Pathogenesis Of Enteric Infections Research Articles

Enterotoxigenic Escherichia coli is phagocytosed by macrophages underlying villus-like intestinal epithelial cells: modeling ex vivo innate immune defenses of the human gut

ABSTRACTThere is a paucity of information on diarrheagenic enterotoxigenic Escherichia coli (ETEC)’s interaction with innate immune cells, in part due to the lack of reliable models that recapitulate infection in human gut. In a recent publication, we described the development of an ex vivo enteroid-macrophage co-culture model using human primary cells. We reported that macrophages residing underneath the epithelial monolayer acquired “resident macrophage” phenotype characterized by lower production of inflammatory cytokines and strong phagocytic activity. These macrophages extended projections across the epithelium, which captured ETEC applied to the apical side of the epithelium and reduced luminal bacterial load. Additional evidence presented in this addendum confirms these findings and further demonstrates that macrophage adaptation occurs regardless of the stage of differentiation of epithelial cells, and that ETEC uptake arises rapidly after infection. The enteroid-macrophage co-culture represents a novel and relevant tool to study host-cell interactions and pathogenesis of enteric infections in humans.

Putative Inv Is Essential for Basolateral Invasion of Caco-2 Cells and Acts Synergistically with OmpA To Affect In Vitro and In Vivo Virulence of Cronobacter sakazakii ATCC 29544

Cronobacter sakazakii is an opportunistic pathogen that causes neonatal meningitis and necrotizing enterocolitis. Its interaction with intestinal epithelium is important in the pathogenesis of enteric infections. In this study, we investigated the involvement of the inv gene in the virulence of C. sakazakii ATCC 29544 in vitro and in vivo. Sequence analysis of C. sakazakii ATCC 29544 inv revealed that it is different from other C. sakazakii isolates. In various cell culture models, an Δinv deletion mutant showed significantly lowered invasion efficiency, which was restored upon genetic complementation. Studying invasion potentials using tight-junction-disrupted Caco-2 cells suggested that the inv gene product mediates basolateral invasion of C. sakazakii ATCC 29544. In addition, comparison of invasion potentials of double mutant (ΔompA Δinv) and single mutants (ΔompA and Δinv) provided evidence for an additive effect of the two putative outer membrane proteins. Finally, the importance of inv and the additive effect of putative Inv and OmpA were also proven in an in vivo rat pup model. This report is the first to demonstrate two proteins working synergistically in vitro, as well as in vivo in C. sakazakii pathogenesis.

Role of Bacterial OmpA and Host Cytoskeleton in the Invasion of Human Intestinal Epithelial Cells by Enterobacter sakazakii

Enterobacter sakazakii is an emerging pathogen in neonates and infants. Interactions of E. sakazakii with intestinal epithelium could be vital in the pathogenesis of enteric infections and in its systemic dissemination. The present study investigated the interaction of E. sakazakii with human intestinal epithelial (INT407) cells and the role of bacterial outer membrane protein A (OmpA) and host cytoskeleton in these interactions. E. sakazakii invaded INT407 cells with moderate efficiency. An ompA mutant of E. sakazakii was significantly attenuated in its invasiveness, and complementation restored the invasive phenotype significantly. Drugs acting on host cell microfilaments (MF) and microtubules (MT) significantly inhibited bacterial invasion. Localization of both microfilaments (MF) and microtubules (MT) was observed in INT407 cells following E. sakazakii infection. The results suggest that E. sakazakii invasion of INT407 cells involves participation of both MF and MT and bacterial OmpA plays a critical role in invasion.

Flagellin/TLR5 responses in epithelia reveal intertwined activation of inflammatory and apoptotic pathways

Flagellin, the primary structural component of bacterial flagella, is recognized by Toll-like receptor 5 (TLR5) present on the basolateral surface of intestinal epithelial cells. Utilizing biochemical assays of proinflammatory signaling pathways and mRNA expression profiling, we found that purified flagellin could recapitulate the human epithelial cell proinflammatory responses activated by flagellated pathogenic bacteria. Flagellin-induced proinflammatory activation showed similar kinetics and gene specificity as that induced by the classical endogenous proinflammatory cytokine TNF-alpha, although both responses were more rapid than that elicited by viable flagellated bacteria. Flagellin, like TNF-alpha, activated a number of antiapoptotic mediators, and pretreatment of epithelial cells with this bacterial protein could protect cells from subsequent bacterially mediated apoptotic challenge. However, when NF-kappaB-mediated or phosphatidylinositol 3-kinase/Akt proinflammatory signaling was blocked, flagellin could induce programmed cell death. Consistently, we demonstrate that flagellin and viable flagellate Salmonella induces both the extrinsic and intrinsic caspase activation pathways, with the extrinsic pathway (caspase 8) activated by purified flagellin in a TLR5-dependant fashion. We conclude that interaction of flagellin with epithelial cells induces caspase activation in parallel with proinflammatory responses. Such intertwining of proinflammatory and apoptotic signaling mediated by bacterial products suggests roles for host programmed cell death in the pathogenesis of enteric infections.

