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Breaching the mucosal barrier by stealth: an emerging pathogenic mechanism for enteroadherent bacterial pathogens
James M. Fleckenstein1 and Dennis J. Kopecko2
1Department of Medicine,University if Tennessee and Veterans Affairs Medical Center, Memphis, Tennessee, USA2Laboratory of Enteric and Sexually Transmitted Diseases, Food and Drug Administration, Center for Biologics Evaluation and Research, Bethesda, Maryland, USA
Address correspondence to: Dennis J. Kopecko, Laboratory of Enteric and Sexually Transmitted Diseases, Food and Drug Administration, Center for Biologics Evaluation and Research, Bethesda, Maryland 20892, USA. Phone: (301) 496-1893; Fax: (301) 480-5047; E-mail: kopecko@cber.fda.gov.
Find articles by Fleckenstein, J. in: JCI | PubMed | Google Scholar
1Department of Medicine,University if Tennessee and Veterans Affairs Medical Center, Memphis, Tennessee, USA2Laboratory of Enteric and Sexually Transmitted Diseases, Food and Drug Administration, Center for Biologics Evaluation and Research, Bethesda, Maryland, USA
Address correspondence to: Dennis J. Kopecko, Laboratory of Enteric and Sexually Transmitted Diseases, Food and Drug Administration, Center for Biologics Evaluation and Research, Bethesda, Maryland 20892, USA. Phone: (301) 496-1893; Fax: (301) 480-5047; E-mail: kopecko@cber.fda.gov.
Find articles by Kopecko, D. in: JCI | PubMed | Google Scholar
Published January 1, 2001 - More info
J Clin Invest. 2001;107(1):27–30. https://doi.org/10.1172/JCI11792.
© 2001 The American Society for Clinical Investigation
The clinical and molecular understanding of bacterial diseases of the gastrointestinal tract, developed largely over the past 50 years, has led to our current assignment of these causative agents into an evolving set of mechanistic categories (see Table 1). Early studies revealed that some organisms (Staphylococcus aureus among them) produce toxins in contaminated foods and that, upon ingestion, these preformed toxins trigger a rapid-onset, net jejunal secretion of electrolytes and water, typically lasting less than 24 hours. In these illnesses, the causative organism need not be present in the intestine. In contrast, the “enterotoxigenic” pathogens, typified by Vibrio cholerae and enterotoxigenic Escherichia coli (ETEC), are noninflammatory and must colonize the gastrointestinal tract in order to deliver toxins to the mucosa. These toxins typically effect cAMP- or cGMP-mediated net Cl– secretion, often resulting in a large-volume diarrhea. Recent studies of certain strains of Bacteroides fragilis indicate that these agents can serve as large bowel mediators of enterotoxigenic disease via a toxin that disrupts the mucosal barrier (1).
Another long-recognized grouping of enteric bacterial diseases is the “invasive” category, which can be subdivided based upon the extent of dissemination within the host. The most superficial invaders, Shigella spp. and enteroinvasive E. coli, have similar, if not identical, virulence properties. These organisms invade the epithelium of the colon and terminal ileum, spread intercellularly via actin-filament projections (2), and cause focal microulcerations of the ileocecal mucosa. The submucosal group is exemplified by Yersinia enterocolitica and Yersinia pseudotuberculosis, which transcytose across the mucosal epithelium and proceed to the regional lymph nodes, causing severe abdominal pain and, occasionally, mesenteric lymphadenitis. Recent clinical and experimental data suggest that Campylobacter jejuni is an invasive pathogen that can translocate across the mucosa, and that may survive submucosally, but does not typically reach the bloodstream (3). The third invasive subgroup is typified by certain Salmonella serovars (e.g., Typhi and Paratyphi) that invade and translocate across the epithelium. These organisms enter and apparently survive for many hours in submucosal monocytes, which may serve to transport these pathogens to distal sites in the host (e.g., spleen, liver, and bone marrow).
The characterization of a previously unrecognized group of pathogenic E. coli (i.e., enteropathogenic E. coli [EPEC], enteroaggregative E. coli [EAEC], diffusely adherent E. coli [DAEC], and enterohemmorhagic E. coli [EHEC]) since the 1950s has led to the establishment of the enteroadherent category of disease. These agents colonize the small and/or large intestine, do not appear to be invasive to any appreciable extent in vivo, and, with the exception of shiga-like toxins of EHEC, are not known to elaborate any of the classical enterotoxins, yet they cause diarrheal illness. Classical EPEC serotypes are known to induce “pedestal” or “cup-like” rearrangements, referred to as attaching and effacing lesions, of epithelial cell membranes. The attaching bacteria erode the microvilli via a rearrangement of cytoskeletal actin into host membrane pedestal structures that support the organism. EAEC and DAEC display characteristic, but different, adherence patterns on cultured intestinal cells and have not been shown to cause any striking membrane effacement. Although certain aspects of EPEC pathogenesis, such as adherence and the attaching and effacing phenotype, have been studied elegantly and in detail (4), the mechanisms by which these and other enteroadherent organisms induce inflammation and diarrhea have only recently started to emerge (5).
