intraluminal osmolality is probably involved in the homeostasis of this ratio. During the postprandial phase, the increased intraluminal osmolarity may activate intestinal "osmotic sensors," with subsequent release of zonulin. After zonulin binding to its target receptor, tight junctions are reversibly opened, and fluid leaks into the intestinal lumen at a higher rate driven by the osmotic gradient. This fluid secretion would allow a better mixing of nutrients with pancreatic and biliary juices and, therefore, a more efficient digestion. The excess fluid that accumulates in the small intestine would be completely reabsorbed in the colon (where the zonulin pathway seems to be nonoperative), preventing intestinal fluid loss and, therefore, diarrhea. If this hypothesis is proved to be correct, it is conceivable to speculate that zonulin-dependent tight junction regulation played a pivotal role in the appearance and conservation of the human race on the Earth stage. At the beginning of the human era, when nutritional resources were scarce and difficult to obtain, a complete digestion of the limited nutrients ingested would be critical for the survival of the Homo sapiens lineage in an environment dominated by more efficient predators.
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PRACTICAL APPLICATIONS OF THE ZOT-ZONULIN PATHWAY |
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The demonstration that Zot activates a complex intracellular cascade of events that regulate intestinal permeability (28, 31) prompted our group to explore the possibility of modulating tight junction permeability to orally deliver molecules normally not absorbed through the gut mucosa (25, 26, 30).
The oral administration of Zot with biologically active ingredients was able to enhance intestinal absorption of the active molecule, and this enhancement seemed to be effective for both relatively small (5,733 Da: insulin) and large molecules (140-160 kDa: IgG) (30). Furthermore, experiments with BB/Wor diabetic rats demonstrated that orally delivered insulin retains its biological activity, since this treatment was as efficient as the parenteral administration of the hormone in controlling the high blood glucose level of treated animals (30).
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WHAT MAKES A PATHOGEN A PATHOGEN? |
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Virulence genes of pathogenic bacteria, which code for toxins that
activate the physiological pathways of eukaryotic cells outlined above,
are acquired via transmissible genetic elements such as transposons,
plasmids, or bacteriophages. In addition, such genes may be parts of
particular regions on the bacterial chromosome termed "pathogenicity
islands." These islands contain DNA sequences for bacteriophage
attachment, suggesting that the genes present within the pathogenicity
islands were previously able to spread among bacterial populations by
horizontal gene transfer (via phage transfection), a process known to
contribute to microbial evolution. This phenomenon has been elegantly
demonstrated to occur in V. cholerae
by Waldor and Mekalanos (94). They have recently shown that the genes
upstream of ctx belong to a
filamentous phage (designated CTX) that replicates as
a plasmid and is responsible for the horizontal transfer of a
pathogenic element (ctx) to
nontoxigenic V. cholerae.
What is the evolutionary advantage for enteric pathogens to acquire toxins that activate host cell pathways leading to intestinal secretion? Diarrhea has always been considered a defense mechanism of the host animal against enteric pathogens. However, a different way to interpret this phenomenon should be considered. For some microorganisms the only way to survive as a species is to spread from one host to another. For other bacteria, such as V. cholerae, the intestine represents a very hostile environment, and the only way to survive is to escape from the intestine and to return to their aquatic reservoir. Therefore, the elaboration of enterotoxic factors that, by different mechanisms of action that activate physiological pathways, may all induce fluid accumulation within the intestinal lumen represents a great advantage to intestinal pathogens, because it increases the chance of survival.
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CONCLUSIONS |
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The few examples of bacteria-host interaction outlined in this review suggest that we can gain new insights in cell biology by studying those microorganisms that know how to communicate with their mammalian host. The most skilled pathogens have been smart enough to 1) learn the physiology of their host, 2) keep this information in their "genetic library," 3) share the "gene books" among other microorganisms, and 4) be "open minded" to acquire new knowledge and to discharge obsolete information. If we want to learn more about ourselves, we should accept with humility the lesson from the "microorganism civilization" and be ready to give up our intellectual prejudice of superior beings. Consider the difference in size between some of the very tiniest and the very largest creatures on Earth. A small bacterium weighs as little as 0.000000000001 grams. A blue whale weighs about 100,000,000 grams. Yet a bacterium can kill a whale. ... Such is the adaptability and versatility of microorganisms, as compared with humans and other so-called `higher' organisms, that they will doubtless continue to colonise and alter the face of the Earth long after we and the rest of our cohabitants have left the stage forever. Microbes, not macrobes, rule the world. [Bernard Dixon (17a)]
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ACKNOWLEDGEMENTS |
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I thank the endless list of collaborators, postdoctoral fellows, and technicians that contributed to the scientific information generated in my laboratory that made this review possible. Special thanks to Vicky Thrash for her secretarial assistance and to Dr. Jo Ann Mackinson for her patient support and unconditioned encouragement.
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FOOTNOTES |
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This paper was partially supported by National Institutes of Health Grants DK-48373 and AI-35740.
Address for reprint requests and other correspondence: A. Fasano, Division of Pediatric Gastroenterology and Nutrition, Univ. of Maryland School of Medicine, 685 W. Baltimore St., HSF Bldg., Rm. 465, Baltimore, MD 21201 (E-mail: afasano{at}umaryland.edu).
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