Type III secretion machines and the pathogenesis of enteric infections caused by Yersinia and Salmonella spp.

Salmonella and Yersinia spp. infect the intestinal tract of humans. Although these organisms cause fundamentally different diseases, each pathogen relies on type III secretion machines to either inject virulence factors into the cytosol of eukaryotic cells or release toxins into the extracellular milieu. Type III secretion machines are composed of many different subunits and export several polypeptides with unique substrate requirements. During Salmonella pathogenesis, the type III machine encoded by the Salmonella pathogenicity island (SPI)-1 genetic element functions to cause invasion of the intestinal epithelium, whereas another type III machine (SPI-2) is required for survival in macrophages. Yersinia enterocolitica and Yersinia pseudotuberculosis employ type III machines to resist macrophage phagocytosis and to manipulate the host's immune response, thereby colonizing intestinal lymphoid tissues. We describe what is known about the pathogenic functions of virulence factors secreted by type III machines. Furthermore, type III secretion machines may be exploited for the injection of recombinant proteins, a strategy that has already been successfully employed to elicit a cell-mediated immune response.

Transmucosal passage of bacteria across rat intestinal epithelium in the Ussing chamber: effect of nutritional factors and bacterial virulence.

Transmucosal passage of bacteria across the intestine, the essential and prerequisite step for bacterial translocation, cannot be effectively studied using in vivo models of translocation. We have adapted the Ussing chamber into a fresh, sterile organ culture system that can facilitate the study of bacterial-epithelial interactions. Intestinal membranes were mounted in the Ussing chamber and perfused with a solution rich in putative mucosal micronutrients. The transmembrane potential difference was constantly monitored as a marker of intestinal integrity. Transmucosal passage of various bacteria across the normal intestinal epithelium was quantitated, and the mucosal membrane was examined by light and transmission electron microscopy. The addition of potassium cyanide to the mucosal perfusate resulted in an irreversible loss of potential difference. Oxygen deprivation also led to a precipitous drop in potential difference, but it was reversible with prompt reoxygenation. In contrast, intestinal membranes perfused with a solution consisting of Dulbecco's modified Eagle's medium + 20 mM glutamine maintained their potential difference for a sustained period (>180 min). Both the viability and structural integrity of the ileal intestinal membrane were maintained in culture ex vivo using this perfusate. Qualitative differences were observed in the mechanism of transmucosal passage of mild to moderately virulent bacteria such as Escherichia coli C-25 and Proteus mirabilis M-13, which pass through the intestinal epithelium without causing overt damage to the mucosa, and more virulent organisms such as Salmonella typhimurium, which cause extensive mucosal damage by light and transmission electron microscopy. The Ussing system should provide a useful model of intact organ culture for the study of the mechanisms of bacterial translocation and the pathogenesis of enteric infections.

Vaccines to prevent enteric infections

Considerable progress has been made in the last decade in developing vaccines against the enteric infections of greatest public health importance. A quadrivalent rotavirus vaccine consisting of rhesus rotavirus vaccine (which contains serotype 3 neutralization antigen) and three reassortant viruses (rhesus virus expressing neutralization antigens of serotypes 1, 2 or 4) is undergoing placebo-controlled field trials of efficacy in the USA and in two developing countries. Two new vaccines against typhoid fever (oral Ty21a and parenteral Vi polysaccharide) have been licensed in many countries. Even newer generations of typhoid vaccines are undergoing clinical testing, including new attenuated S. typhi strains and Vi polysaccharide-carrier protein conjugate vaccines. Two inactivated oral cholera vaccines, consisting of inactivated V. cholerae 01 bacteria alone or in combination with purified B subunit of cholera toxin, each conferred 50–53% protection over 3 years in a field trial in Bangladesh where subjects were immunized with a three-dose regimen. In extensive clinical trials in adults and children in less-developed countries, an engineered live oral cholera vaccine, strain CVD 103-HgR, has been shown to be well tolerated and highly immunogenic following administration of just a single oral dose; a large-scale field trial in 70000 subjects is underway to investigate the efficacy of this vaccine. Several candidate vaccines against Shigella and enterotoxigenic Escherichia coli are in clinical trials. Accumulating knowledge on pathogenesis of enteric infections and advances in mucosal and cellular immunology, coupled with the application of modern biotechnology, have resulted in a plethora of vaccine candidates. It is expected that in future years efforts will be directed to construct vaccines against other enteric pathogens.

