AbstractClostridioides difficile is an opportunistic, anaerobic, spore-forming bacterium which causes healthcare-associated infection, following the use of broad-spectrum antibiotics. Approximately 30% of cases of C. difficile infection (CDI) are recurrent, with the rate of recurrence increasing with each repeated infection. The cause of CDI recurrence has been attributed to biofilm formation and sporulation in the gut.
Previous work found that mild, clinically relevant heat stress of 41°C increased expression of stress responsive genes in C. difficile 630 including the dnaK gene encoding the molecular chaperone protein, DnaK. Subsequent ClosTron mutagenesis of the dnaK gene in C. difficile 630∆erm resulted in a mutant strain 630∆erm:dnaK which presented with approximately 50% cell elongation, increased cell hydrophobicity and significantly increased biofilm biomass (p <0.05) when compared to the parent strain 630∆erm. Considerable transcriptional differences were also reported between the dnaK mutant strain and parent 630Δerm strain when grown planktonically at 37°C.
These preliminary observations led to the research question of what changes are seen transcriptionally within biofilms of C. difficile 630∆erm:dnaKcompared to the parent strain? To achieve this overall objective, an appropriate biofilm model for transcriptional analysis was selected by further characterisation of biofilm formation in C. difficile 630, 630Δerm and 630Δerm:dnaK.
Identification of colony biofilms as a suitable model for all strains led to stringent development of an RNA extraction procedure from a Qiagen RNeasy PowerBiofilm kit. Analysis of gene expression in C. difficile colony biofilms compared to planktonic cells by RNA sequencing revealed extensive transcriptional changes, some linked to the global master regulator codY with further effects on the sin locus, and sporulation master regulator Spo0A in C. difficile. Increased nutrient starvation in 630Δerm:dnaK colony biofilms was linked to increased matrix production by limiting nutrient diffusion and further investigation into the DnaK protein as a moonlighting protein in C. difficile is proposed.
|Date of Award||Oct 2023|
|Sponsors||Department for the Economy|
|Supervisor||James Dooley (Supervisor), Patrick Naughton (Supervisor), Nigel Ternan (Supervisor), William Snelling (Supervisor) & Roberto La Ragione (Supervisor)|
- Colony biofilm