Salmonella enterica serovar Typhimurium (now abbreviated S. Typhimurium) have previously been considered members of the invasive category, because of their invasiveness in cultured cells and their ability to cause a typhoid-like illness in mice. Indeed, S. Typhimurium may occasionally be isolated from extraintestinal sites in humans including the bloodstream, particularly in immunocompromised hosts. However, these pathogens are more typically associated in humans with diarrheal illness of mild to moderate severity accompanied by fever and the appearance of fecal leukocytes. Although S. Typhimurium may trigger limited invasion of the intestine, as may occur with other enteroadherent pathogens, mucosal invasion does not appear to be a prominent aspect of normal disease. As now shown in this issue of the JCI (6), S. Typhimurium appear to be prime examples of organisms that cause diarrheal disease through a general mechanism that may characterize many bacteria commonly placed in the enteroadherent category of intestinal diseases.
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Induction of epithelial cell IL-8 production by bacterial flagellin
Recently, in the JCI, Steiner et al. demonstrated that a unique bacterial flagellin protein produced by EAEC is a potent stimulus for IL-8 secretion in cultured epithelial cells (13). These authors also demonstrated that purified flagellin from selected EPEC and EHEC strains also stimulated IL-8 secretion by target cells. In the current issue of the JCI, Gewirtz and coworkers (6) have expanded upon their earlier studies showing that S. Typhimurium triggers basolateral IL-8 secretion from cultured model epithelia (14) via Ca++-mediated activation of the NF-κB pathway. Previous work showed that S. Typhimurium only associates with a small proportion of model epithelial cells but that it induces a robust IL-8 release, as if all cells were somehow responding. Gewirtz et al. hypothesized that S. Typhimurium might trigger epithelial exocytosis of a proinflammatory mediator that might activate IL-8 synthesis via a mechanism similar to TNF-α (6).
Indeed, these researchers found a soluble mediator, present after 1 hour in the basolateral fluids of monolayer cells infected apically with S. Typhimurium. This soluble mediator could trigger IL-8 synthesis when applied basolaterally, but not apically, to fresh model epithelial cells. Further study revealed that the IL-8 mediator was not of eukaryotic origin, but rather represented bacterial flagellin, and that supernatants of flagellated, but not of nonflagellated mutants of, S. Typhimurium cultures could induce IL-8 synthesis when applied basolaterally to model epithelia. Moreover, the culture supernatants of five out of seven strains of flagellated, but nonpathogenic, commensal E. coli induced IL-8 in this basolateral exposure system.
Purified flagellin, added apically in the absence of bacteria, was unable to translocate across model epithelia or to elicit IL-8 secretion, emphasizing the basolateral localization of this activation. As one might expect, flagellin translocation across model epithelia was not mediated by flagellated strains of commensal E. coli. Limited studies of S. Typhimurium mutants indicate that type III secretory–mediated interactions of bacteria with host cells are not essential for flagellin translocation. While the mechanism of flagellin translocation remains undefined, the ability to translocate flagellin across the mucosa appears to be an important determinant of virulence in certain bacteria that elicit IL-8 in the absence of a physical break in the mucosa (see Figure 1).
Figure 1Working model for flagellin-induced stimulation of inflammatory diarrhea by S. Typhimurium. Schematic shows monolayer of intestinal epithelial cells with an adherent S. Typhimurium. Flagellin is translocated by an undetermined mechanism (depicted in red) to the basolateral surface, where it interacts with putative receptors (R) that trigger IL-8 production (depicted in dark green) and basolateral recruitment of PMNs. S. Typhimurium has recently been reported to stimulate production of an apical chemoattractant (depicted in light green) that aids in PMN transmigration to the luminal surface, where these leukocytes trigger fluid secretion (pathway depicted in blue).
Flagellin purified from wild-type S. Typhimurium induced detectable IL-8 secretion at concentrations below 1 ng/ml, more potent than the proinflammatory agonist TNF-α. Although LPS is a key activator of subepithelial macrophages, flagellin now appears to play a major role in triggering the intestinal mucosal stimulation of an inflammatory response.
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