Interactions between bacteria and the surface of the small intestine

Many bacterial species are closely associated with epithelial surfaces of the gastrointestinal tract. The physicochemical interactions between bacteria and the intestinal surface are complex and include both stereospecific and chemotactic effects. Such mechanisms of interaction and attachment of bacteria to the gut have received considerable attention over the last decade as they have been recognised as an important initial event in colonisation and the pathogenesis of enteric infections. Many disease-causing bacteria possess surface fimbriae or fibrillae with lectin-like adhesins which interact with peptides or carbohydrates present on surface glycosphingolipids and/or glycoproteins. Research on the structural features of fimbrial receptors and on their expression on the surfaces of the mammalian intestine has led to new ideas for nutritional therapy and/or prophylaxis based on the prevention of adherence of pathogenic bacteria.

Summary of the 27th United States-Japan Joint Conference on Cholera and Related Diarrheal Diseases.

The 31 st United States-Japan Cholera and Related Diarrheal Diseases Conference was held in conjunction with the United States-Japan Malnutrition Conference at Kiawah Island, South Carolina, 30 November to 3 December 1995. In addition to the overall conference theme of the role of cytokines in the pathogenesis of enteric infections and malnutrition, researchers described substantial advances in cholera epidemiology, detection, molecular mechanisms, and pathophysiology plus new mechanisms for enterotoxigenic, enteroadherent, enterohemorrhagic, and enteroinvasive Escherichia coli. There was also emphasis on new work with and vaccine development with Bacteroidesfragilis and Yersinia, Shigella, and Salmonella species.

RGD-dependent entry of coxsackievirus A9 into host cells and its bypass after cleavage of VP1 protein by intestinal proteases

The recently reported nucleotide sequence of coxsackievirus A9 (CAV-9) showed that unlike other enteroviruses, CAV-9 has an insertion of about 17 amino acids at the C-terminal end of VP1 (K. H. Chang, P. Auvinen, T. Hyypiä, and G. Stanway, J. Gen. Virol. 70:3269-3280, 1989). This sequence includes the RGD (arginine-glycine-aspartic acid) motif which is known to be important in certain protein-protein interactions. We studied the inhibitory effect of RGD-containing peptides in the attachment of CAV-9 to African green monkey kidney cells. A peptide corresponding to the RRGDM sequence derived from the inserted segment of CAV-9 was found to block virus attachment effectively, and the inhibition was dose dependent. Substitution of glutamic acid for the homologous aspartic acid completely abolished the inhibitory effect, indicating great specificity of the action. During replication in the gut, all enteroviruses are exposed to host proteolytic enzymes. Exposure of CAV-9 to purified trypsin or human intestinal fluid resulted in selective cleavage of the VP1 capsid protein. Intact and trypsin-cleaved VP1 proteins gave identical N-terminal sequences, indicating that cleavage of VP1 takes place near the C terminus. Attachment of proteolytically cleaved infectious CAV-9 to green monkey kidney cells was not prevented by RGD-containing peptides, indicating that cleaved CAV-9 is able to bypass RGD-dependent entry. The altered receptor specificity of proteolytically cleaved viruses may have important consequences in the pathogenesis of enteric infections.

Summary of the thirty-first United States-Japan Joint Conference on Cholera and Related Diarrheal Diseases.

The 31st United States-Japan Cholera and Related Diarrheal Diseases Conference was held in conjunction with the United States-Japan Malnutrition Conference at Kiawah Island, South Carolina, 30 November to 3 December 1995. In addition to the overall conference theme of the role of cytokines in the pathogenesis of enteric infections and malnutrition, researchers described substantial advances in cholera epidemiology, detection, molecular mechanisms, and pathophysiology plus new mechanisms for enterotoxigenic, enteroadherent, enterohemorrhagic, and enteroinvasive Escherichia coli. There was also emphasis on new work with and vaccine development with Bacteroides fragilis and Yersinia, Shigella, and Salmonella species.

