Identification of Host Receptors for Clostridium difficile Bacteriophage
Abstract:
Clostridium difficile is responsible for a range of gastrointestinal diseases commonly known as C. difficile infection (CDI). The emergence of antibiotic resistance has triggered the quest for alternative treatments for CDI. Phages could provide an alternative source of antimicrobials for this pathogen due to their specificity and ability to amplify at the site of infection. The ability of phages to specifically adsorb to receptors on the bacterial surface is essential to the infection process and underpins its specificity and selection for therapeutic purposes. Here, we aim to identify C. difficile phage receptors for phiCDHS1 (siphovirus) and phiCDHM1 (myovirus), which lytically infect different strains. Analysis of morphologically distinct phages would enable us target potentially different receptors for C. difficile phages. Two main approaches were employed to identify the phages receptors. Firstly, the chemical composition of the receptors was ascertained by digesting the C. difficile surface proteins with proteinase K. Subsequently, the treated cells were incubated with the phages and adsorption was monitored over a time course. Secondly, we over-expressed the predicted phage tail fiber proteins which are presumably involved in phage host binding. The expressed proteins were chosen as targets to identify the corresponding receptors on the C. difficile surface. After several optimisations, we observed that the phages continued to bind to the bacterial hosts despite treatment with proteinase K. This strongly suggests that the receptors are carbohydrate in nature. Work is currently ongoing to identify the phage tail fiber proteins potentially involved in phage bacterial attachment by tagging the proteins with Green Fluorescent proteins and monitoring the proteins binding through binding assays on C. difficile. These findings will lead to greater understanding of bacteriophage biology, the mechanisms behind the predator-prey interaction between the phages and their hosts and their potential application in formulation of cocktails for CDI.
Clostridium difficile is responsible for a range of gastrointestinal diseases commonly known as C. difficile infection (CDI). The emergence of antibiotic resistance has triggered the quest for alternative treatments for CDI. Phages could provide an alternative source of antimicrobials for this pathogen due to their specificity and ability to amplify at the site of infection. The ability of phages to specifically adsorb to receptors on the bacterial surface is essential to the infection process and underpins its specificity and selection for therapeutic purposes. Here, we aim to identify C. difficile phage receptors for phiCDHS1 (siphovirus) and phiCDHM1 (myovirus), which lytically infect different strains. Analysis of morphologically distinct phages would enable us target potentially different receptors for C. difficile phages. Two main approaches were employed to identify the phages receptors. Firstly, the chemical composition of the receptors was ascertained by digesting the C. difficile surface proteins with proteinase K. Subsequently, the treated cells were incubated with the phages and adsorption was monitored over a time course. Secondly, we over-expressed the predicted phage tail fiber proteins which are presumably involved in phage host binding. The expressed proteins were chosen as targets to identify the corresponding receptors on the C. difficile surface. After several optimisations, we observed that the phages continued to bind to the bacterial hosts despite treatment with proteinase K. This strongly suggests that the receptors are carbohydrate in nature. Work is currently ongoing to identify the phage tail fiber proteins potentially involved in phage bacterial attachment by tagging the proteins with Green Fluorescent proteins and monitoring the proteins binding through binding assays on C. difficile. These findings will lead to greater understanding of bacteriophage biology, the mechanisms behind the predator-prey interaction between the phages and their hosts and their potential application in formulation of cocktails for CDI.
Reference:
Poster Day 3-T08-Pos-38
Session:
Posters: Virus host cell interactions, Structure/Function, Viral control of the host
Presenters:
Ahmed Dowah
Session:
Day 3 Posters Covering: Virus host cell interactions, Structure/Function, Viral control of the host
Presentation type:
Poster presentation
Room:
Poster Halls
Date:
Wednesday, 20 July 2016
Time:
12:05 - 15:30