Caboni, Mariaelena (2013) Identification and characterization of a capsule in Shigella and its role in the pathogenesis. [Tesi di dottorato]

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Abstract

Shigella spp. are the etiologic agent of bacillary dysentery and one of the most frequent causes of diarrhea in developing countries, especially in children under five years of age. 50 different serotypes have been defined by the O antigen of the LPS. A key objective of the Novartis Vaccines Institute for Global Health is to develop a broadly-protective vaccine against Shigella based on outer membrane particles. Inactivation of OAg synthesis is of interest to avoid serotype-specific responses. Deletion of the gene galU results in OAg-deficiency in S. flexneri. Surprisingly, we found that immunization with Delta_galU S. sonnei 53G outer membrane particles or inactivated bacteria raised an OAg-specific response in mice. The presence of OAg on the surface of Delta_galU S. sonnei was confirmed by flow cytometry. GalU is involved in the synthesis of the LPS core but not in the synthesis of the OAg repeating units. A group 4 capsule (G4C) consisting of the same repeating units as the OAg side chain of LPS has been described in enteropathogenic E. coli. Shigella is reported to posses a homologous G4C operon. Size-Exclusion-HPLC analyses of exopolysaccharide extracts showed that S. sonnei WT and Delta_galU strains both expressed a polysaccharide with higher molecular weight than the predominant wild-type OAg chain of 20-25 units. NMR analysis identified the presence of the S. sonnei OAg in the high molecular weight polysaccharide (HMW-PS). The absence of this HMW-PS was confirmed by generating isogenic mutants in the G4C operon. Together, our results indicate for the first time that the G4C gene cluster is functional in S. sonnei and encodes the synthesis of a group 4 capsule and that this capsule is still present in the S. sonnei Delta_galU mutant. The function of the G4C as potential virulence factor of S. sonnei was investigated both in vitro and in vivo. Our results showed that the presence of the capsule polysaccharide accounted for changes in cell invasion ability both in vitro and in vivo. Moreover, the G4C modulated the inflammatory host responses during the infection and the abilities of bacteria to translocate and disseminate. In conclusion, this study describes a new and interesting finding in the Shigella field to achieve a better understanding of the pathogenic mechanism of Shigella and in addition of the function of extracellular polysaccharides as virulence factors. Capsular polysaccharides are important factors in bacterial pathogenesis and have been the target of a number of successful vaccines. Therefore, the identification of a capsule in Shigella could have implications on the design of new-generation anti-Shigella drugs and also on the development of preventive measures, as a vaccine against this enteric disease.

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