Summary of the 25th United States-Japan Joint Conference on Cholera and Related Diarrheal Diseases

The 31 st United States-Japan Cholera and Related Diarrheal Diseases Conference was held in conjunction with the United States-Japan Malnutrition Conference at Kiawah Island, South Carolina, 30 November to 3 December 1995. In addition to the overall conference theme of the role of cytokines in the pathogenesis of enteric infections and malnutrition, researchers described substantial advances in cholera epidemiology, detection, molecular mechanisms, and pathophysiology plus new mechanisms for enterotoxigenic, enteroadherent, enterohemorrhagic, and enteroinvasive Escherichia coli. There was also emphasis on new work with and vaccine development with Bacteroidesfragilis and Yersinia, Shigella, and Salmonella species.

EFFECT OF MALNUTRITION ON THE ADHERENCE OF SALMONELLA TYPHIMURIUM (ST) TO RAT ENTEROCYTE

The adherence of bacteria to intestinal mucosa is an important initial event in the pathogenesis of enteric infections. Such infections occur frequently in association with undernutrition. We investigated the adherence of two strains of ST to enterocytes from ad-libitum fed and nutrient restricted rats in order to study the effects of nutrient restriction on the adherence of ST to enterocytes. An animal model of malnutrition was produced by feeding young male adult rats 50% of the daily intake in control ad-libitum fed rats. Enterocytes were isolated from proximal and distal small intestinal segments by agitation in isolation medium. Adherence was studied using fimbriated (S7471F) and non-fimbriated (S7471N) strains of ST labelled with 3H-adenine. Labelled bacteria were incubated with freshly isolated enterocytes for 30 min. at 37 C. and the free bacteria were separated from those bound to enterocytes by membrane filtration and washing. The bound bacteria was quantitated by liquid scintillation. Adherence was significantly higher with S7471F both with proximal and distal intestinal enterocytes. There was no significant difference between the malnourished and control rats with respect to degree of adherence despite significant differences in body and organ weights and serum albumin. Prolongation of the period of nutrient restriction (to 12 wks) did not produce any change in pattern of adherence. We conclude that the adherence patterns of these strains of ST to enterocytes are not significantly altered by malnutrition.

Studies on the pathogenesis of enteric infections caused by invasive bacteria.

Salmonellae, shigellae and some Escherichia coli must invade the intestinal epithelial cell and multiply within the mucosa to cause disease. Although the bacterial cell most likely possesses several properties essential to this invasive ability, the nature of the cell envelope complex is at present the only characteristic which has been implicated in this process. While a number of pathophysiological events result from invasion, some of our recent efforts have concerned the site and mechanism of intestinal fluid loss in salmonellosis and shigellosis. In both these disorders, bacterial invasion of the colonic mucosa, associated with an acute inflammatory reaction and mucosal damage, is regularly seen and colonic salt and water transport is abnormal. These defects may account for mild diarrhoea in salmonellosis and the dysenteric stools of shigellosis. However, in salmonella-infected animals with severe watery diarrhoea and in shigella-infected animals with diarrhoea alone or in combination with dysentery, the jejunum is in a net secretory state. This secretion occurs in the absence of bacterial invasion or morphological abnormalities. Thus, the diarrhoea caused by invasive bacteria may result from the inability of the colon to reabsorb the increased volume of fluid entering it from the small intestine. Although colonic mucosal damage is a feature of invasive-type diarrhoeas, the permeability of both the colon and small intestine to small molecules, mannitol and erythritol, is not altered. Thus intestinal fluid loss cannot be ascribed to transudation. In addition, the results of our Ussing chamber experiments, employing salmonella-infected rabbit ileum, reveal that salt and water secretion is an active process. Since secretion occurs in the jejunum in the absence of bacterial invasion, this might suggest the participation of an enterotoxin. Shigella dysenteriae I is the best-studied invasive organism in which an enterotoxin has been found, yet mutant strains which do not invade but retain the ability to elaborate enterotoxin fail to cause disease in either monkeys or man. Thus, the physiological relevance of Shiga enterotoxin and the mechanism of jejunal secretion in these disorders remain unclear. Recent data suggest that invasive enteropathogens, like the enterotoxin-producing bacteria, activate the mucosal adenylate cyclase-cyclic AMP system and that this activation may play a role in intestinal fluid secretion.