lipid-a and Dysentery--Bacillary

LPS is a potent bacterial effector triggering the activation of the innate immune system following binding with the complex CD14, myeloid differentiation protein 2, and Toll-like receptor 4. The LPS of the enteropathogen Shigella flexneri is a hexa-acylated isoform possessing an optimal inflammatory activity. Symptoms of shigellosis are produced by severe inflammation caused by the invasion process of Shigella in colonic and rectal mucosa. Here we addressed the question of the role played by the Shigella LPS in eliciting a dysregulated inflammatory response of the host. We unveil that (i) Shigella is able to modify the LPS composition, e.g., the lipid A and core domains, during proliferation within epithelial cells; (ii) the LPS of intracellular bacteria (iLPS) and that of bacteria grown in laboratory medium differ in the number of acyl chains in lipid A, with iLPS being the hypoacylated; (iii) the immunopotential of iLPS is dramatically lower than that of bacteria grown in laboratory medium; (iv) both LPS forms mainly signal through the Toll-like receptor 4/myeloid differentiation primary response gene 88 pathway; (v) iLPS down-regulates the inflammasome-mediated release of IL-1β in Shigella-infected macrophages; and (vi) iLPS exhibits a reduced capacity to prime polymorfonuclear cells for an oxidative burst. We propose a working model whereby the two forms of LPS might govern different steps of the invasive process of Shigella. In the first phases, the bacteria, decorated with hypoacylated LPS, are able to lower the immune system surveillance, whereas, in the late phases, shigellae harboring immunopotent LPS are fully recognized by the immune system, which can then successfully resolve the infection.

Topics: Blotting, Western; Cell Line; Dysentery, Bacillary; Enzyme-Linked Immunosorbent Assay; Humans; Immune Evasion; Immunity, Innate; Inflammasomes; Intestinal Mucosa; Lipid A; Lipopolysaccharides; Models, Biological; Shigella flexneri

The structure of lipid A released by mild acid hydrolysis from lipopolysaccharide from two strains of Shigella flexneri with different degrees of acylation was characterized using electrospray ionization (ESI) and ion-trap mass spectrometry. The lipid A was analyzed underivatized with ESI in negative-ion mode. With multiple stages of fragmentation (MS(n)), both the degree of acylation and the positions of the fatty acids on the disaccharide backbone could be determined. It was possible to determine the degree of acylation by the MS(n) technique, where in each MS stage the parent ion was an ion where one fatty acid had been eliminated. One way to determine the location of the fatty acids was by identifying cross-ring fragments of the reducing sugar from parent ions containing different numbers of fatty acids. Another was by identifying a possible charge-driven release of fatty acids situated close to a phosphate group. The fatty acids were otherwise eliminated by a charge-remote fragmentation mechanism. The combined data show the usefulness of ion-trap mass spectrometers for this type of analysis.

Topics: Acylation; Dysentery, Bacillary; Fatty Acids; Lipid A; Lipopolysaccharides; Shigella flexneri; Spectrometry, Mass, Electrospray Ionization; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Shigella flexneri is a Gram-negative pathogen that invades and causes inflammatory destruction of the human colonic epithelium, thus leading to bloody diarrhea and dysentery. A type III secretion system that delivers effector proteins into target eukaryotic cells is largely responsible for cell and tissue invasion. However, the respective role of this invasive phenotype and of lipid A, the endotoxin of the Shigella LPS, in eliciting the inflammatory cascade that leads to rupture and destruction of the epithelial barrier, was unknown. We investigated whether genetic detoxification of lipid A would cause significant alteration in pathogenicity. We showed that S. flexneri has two functional msbB genes, one carried by the chromosome (msbB1) and the other by the virulence plasmid (msbB2), the products of which act in complement to produce full acyl-oxy-acylation of the myristate at the 3' position of the lipid A glucosamine disaccharide. A mutant in which both the msbB1 and msbB2 genes have been inactivated was impaired in its capacity to cause TNF-alpha production by human monocytes and to cause rupture and inflammatory destruction of the epithelial barrier in the rabbit ligated intestinal loop model of shigellosis, indicating that lipid A plays a significant role in aggravating inflammation that eventually destroys the intestinal barrier. In addition, neutralization of TNF-alpha during invasion by the wild-type strain strongly impaired its ability to cause rupture and inflammatory destruction of the epithelial lining, thus indicating that TNF-alpha is a major effector of epithelial destruction by Shigella.

Topics: Acylation; Acyltransferases; Animals; Bacterial Proteins; Caco-2 Cells; Disease Models, Animal; Dysentery, Bacillary; Escherichia coli Proteins; Genes, Bacterial; Guinea Pigs; Humans; Intestinal Mucosa; Ligation; Lipid A; Monocytes; Mutagenesis; Rabbits; Shigella flexneri; Tumor Necrosis Factor-alpha; Virulence

The isolation and properties of endotoxin protein, or lipid A-associated protein (LAP), from Shigella sonnei were described earlier (Zh. mikrobiol. epidemiol. immunobiol., 1991, No. 4, pp. 11-17, and No. 7). In this report the data on its protective activity are presented. In experiments on mice one nanogram of LAP injected i. v. protected 50% of the animals against i. p. challenge with 40 LD50 of virulent S. sonnei. Guinea pigs injected s. c. with 10 micrograms of LAP were protected against local (keratoconjunctival) challenge with S. sonnei, the efficiency of immunization being 58%. LAP preparations containing no detectable amounts of O-antigen (less than 0.003%) were found to have a protective effect. Hyperimmune anti-LAP rabbit serum prevented local infection when incubated with S. sonnei challenge inoculum before injection into guinea pigs. Both active and passive protection induced by LAP was specific since no effect was observed in animals challenged with Shigella flexneri. In the homologous system the protective effect of anti-LAP serum was abolished by the addition of protein-free LPS. These results are compatible with the hypothesis that the protective activity of LAP depends on the presence of minute amounts of O-antigen whose immunogenic effect is greatly amplified by the protein component of the natural endotoxin complex.

Topics: Animals; Bacterial Proteins; Drug Evaluation, Preclinical; Dysentery, Bacillary; Endotoxins; Guinea Pigs; Immunization, Passive; Keratoconjunctivitis; Lipid A; Mice; Mice, Inbred C57BL; Mice, Inbred CBA; Shigella flexneri; Shigella sonnei; Vaccination