Debaffling Biofilms

CAUGHT ON FILM:Courtesy of David Davies and Peg DyrckxPseudomonas aeruginosa biofilm development occurs in five stages. 1. Reversible attachment: Cells transiently affix to substratum, and surface induced gene expression results in a protein profile significantly different from planktonic bacteria. 2. Irreversible attachment: Cells reorient themselves, clusters develop, motility is lost, and the las quorum sensing regulon becomes activated. 3. Maturation I: Cell clusters become thicker than 10 &

Nicole Johnston
Aug 1, 2004
<p>CAUGHT ON FILM:</p>

Courtesy of David Davies and Peg Dyrckx

Pseudomonas aeruginosa biofilm development occurs in five stages. 1. Reversible attachment: Cells transiently affix to substratum, and surface induced gene expression results in a protein profile significantly different from planktonic bacteria. 2. Irreversible attachment: Cells reorient themselves, clusters develop, motility is lost, and the las quorum sensing regulon becomes activated. 3. Maturation I: Cell clusters become thicker than 10 υm and the rhl quorum sensing system becomes active. 4. Maturation II: Cell clusters reach maximum thickness (100 υm) with a protein profile most different from planktonic cells. 5. Dispersion: Cluster structures change, and pores and channels form. Motile and non-motile bacteria are present as the protein profile begins to resemble planktonic cells once again.

In nature, bacteria exist in highly-coordinated structures known as biofilms. These densely-packed microbial communities develop when free-swimming (planktonic) cells attach to a surface and form mushroom-like...

CAUGHT CHANGING

While scientists have long been studying either mature biofilm forms or free-floating planktonic forms, what has been missing is everything that occurs in between these extreme physiological states, says Karin Sauer, a microbiologist at Binghamton University. To address this, she and colleagues decided to look at different changes within the P. aeruginosa biofilm and correlate microscopic observations with protein expression and subsequent protein analysis. "Nobody had done a really comprehensive study before. We've completed the full cycle, right up to when they disperse and convert back to planktonic cells," says Sauer.

Using microscopy and gene analysis, combined with protein-expression analysis to correlate observable changes with protein expression, they followed developmental changes while they occurred. The radical changes they found took them completely by surprise. They discovered five distinct stages in the biofilm process, during which more than 800 proteins (more than 50% of the proteome) showed a sixfold or greater expression level change. "We thought 'Oh my god, we have contaminated it,"' recalls Sauer. "So we repeated it three or four times."

"The protein profile from mature biofilm, when compared to planktonic cells, was as different as comparing two different strains [of Pseudomonas]," she says. Upregulated proteins included those involved in anaerobic processes, denitrification, many efflux pumps, and some quorum sensing proteins. One major player they found upregulated was a known transcriptional regulator that turns on antibiotic resistance by inducing transcription of efflux pumps. An Achille's heel of sorts, mutant versions render the biofilm completely susceptible to antibiotics, says Sauer.

"It was surprising to us," says Davies. "The magnitude of differences from one stage of development to the next was unexpected. We've established that there is a difference [between biofilm and planktonic forms] and that the difference is quite profound." Furthermore, the repercussions of these differences may be far-reaching, both in terms of what we know about bacteria and how we develop therapies and strategies to manage them. "If all management strategies are based on free-floating [bacterial physiology] then clearly we have to go back and reexamine their behavior."

"It's a nice paper because they really follow through the process in a sequential way," says Surette. "It's the most thorough analysis at a proteomic level for P. aeruginosa biofilms that I know of." Matthew Parsek, a microbiologist at the University of Iowa in Iowa City says, "Instead of just looking at single stages in the process, they looked experimentally at processes associated with different steps in the developmental cycle. This is where this paper stands out and is unique." And due to the connection with chronic infections in patients with cystic fibrosis, says Parsek. "there's a strong medical relevance to [this] work."

For scientists trying to understand cell-cell signaling within biofilms, however, the structure of AI-2 had been elusive. It's a feat that scientists originally thought would be relatively easy but proved to be anything but, owing to the notorious instability of AI-2 and its tendency to spontaneously morph into a different rearrangement.

A SLIPPERY MOLECULE

Unlike all other known autoinducers, which are species-specific, AI-2 can carry out communication between different species, making it an attractive target for antibiofilm strategies. "Well over 50 species of bacteria make this molecule; it's not really known why," says Fred Hughson, a structural biologist at Princeton University, and principle author on this issue's second Hot Paper, in collaboration with Bonnie Bassler's lab also at Princeton.

<p>A SUBLIME SIGNAL:</p>

© 2002 Nature Publishing Group

LuxP, the primary AI-2 receptor, with AI-2, a boron containing molecule, bound at the center. (Nature 415:545–9, 2002.)

AI-2 is a small, shape-shifting molecule that proved difficult to crystallize. Bacteria, however, have receptors that bind and trap the signal. Hughson and colleagues decided to take advantage of this process and trap the active form of the molecule through its interaction with the cellular receptor. They achieved a 1.5 Å image of AI-2, bound to its receptor, the LuxP sensor protein, from the marine bacterium Vibrio harveyi.

But identity of the center atom was elusive. "Initially we modeled it with the center atom as a carbon, but that was not chemically plausible," says Hughson. "That was a huge conundrum for a long time. So we started imagining something else."

And that's when boron entered the picture. "It made chemical sense even if it made no biological sense," he says. "The only well-defined reference to boron in biology is its role in stabilizing cell wall structures of plants. But it's ubiquitous in the biosphere, especially in oceans."

Millimolar quantities of borate are found in seawater, meaning that a role for boron in communication among the marine V. harveyi, makes sense in hindsight, reasons Surette, "yet nobody would have ever thought that boron would be part of this molecule." he adds. "There are only a handful of borate papers in biology." Unlike most biological organisms, which don't use boron, V. harveyi clearly adapted to take advantage of its abundance in the oceans.

To test the hypothesis, Stephan Schauder, a postdoc in Bassler's lab, added borate to some of the bacteria, resulting in a 1000-fold increase in signaling. They confirmed boron's identity by nuclear magnetic resonance. What they found was chemically and biologically unprecedented, says Hughson. "It was different from previously characterized signals, but finding boron was the kicker. It is one of the first biochemically defined roles for boron in biology."

In addition to this discovery, another key aspect of the study that stands out, says Surette, is the nature of the crystallography itself. "Getting the structure of a small molecule embedded in a protein is a feat in itself. It's beautiful crystallography," he explains. "And the fact that there's boron in there – that's a very unusual atom to be associated with a biological molecule."

Knowing what the AI-2 autoinducer looks like will help scientists devise strategies to cripple biofilm formation to eradicate unwanted bacterial guests. "Many bacteria regulate virulence and pathogenicity as a function of cell density," explains Hughson. "If you antagonize quorum sensing, you might block virulence." Targeting a general signal such as AI-2 may antagonize different biofilm producers all at once, killing many birds with the same stone.

Nicole Johnston

njohnston@the-scientist.com