by Alaina McGinley
What is a biofilm dependent on?
The
infection is often without symptoms, but can be severe with an estimated 3-5
million cases and over 100,000 deaths each year around the world (Center For
Disease Control). Vibrio cholera spends most of its time outside of its
host in mature biofilms (a thin slimy layer of bacteria). After a person
ingests water contaminated with Vibrio cholera (because of inadequate
treatment of sewage and drinking water), the virulent pilus adheres to the
intestinal lining to secrete the cholera toxin, which releases V. cholera
through severe diarrhea. Since biofilm formation is a large part of V.
cholera’s life
cycle, the mechanisms and signals that trigger biofilm assembly are important
to investigate. Mature biofilms help the pathogen persist in stressful environments,
but the biofilm is dependent on the production of an exopolysaccharide (EPS).
There are multiple signaling pathways to produce EPS in V. cholera,
including a flagellum-dependent pathway, a phase variation pathway, and a
quorum sensing (QS) pathway (Heithoff and Mahan 2004).
The QS pathway is known to be
regulated by the transcriptional factor HapR. Biofilm formation, vps (Vibrio
polysaccharide) genes, and virulence formation are repressed when HapR is
produced (Heithoff and Mahan 2004). HapR represses transcription of the aphA
gene, which encodes an activator of the virulent pilus. HapR also controls
transcription of genes which code for proteins that manage c-di-GMP. The
intracellular second messenger called c-di-GMP contains information about the
environmental conditions, and in V. cholera, it also activates biofilm
formation. At high cell density, c-di-GMP levels are reduced, and consequently
biofilm formation stops (Zhao et al. 2013).
The QS pathway uses small RNAs with
a RNA chaperone (Hfq) to stop the activation of HapR (Figure
1A), and therefore control biofilm formation. Use of QS sRNAs at low cell
density correlates with high levels of c-di-GMP, and thus high levels of
biofilm. Since QS sRNAs repress HapR, it was previously thought that the result
was solely because of the base-pairing (complementary structures in nucleic
acid) between the sRNA and HapR (Zhao et al. 2013).
Figure 1.
The vca0939 gene
The QS
sRNAs alter translation of mRNA besides hapR mRNA, such as mRNA of the gene vca0939.
This gene synthesizes c-di-GMP, but it had not been experimentally
documented to participate
in the QS pathway before the 2013 paper:
Post-transcriptional activation of a diguanylate cyclase by quorum sensing
small RNAs promotes biofilm formation in Vibrio cholerae by Zhao, Koestler,
Waters, and Hammer. They wanted to look into other ways in which QS sRNAs
control biofilm formation, and so the purpose of their study was to define the
regulation of vca0939 expression in V. cholera. They hoped to
show that QS sRNAs positively regulate Vca0939, and that QS sRNAs directly bind
to vca0939. These results would be important because it would
demonstrate for the first time the post-transcriptional activation of Vca0939 by
direct pairing with a QS sRNA (Zhao et al. 2013).
Figure 2.
The Results: sRNAs are in, and HapR
is out
They
used a tagged version of vca0939 on a plasmid to test whether
QS-dependent activation of Vca0939 was enough for biofilm formation. They found
that QS sRNAs also basepair with vca0939 to activate translation of its
protein. Mutation of a nucleotide in the QS sRNA resulted in no binding, and
therefore prevented translation of the protein. The QS sRNA dependent
activation led to c-di-GMP accumulation and biofilm formation. They confirmed
their original research question, that vca0939 is a gene that is
positively regulated by the QS sRNAs through base-pairing, which frees the
ribosome binding site. They also found that Vca0939 is a diguanylate cyclase
(DGC), which promotes early biofilm formation in V. cholera (Figure 2). Most importantly, they found that this base-pairing
repressed translation of HapR. The same QS sRNAs are paired with and regulate
Vca0903 and HapR to control biofilm formation, except HapR is repressed while
the other is not (Zhao et al. 2013).
This result is significant because
Vca0939 controlled biofilm development is the first QS-mediated process in V. cholera
without HapR. Vca0939 is also the only other protein of its kind (the other is
in E. coli) shown to be post-transcriptionally regulated because of direct
pairing with a QS sRNA (Zhao et al. 2013).
A future study is to determine the
remaining genes under QS sRNA control in V.
cholera. Their results exactly agreed with
previous assumptions; the paper could have been more full and intriguing if
they had included something different, such as more analyzation of the vca0939
structure to look closer at the activation by the QS sRNAS (Zhao et al. 2013).
Nevertheless, the results are a step further into understanding the persistence
of V. cholera - it just doesn’t want to play by the rules.
Cited Sources:
Heithoff D., and M. Mahan. 2004.
Vibrio cholerae biofilms: stuck between a rock and a hard place. Journal of Bacteriology.
186:4835-4837.
Zhao X., B. Koestler, C. Waters, B.
Hammer. 2013. Post -transcriptional activation of a diguanylate cylase by quorum sensing small RNAs
promotes biofilm formation in Vibrio cholerae. Molecular Microbiology. 89:989-1002.
First Image found at: https://microbelog.wordpress.com/2012/09/06/getting-out-of-a-sticky-situation/ from Pacifi Northwest National Laboratory
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