Sandia LabNews

Dangerous bacteria may evolve from a single cell, not many, Sandia/UNM researchers find

Most scientists believe that staph infections causing inflammation or worse are produced by a large community of bacterial cells signaling each other to emit toxins and biodegradatory enzymes. The signaling process is called “quorum sensing” because many bacteria — a quorum — are thought to be present to start the process.

Jeff Brinker sits next to a cell-suspension wheel that contains bacteria suspended in media. (Photo by Randy Montoya)

Jeff Brinker sits next to a cell-suspension wheel that contains bacteria suspended in media. (Photo by Randy Montoya)

Contrary to this opinion, in the Nov. 22 Nature Chemical Biology journal, a research group led by Jeff Brinker (1004) has determined that the very first stage of staph infection — a bacteria’s switch from a harmless to a virulent form — can occur in a single cell independent from the behavior of other cells around it.

“The good news is that by inhibiting the single cell’s signaling molecules with a small protein, we were able to suppress any genetic reprogramming into the bacterium’s more virulent form,” says Jeff. “Our work clearly showed the strategy worked.”

While staph are often harmless bacteria that commonly live in and on the body, the Brinker group’s nonantibiotic approach may make it easier to treat staphylococci strains that have mutated to become drug resistant like the methicillin-resistant Staphylococcus aureus MRSA, control of which is a formidable problem in modern hospitals.

In the course of their experiments, the Brinker team achieved several interesting firsts.

First, they isolated Staphylococcus aureus bacteria in individual nanoscale compartments self-assembled by silica and lipids. Isolation of an individual bacterium previously had been achieved only computationally, leaving open questions of how a physically and chemically isolated bacterium would actually behave.

Second, the team demonstrated that it was the release of signaling peptides from a single cell — not a group — that acted as a trigger to reprogram that same cell so that it released toxins.

The finding challenges a generally accepted but unproven biological hypothesis that it takes a number of cells, called a quorum, to produce enough peptides to stimulate bacterial transformations. So settled is this belief that the process is referred to in technical literature as “quorum sensing.”

But the term may prove a misnomer, the result of observations made in cell cultures rather than in the body, says Jeff. Because signaling molecules diffuse away in liquid, a culture of cells would naturally require many bacteria corralled together to produce enough signaling bacteria to begin reprogramming. The situation is otherwise in nature, where even a single cell may be sufficiently isolated that its own manufactured peptides would remain in its vicinity.

“Also, it’s hard to believe that one cell’s evolution could be based on what a whole bunch of cells do,” says Jeff. “When we instead consider that an individual cell will do what’s best for it, we can more clearly understand the benefits of that cell’s behavior.”

For example, by reprogramming itself to produce toxins or enzymes, a bacterium can break out of its confinement to access external nutrients and survive longer, the Brinker group showed.

This aspect of the research has drawn favorable comment from researchers in the field.

In an email, University of Illinois professor of microbiology and immunology Mike Federle wrote to Jeff, “I am often asked when and where during the infection process quorum sensing starts. I often suggest that shortly after colonization, small numbers of cells may signal to initiate virulence factor expression, but this hypothesis is not always received well since many assume large groups need to be involved.

“Thank you for providing evidence this is not just a theoretical possibility.”

Also at the University of Illinois, Professor Linda Kenney emailed, “. . . that the term quorum sensing is actually not an accurate description of [bacterial] behavior . . . [is] likely to have significant impact on the field as well as enhance our understanding of how biofilms [the relevant bacterial lifestyle in most infections] form.”

Third, and equally startling, the Brinker group demonstrated that merely by introducing an inexpensive, very low-density lipoprotein (VLDL) to bind to the messenger peptide, they could stop the single cell from reprogramming itself.

One aspect of experimental rigor was the team’s ability to organize living cells into a nanostructured matrix. “We’ve already done this with yeast,” says Jeff. “We just extended the process to bacteria.”

By compartmentalizing the bacteria individually, the Brinker group had set the stage to determine whether a single bacterium could reprogram itself without a quorum present.

A key question was whether a cell could distinguish between peptides emitted by itself from those sent by other cells. If the specific signaling peptides were chemically the same, what would it matter which bacterium emitted it?

It turned out, says Jeff, “Peptides could bond to surface receptors on their own [generating] cell. So a single cell’s peptide molecules could activate its own genes into an expression that makes staphs virulent.”

One indication that the experiment had isolated the actual cause of the transformation was that when the number of peptides produced by a cell ultimately came to exceed the number of VLDL molecules in solution the stalled quorum-sensing procedure started up again.

The researchers also demonstrated that if more signaling molecules were added to the mix, the cell’s transformation occurred more rapidly.

A green fluorescent protein inserted in the cell’s DNA showed, in its operation, that proteins were being manufactured by the cell itself when the transformation was permitted to occur.

Among the problems remaining for researchers is to find a mechanism to locate bacteria in the body starting to reprogram and deliver the antidote in time.

The problem could be solved, suggests Jeff, by the insertion of VLDL-bearing nanospheres (another Brinker-group creation) into the bloodstream, linked to a ‘searcher’ molecule designed to find and link to suspect peptides or cells that produce them.

“Inhibiting this species-specific signaling molecule from turning on the virulence wouldn’t inhibit other bacteria,” Jeff says.

Targeting is important because the human gut contains many useful bacteria. These are often decimated by conventional antibiotics but would be spared by the Brinker group’s method.

Extending implications of the work to bacterial pathogenicity in general, Jeff says, “Our results imply that shortly after bacteria colonize the gut, respiratory tract, or other enclosed spaces, small groups of cells or even individual cells initiate an expression of virulence through use of these signaling molecules. So therapies aimed at inhibiting this behavior are promising strategies for eradication of infection at its outset.”

Jeff, a Sandia Fellow and distinguished professor of chemical engineering and molecular genetics and microbiology at UNM, performed this work with Eric Carnes and DeAnna Lopez at the UNM Department of Chemical and Nuclear Engineering (DeAnna is now a Sandia technologist), Graham Timmins at the UNM College of Pharmacy, Niles Donegan and Ambrose Cheung at Dartmouth Medical School, and Hattie Gresham at the New Mexico Veterans Administration Health Care System.

The Sandia work is supported by the DOE Basic Energy Science/Division of Materials Science and Engineering and Sandia’s Laboratory Directed Research and Development (LDRD) program. Other project work is supported by the Air Force Office of Scientific Research, the National Science Foundation, the Defense Threat Reduction Agency, and the National Institutes of Health.

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