Abstract
Introduction.
Clostridioides difficile
is an opportunistic enteric pathogen posing significant challenges in human and animal healthcare due to the recurrent nature of infections and associated financial burden. The molecular chaperone protein, DnaK (Hsp70), encoded by
dnaK
, plays a central role in the heat stress response and protein homeostasis. Disruption of
dnaK
in
C. difficile
630Δ
erm
has previously been associated with increased biofilm formation and multiple phenotypic alterations.
Gap Statement.
Although disruption of
dnaK
has been shown to alter biofilm formation and multiple phenotypes in
C. difficile
, the global transcriptional mechanisms linking DnaK to biofilm development and stress adaptation remain poorly understood.
Aim.
This study aimed to characterize the global transcriptional response associated with
dnaK
disruption during planktonic and biofilm growth to identify pathways linking the DnaK-mediated stress response with biofilm formation.
Methods.
This study investigated the global transcriptional changes associated with
dnaK
disruption during planktonic and colony biofilm culture. Colony biofilms of
C. difficile
strains 630, 630Δ
erm
and 630Δ
erm:dnaK
were cultured on mixed nitrocellulose ester semi-permeable membranes and examined structurally using low-vacuum scanning electron microscopy. Total RNA was extracted from both planktonic cultures and 24 h colony biofilms, followed by Illumina RNA sequencing. Differential gene expression analysis was performed using DESeq (
P
adj
Results.
Disruption of
dnaK
resulted in global transcriptomic remodelling, particularly during biofilm culture, where 72.4% of genes were significantly differentially expressed relative to planktonic culture. A core number of pathways were involved in biofilm formation in all strains, including increased sporulation with reduced translation, amino acid biosynthesis and flagellar assembly. The
dnaK
mutant exhibited a unique pattern of enrichment of pathways associated with cell division, peptidoglycan synthesis, carbon metabolism and amino acid biosynthesis, alongside a coordinated reduction of motility and chemotaxis pathways.
Conclusion.
Disruption of the
dnaK
gene appears to link the stress response to biofilm formation through a number of pathways associated with biofilm development. Understanding this link may identify systems relevant to persistent and recurrent infections.