PLoS One. 2014 Jan 9;9(1):e85211. doi: 10.1371/journal.pone.0085211.

Effect of wild-type Shigella species and attenuated Shigella vaccine candidates on small intestinal barrier function, antigen trafficking, and cytokine release.

Fiorentino M1, Levine MM2, Sztein MB2, Fasano A1.

1Mucosal Immunology and Biology Research Center, Massachusetts General Hospital, Charlestown, Massachusetts, United States of America.
2Center for Vaccine Development and the Departments of Pediatrics and Medicine, University of Maryland School of Medicine, Baltimore, Maryland, United States of America.



Bacterial dysentery due to Shigella species is a major cause of morbidity and mortality worldwide. The pathogenesis of Shigella is based on the bacteria’s ability to invade and replicate within the colonic epithelium, resulting in severe intestinal inflammatory response and epithelial destruction. Although the mechanisms of pathogenesis of Shigella in the colon have been extensively studied, little is known on the effect of wild-type Shigella on the small intestine and the role of the host response in the development of the disease. Moreover, to the best of our knowledge no studies have described the effects of apically administered Shigella flexneri 2a and S. dysenteriae 1 vaccine strains on human small intestinal enterocytes. The aim of this study was to assess the coordinated functional and immunological human epithelial responses evoked by strains of Shigella and candidate vaccines on small intestinal enterocytes. To model the interactions of Shigella with the intestinal mucosa, we apically exposed monolayers of human intestinal Caco2 cells to increasing bacterial inocula. We monitored changes in paracellular permeability, examined the organization of tight-junctions and the pro-inflammatory response of epithelial cells. Shigella infection of Caco2 monolayers caused severe mucosal damage, apparent as a drastic increase in paracellular permeability and disruption of tight junctions at the cell-cell boundary. Secretion of pro-inflammatory IL-8 was independent of epithelial barrier dysfunction. Shigella vaccine strains elicited a pro-inflammatory response without affecting the intestinal barrier integrity. Our data show that wild-type Shigella infection causes a severe alteration of the barrier function of a small intestinal cell monolayer (a proxy for mucosa) and might contribute (along with enterotoxins) to the induction of watery diarrhea. Diarrhea may be a mechanism by which the host attempts to eliminate harmful bacteria and transport them from the small to the large intestine where they invade colonocytes inducing a strong inflammatory response.

PMID: 24416363



Shigellosis is an invasive infection of the colon and represents a global human health problem. Infection is transmitted by faecal-oral contact and is highly contagious. As low as 10 ingested organisms are sufficient to cause the disease1 typically characterized by watery diarrhea episodes thought to arise in the jejunum, followed by bloody stools containing mucus and pus, as a consequence of bacterial invasion of the colonic epithelium.

Many studies have described the pathogenesis of Shigella spp, especially in the colon using a variety of cell and animal models and also humans immunized and/or challenged with wild-type strains but to date the effect of Shigella on the small intestine and the distinct role played by the pathogens and/or the host in the onset of the watery diarrhea typical of early stages of shigellosis, is still poorly understood. Therefore, by using a Caco2 cell monolayer model apically exposed to bacteria, we sought to understand the possible role the host plays in the establishment of the disease. Our apically infected polarized Caco2 monolayer system, allowed us to directly evaluate the effect of infection on paracellular permeability, tight-junctions, and inflammatory molecules release. We measured changes in the monolayer transepithelial electric resistance (TEER) and observed that wild-type Shigella infection induces an important decrease of TEER at all infection inocula applied, indicating an increased paracellular permeability that we further confirmed by measuring increased FITC-dextran flux through the cellular monolayer and the disengagement of tight-junction proteins from the cell-cell boundary.

Results from infections with heat killed bacteria and bacteria culture supernatants indicated that the direct interaction of viable bacteria with the host epithelium is necessary to elicit these effects suggesting that virulence effectors are secreted upon contact with host cells. This observation did not apply to S. dysenteriae-1 whose bacteria filtered supernatant alone was sufficient to induce the loss of the monolayer integrity, presumably due to the secretion of the potent Shiga toxin. Monolayers infected with guanine auxotroph attenuated Shigella strains2 (capable of multiplying only in the presence of guanine), lacking their virulence effectors with described enterotoxic activity (i.e. SHigella EnteroToxins-SHET1/2)3,4 on the contrary, were little or not affected except when exposed to the highest bacteria inoculum (107), suggesting a possible, concurrent specific response of the host to infection, aimed to establish a water flow towards the apical side (intestinal luminal side) to both dilute bacterial toxins and flush away the harmful bacteria. To corroborate these findings, we have exposed Caco2 monolayers to the attenuated S. flexneri strain CVD 1208S in the presence of guanine (unpublished, preliminary), to allow for bacterial multiplication following invasion and, as shown in Fig. 1 even at the lowest inoculum (105) the enterotoxin-depleted mutant elicits the same destructive effect of wild-type S. flexneri on the epithelial barrier integrity, further indicating that in the presence of what the host perceives as enemies, although deprived of their weapons, the host fights its battle regardless.

Furthermore, we showed a significant basolateral secretion of IL-8 not only upon infection with live wild-type bacteria, as expected, but also with killed bacteria, bacteria culture supernatants and attenuated strains, demonstrating that the host epithelial inflammatory response is independent of both barrier integrity and Shigella enterotoxins.

Overall our data point to a biphasic model of Shigella infection with an important role played in the small intestine where watery diarrhea is initiated as a mechanism for bacteria to reach their final destination niche in the colon for invasion and multiplication and for the host in the attempt to neutralize toxins and flush away the harmful microorganisms.



1. DuPont HL, Levine MM, Hornick RB & Formal SB (1989) Inoculum size in shigellosis and implications for expected mode of transmission. J Infect Dis 159(6): 1126-1128.

2. Kotloff KL, et al (2004) Deletion in the shigella enterotoxin genes further attenuates shigella flexneri 2a bearing guanine auxotrophy in a phase 1 trial of CVD 1204 and CVD 1208. J Infect Dis 190(10): 1745-1754.

3. Fasano A, et al (1995) Shigella enterotoxin 1: An enterotoxin of shigella flexneri 2a active in rabbit small intestine in vivo and in vitro. J Clin Invest 95(6): 2853-2861.

4. Nataro JP, et al (1995) Identification and cloning of a novel plasmid-encoded enterotoxin of enteroinvasive escherichia coli and shigella strains. Infect Immun 63(12): 4721-4728.



Maria Rosaria Fiorentino, PhD
Mucosal Immunology and Biology Research Center
Massachusetts General Hospital East
Building 114, 16th Street (Mail Stop 114-3503)
Charlestown, MA 02129-4404

Figure_11Figure 1. Trans-epithelial electrical resistance (TEER) responses of Caco-2 cell monolayers to Shigella flexneri 2a attenuated vaccine candidate CVD1208S in the absence (A) or presence (B) of guanine. Wild-type Shigella served as positive control. Bacterial strains were applied at a concentration of 105.

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