Shigella flexneri is an entero-pathogen that is considered a significant public health risk, causing shigellosis or bacillary dysentery, and accounts for the highest percentage of diarrheal deaths annually. Absence of a licensed global vaccine against Shigella, compels us to enormously depend on antibiotics as the major treatment option and with the surge of antimicrobial resistant Shigella strains it makes treatment strategies even more limited. The recent advent of large-scale whole-genome sequencing projects have identified numerous uncharacterized protein-encoding genes which constitute about two-thirds of the genome. These are annotated as hypothetical proteins (HP), known to be conserved in their expression but are poorly understood in terms of their presence and functional role in the bacteria. It’s believed that these proteins might be involved in essential functions like the growth, survival, and virulence of the pathogen and are hiding crucial information which could be utilized in combatting bacterial infections. Functional studies of HPs can provide an understanding of its functions, associated pathways, identification of new structures which can act as targets for drug and vaccine studies, and eventually improve our knowledge about the pathogen. The overall aim of this study was to examine the HPs present in the S. flexneri 1c strain, to carry out functional elucidation of these proteins in terms of their involvement in host-pathogen interactions, growth, intracellular survival, and virulence of Shigella.

An in-silico functional characterization of the HP’s present in the genome of S. flexneri serotype 1c, utilizing various bioinformatics tools and protein databases was performed. This contributed to a deeper understanding of the functional involvement of 721 identified HPs and assisted in the priority selection of gene targets for additional in-vitro and in-vivo assays to experimentally validate these predicted functions. The selected gene targets were deleted from the genome and the knockout (KO) mutants along with the wild type 1c strain were subjected to various virulence assays evaluating the phenotypic differences if any between them. Further in-depth functional elucidation of 2 HPs was also carried out in this study: 1) YnfA: an inner membrane multidrug transporter protein, belonging to the small multidrug resistance family and its functional role in promoting antimicrobial resistance in S. flexneri and 2) YfiB: an outer membrane OmpA family protein, part of the c-di-GMP regulating YfiBNR system and its involvement in the biofilm formation, host cell adhesion and invasion and the overall virulence of S. flexneri. The outcomes of this study have broadened our understanding of the functional roles which the hypothetical proteins play in S. flexneri serotype 1c. Additionally identifying and characterizing two HPs that are involved in pathogenicity and antimicrobial resistance mechanisms of Shigella. This study can further aid in finding novel targets for improved drugs and developing diverse vaccine strategies, thereby assisting in the treatment and prevention of shigellosis.