(Synopsis) Structural Insight into a Biofilm Signaling Molecule

Liza Gross

Citation: Gross L (2008) Structural Insight into a Biofilm Signaling
Molecule.  PLoS Biol 6(3): e89 doi:10.1371/journal.pbio.0060089

Published: March 25, 2008

Life as a bacterium presents special challenges--primary among them the
need to sense and respond to the environment--which for an organism
just 3 micrometers long, like Pseudomonas aeruginosa, might seem a
daunting prospect. One way Pseudomonas and other bacteria cope with
the rigors of existence is by forming highly organized communities
called biofilms. As members of a biofilm, microbes gain access to
nutrients, genetic traits, and metabolic processes that are
unavailable to them as individuals. They also find protection from the
elements in the sticky extracellular matrix that holds the cells
together.

Unfortunately, when biofilms underlie chronic infections, as P.
aeruginosa does in the lungs of patients with cystic fibrosis, the
matrix also protects its bacterial inhabitants from host immune
defenses and antibiotic therapies. In a new study, Holger Sondermann
and his colleagues reveal a novel mode of regulation in bacterial
biofilm colonization by solving the structure of an enzyme bound to a
"second messenger" that triggers the cell responses necessary for
biofilm formation.

In bacteria, increased levels of a small signaling molecule with the
ungainly name of bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-
di-GMP) induce cells to abandon the single life in favor of
multicellular, "social" living as a biofilm colony. Proteins that make
c-di-GMP--a so-called second messenger that triggers cellular responses
by accumulating in the cell--from GTP have a highly conserved module
containing the amino acid motif GGDEF and are called diguanylate
cyclases. Nabanita De et al. first solved the structure of the
diguanylate cyclase WspR, a potent regulator of biofilm formation,
bound to c-di-GMP and then used a series of biochemical experiments to
shed light on the mechanism that WspR uses to regulate its own
activity along with the production of the second messenger.

Bacteria sense and respond to signals in their environment, including
cues from other bacteria, to form biofilms through "two-component
signaling" systems. The first component, a membrane-bound sensor,
receives the signal and transmits it to the second component, the
response regulator protein, which triggers a cellular response, such
as gene expression. These messages are transferred via a chemical
reaction called phosphorylation, which involve the transfer of
phosphates between the proteins.

Response regulators can also change cell behavior by controlling the
second messenger c-di-GMP. Low concentrations of c-di-GMP are found in
motile cells, while high c-di-GMP concentrations lead to the
production of extracellular and adhesive matrix components,
multicellular behavior, and aggregation into a biofilm. In addition to
the most common regulation through phosphorylation, this new study
shows that the response regulator WspR exists in at least three
different states that determine its activity: an active and an
inactive state, bridged by an intermediate form.

Previous studies had identified another response regulator, PleD, that
binds to c-di-GMP and controls the switch from a free-swimming to
stationary lifestyle in marine bacteria. PleD has a similar domain
organization to WspR, containing a diguanylate cyclase module and a
response regulator domain that is regulated by phosphorylation. PleD
is also regulated by c-di-GMP, the product of the enzymatic reaction.
c-di-GMP binds to regions at the diguanylate cyclase and regulatory
domains to inhibit its activity. While both domains occur in WspR,
which formed a four-unit complex (or tetramer) in the solved
structure, they were in a different configuration, leading the
researchers to suspect that WspR might be regulated through a
different mechanism.

To understand what this mechanism might be, the researchers subjected
the enzyme to a number of biochemical analyses. They show that WspR
exists in different configurations depending on its c-di-GMP binding
state. Both nucleotide-tethered and nucleotide-free WspR existed as
bound pairs (called dimers), though with different conformations: the
nucleotide-bound enzyme appeared elongated, while the free species
appeared more compact. The tetramer state emerged spontaneously over
time from the compact dimer.

By analyzing the catalytic capacities of the enzyme's different
states, the researchers demonstrate that the compact configuration is
most active while the elongated c-di-GMP-bound state shows the least
activity. They go on to propose a model through which WspR modulates
its own activity, with a feedback mechanism in which the tetramer
assembles from the active compact species and provides a platform for
disbanding into the inactive elongated species, which is inhibited by
the bound c-di-GMP. The removal of c-di-GMP (by phosphodiesterases
that degrade it) leads to WspR reactivation as the enzyme switches to
the compact state.

By elucidating a novel regulatory mechanism for an enzyme that
controls the synthesis and degradation of a key player in biofilm
formation, these findings suggest new approaches to controlling the
behavior of bacteria that are responsible for chronic infections.
Given that bacteria commonly found in cystic fibrosis patients with
lung infections carry a WspR mutation in a critical site for c-di-GMP
binding, the researchers suspect that this mutation might underlie the
virulence of these pathogenic strains. Future studies can test this
possibility. Since c-di-GMP signaling is found only in bacteria and is
unknown in eukaryotic organisms like humans, the prospect of
developing therapies aimed at disrupting this second messenger to
fight biofilm-mediated infections appears particularly promising.
Tetrameric assembly of the response regulator diguanylate cyclase WspR
from Pseudomonas aeruginosa. The inset shows a close-up of cyclic di-
GMP bound to the inhibitory site.

  1. De N, Pirruccello M, Krasteva PV, Bae N, Raghavan RV, et al.
(2008) Phosphorylation-independent regulation of the diguanylate
cyclase WspR. doi:10.1371/journal.pbio.0060067.

Full text etc. available via:-
http://biology.plosjournals.org/perlserv/?request=get-document&doi=10.1371/journ
al.pbio.0060089

On 3/25/08 5:22 PM, in article
af007ac3-ae97-4de9-8e35-16325d6c054a@e60g2000hsh.googlegroups.com, "Michael"
<mfrpersonal@gmail.com> wrote:

Enzymes administered systemically  are known to thin mucus. Perhaps the are
also loosening up biofilm too? Inhallation of Dornase for CF is therapeutic
for example. .
Systemic enzymes also increase the effectiveness of antibiotics by opening
channels for better penetration.