Study of bacteriophage encoded glucosyltransferase (gtr) genes in Shigella flexneri serotype 1c

Shigella flexneri is the primary cause of bacillary dysentery or shigellosis in developing countries. S. flexneri serotype 1c is a novel serotype which is emerging as the most prevalent serotype in many developing nations. It has a unique and highly modified O-antigen structure comprising of at least two distinct O-antigen modifying glucosyltransferase gene clusters (gtrI and gtrIc) that adds two-glucosyl groups to the N-acetylglucosamine residue of O-antigen. These genes are encoded by two different bacteriophages; gtrI gene cluster mediates the addition of the first glucosyl group whereas the gtrIc gene cluster mediates the addition of the second glucosyl group resulting in the serotype 1c-specific O-antigen modification. Despite being an important serotype, very little is known about its genomic architecture and virulence signatures. The overall aim of this study was to understand how these O-antigen modifying bacteriophage are arranged in S. flexneri 1c strains using whole genome sequencing approach and to further understand the role of these two gtr gene clusters in Shigella flexneri virulence using various functional assays. The first aim of my PhD involved developing a reference genome of S. flexneri serotype 1c using a clinical isolate from Bangladesh (Y394). We generated a high-quality reference genome of S. flexneri serotype 1c using the hybrid methods of long-read single-molecule real-time (SMRT) sequencing technology and short-read MiSeq (Illumina) sequencing technology. The Y394 chromosome was 4.58 Mb in size and shared the basic genomic features with other S. flexneri complete genomes. However, it possesses a unique and highly modified O-antigen structure comprising of three distinct O-antigen modifying gene clusters (gtrI, gtrIc and oacB) that came from three different bacteriophages. It also possesses a large number of hypothetical unique genes compared to other S. flexneri genomes. The second aim was to gain insight into the diversity and evolution of S. flexneri serotype 1c strains using whole-genome sequences (MiSeq) of 85 different clinical isolates obtained from different geographical locations of the world including Bangladesh, Egypt, Japan, United Kingdom (UK) and Vietnam. We identified a large Shigella flexneri pan-genome with less than 30% of genes in the core genome. The phylogeny based on core genes suggested two distinct lineages of serotype 1c one specific to Bangladesh and the other representing remaining isolates. We also identified a large number of isolates having multidrug resistance genes in them, which is very alarming. Finally, in order to understand the role of gtrI and gtrIc gene clusters in Shigella flexneri serotype 1c virulence, we generated isogenic mutants: gtrI- knockout and gtrIc-knockout using Y394. These mutants were evaluated using different virulence assays including biofilm assay, in vivo accumulation assay using Caenorhabditis elegans and in vitro invasion assay using HeLa cells. All strains showed biofilm formation in the presence of bile salts but the biofilm formation was significantly reduced in two gtr mutants compared to Y394. We were also able to see the differences in the ability of the wild type and mutants to colonize in the intestine of C. elegans. These differences are also consistent with the in vitro invasion assay using HeLa cells. Thus, this study showed that the addition of an extra glucosyl group at the N-acetylglucosamine residue of O-antigen has a significant impact on the virulence of S. flexneri serotype 1c. Thus, these findings will increase our understanding of the genomic features, evolution and virulence signatures of S. flexneri serotype 1c. The knowledge of the entire repertoire of the genes encoded in S. flexneri serotype 1c and their functional genomic studies offers the opportunity to identify new targets that are potential for cross-protective Shigella vaccine