Transcriptomic analysis of colony biofilm formation by a dnaK deficient C. difficile 630 mutant

The opportunistic enteric pathogen Clostridioides difficile causes significant healthcare problems due to the recurrent nature of infections attributed to the ability of C. difficile to form biofilms and spores within the gut. Disruption of the key chaperone gene, dnaK, by ClosTron mutagenesis yielded a DnaK deficient strain (630Δerm:dnaK) which exhibited 50 % greater cell elongation, increased cell hydrophobicity, and increased biofilm production ).
We used a colony biofilm model to investigate gene expression in 24h C. difficile biofilms and in planktonically grown cells comparing mutant strain (630Δerm:dnaK), parent strain (630Δerm) and wild-type (630). RNA sequencing revealed major transcriptional differences caused by disruption of the dnaK gene in 630Δerm:dnaK. Gene set enrichment analysis identified increased expression of peptidoglycan biosynthesis in 630Δerm:dnaK biofilm formation that was not observed in the other strains. However, all strains showed a similar increased expression of the sporulation pathway and a decrease in flagellar assembly when comparing 24h biofilms to planktonically cultured cells. Other indications of cell wall restructuring in 630Δerm:dnaK included over representation of vancomycin resistance genes and glycerophospholipid metabolism KEGG pathways. Further biological processes such as cell division, cell wall organization, regulation of cell shape and peptidoglycan biosynthetic process were also overrepresented.
The novel transcriptional changes in cell wall related processes and pathways may explain phenotypical changes in cell morphology including cell elongation, increased hydrophobicity and recalcitrance to antibiotics previously reported by our group.