Molecular characterization of novel serotype 1c of Shigella flexneri

Abstract

Shigellosis, or bacillary dysentery, is a major disease burden worldwide. It is responsible for 165 million cases annually resulting in approximately 1.1 million deaths. Vaccine development remains a high priority as there are currently no licensed vaccines available. The emergence of several novel serotypes over the last two decades, and the variety of prevalent serotypes in different geographic locations, require the development of a S. flexneri vaccine to target each of these novel and prevalent serotypes. In order to develop a multivalent vaccine which is effective against all S. flexneri serotypes, an understanding of the molecular underpinnings of the serotypes is essential. The novel serotype 1c identified in late 1980s has not been characterised with regard to its origin or virulence properties. Thus this study aims to characterize the serotype 1c at molecular level by (i) examining the genetic organization of the gtrIC cluster and surrounding sequences, (ii) characterizing the cluster for functionality of gtrAIc and gtrBIc and their contribution in serotype conversion of serotype 1c, and (iii) analysing the role of the Type Ic modification in Shigella virulence. Key results: The gtrIC nucleotide sequence is highly conserved among wild type serotype 1c strains. Prophage intergrase and tRNAPro (should it be in italics?) genes upstream of gtrIC cluster revealed that of the 20 kb sequence surrounding the gtrIC cluster is reminiscent of a prophage. Therefore the gtrIC gene cluster most likely entered serotype 1c via the bacteriophage SfIc. It seems the integrated prophage sequence was later rearranged by mobile elements resulting in more than half of the phage genome being deleted. Southern blot assays performed in all serotype 1c isolates collected from several geographical locations revealed that the organization of gtrIC gene cluster is universal in all S. flexneri serotype 1c strains, indicating they all evolved from a single parental strain. Southern blot assays on serotype 1a and 1b strains indicated that all serotype 1c strains originated from a single parental serotype 1a strain. The gtrAIc and gtrBIc from gtrIC cluster were found to be functional while they are redundant because of the presence of gtrI cluster in serotype 1c genome. However, they do not appear to increase the efficiency of Type Ic modification. Interestingly, the serotype 1c wild type strains also showed an incomplete modification which was an unexpected finding. The plaque assays results demonstrated a significant difference in the virulence of the wild type 1c strain (SFL1613) and its isogenic gtrIC-KO strain (SFL2349), indicating the Type Ic modification contributes to Shigella virulence. The change in the size of the plaques formed also indicated that Type Ic modification positively affect the ability of S. flexneri to spread inter and intracellularly. Conclusions and Implications: The findings of this study open up new avenues for future research which could potentially increase knowledge of how serotype conversion occurs and could potentially be utilised in multivalent vaccine development. Show more