The anaerobic gut pathogen,
Clostridioides difficile
, forms adherent biofilms that may play an important role in recurrent
C
.
difficile
infections. The mechanisms underlying
C
.
difficile
community formation and inter-bacterial interactions are nevertheless poorly understood.
C
.
difficile
produces AI-2, a quorum sensing molecule that modulates biofilm formation across many bacterial species. We found that a strain defective in LuxS, the enzyme that mediates AI-2 production, is defective in biofilm development
in vitro
. Transcriptomic analyses of biofilms formed by wild type (WT) and
luxS
mutant (
luxS
) strains revealed a downregulation of prophage loci in the
luxS
mutant biofilms compared to the WT. Detection of phages and eDNA within biofilms may suggest that DNA release by phage-mediated cell lysis contributes to
C
.
difficile
biofilm formation. In order to understand if LuxS mediates
C
.
difficile
crosstalk with other gut species,
C
.
difficile
interactions with a common gut bacterium,
Bacteroides fragilis
, were studied. We demonstrate that
C
.
difficile
growth is significantly reduced when co-cultured with
B
.
fragilis
in mixed biofilms. Interestingly, the absence of
C
.
difficile
LuxS alleviates the
B
.
fragilis
-mediated growth inhibition. Dual species RNA-sequencing analyses from single and mixed biofilms revealed differential modulation of distinct metabolic pathways for
C
.
difficile
WT,
luxS
and
B
.
fragilis
upon co-culture, indicating that AI-2 may be involved in induction of selective metabolic responses in
B
.
fragilis
. Overall, our data suggest that
C
.
difficile
LuxS/AI-2 utilises different mechanisms to mediate formation of single and mixed species communities.
The anaerobic pathogen Clostridioides difficile, which is a primary cause of antibiotic-associated diarrhoea, faces a variety of stresses in the environment and in the mammalian gut. To cope with environmental stresses, it uses the alternative sigma factor B (σB) to modulate gene transcription, which is regulated by an anti-sigma factor, RsbW. To understand the role of RsbW in C. difficile physiology, a rsbW mutant (ΔrsbW) where σB is always on, was generated. ΔrsbW did not have deleterious fitness defects but tolerated acidic environments and detoxified reactive oxygen and nitrogen species better. ΔrsbW was defective in spore and biofilm formation, adhered better to human gut epithelia and was less virulent in a Galleria mellonella infection model. A transcriptomic analysis to understand this unique phenotype showed a change in expression of some σB-controlled genes along with several non-σB controlled genes. Interestingly, the sinRR locus that encodes a pleiotropic regulator, was highly upregulated in ΔrsbW indicating a potential indirect role for σB or RsbW in control of sinRR. Furthermore, the unexpected lower intracellular levels of σB observed suggest post translational control mechanisms. Our study thus provides new insight into the regulatory role of RsbW and the complexity of regulatory networks in C. difficile.
Pathogens like
Clostridioides difficile
face a range of stresses in the environment and within the host. Alternative transcriptional factors like sigma factor B (σ
B
) enable the bacterium to respond quickly to different stresses.
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