Let’s talk, cell-to-cell

Just as a football game cannot be played with just one person, many tasks performed by cells require multiple players, too.  For many bacteria to act as one, proper communication is key. One way of talking to a neighboring bacterium is called quorum sensing, in which one bacterium releases special molecules, called autoinducers, into the environment so that other bacteria can sense and respond to them [1]. Another way, used not only in bacteria but also in plants and animals, involves physically bridging two cells and exchanging specific molecules with each other through a microscopic tunnel.

Getting molecules directly from one cell into another is no trivial task, though. All cells are enveloped by a membrane: a double-layer of lipids, or fat-like molecules, that acts as a selectively permeable barrier between the inside and outside of a cell. Bacteria also have cell walls, additional sugar-rich layers that make these barriers even stronger. Many molecules that could be great gifts to a neighbor cannot pass through the membrane barrier. DNA (deoxyribonucleic acid), which contains the genetic “recipes” cells use to make protein molecules, is one example. Proteins, molecules that facilitate most of the important biological reactions in a cell, also cannot pass through cell membranes freely.

One way bacteria have gotten around the challenge of moving DNA across a membrane is through a process called conjugation. During conjugation, plasmids — small, circular pieces of DNA — are shuttled from one cell into another through a pilus, a tube-like structure constructed of proteins. While most plants and some animal cells also have tube-like transport systems, those tubes are more like extensions of the cell membrane, being made of lipids rather than proteins [2,3].

Nanotubes: a new transport highway in bacteria

In a recent report by Gyanenedra P. Dubey and Sigal Ben-Yehuda of the Hebrew University of Israel [4], Bacillus subtilis, a species of bacteria used widely as a tool to study bacterial biology, was found to form “membrane nanotubes” that allow intercellular exchange. These nanotubes resemble the tube-like connections seen in plants and animals, rather than the protein-based pili formed during bacterial conjugation. This is the first report of such membrane-based transport highways in bacteria.

The scientists made this discovery when they noticed bacterial cells were able to exchange protein molecules with each other under a microscope. To observe this protein movement between cells, they used a tool called green fluorescent protein (GFP). GFP is a protein that glows bright green when exposed to blue light, making it easy to see using a fluorescence microscope. It is also easy to artificially introduce the genes for GFP into Bacillus bacteria so the cells produce this bright fluorescent protein. Dubey and Ben-Yehuda therefore mixed some bacteria harboring the GFP gene (“GFP-positive”) with bacteria lacking it (“GFP-negative”). Interestingly, they found that GFP-negative cells growing next to GFP-positive ones appeared to acquire green fluorescence over time. This suggested that the fluorescent bacteria were transporting GFP molecules into their neighbors.

Speculating that a direct connection might be enabling this transport, the researchers used high-resolution scanning electron microscopy, a type of high-magnification microscope, to look at the fine features of the bacterial surface. To their surprise, they saw what looked like tiny tubes extending from the membrane of one bacterium to another. Were these tubes made of membrane lipids? Additional experiments done by the authors using membrane-disrupting chemicals suggest this may be the case, although the possibility still remains that other types of molecules, like proteins or sugars, are involved.

These “nanotubes” appeared large enough to allow the passage of GFP molecules, so the authors did experiments to see if GFP could be observed within the tubes. They took advantage of the high-magnification capabilities of electron microscopy to visualize cross-sections of bacterial cells. Then, by employing a special technique to locate GFP molecules under an electron microscope, they were able to spot some GFP in the tubes connecting the cells, thus supporting their hypothesis.

Since one cannot use electron microscopy to observe living cells, it is important to remember that what they saw was a snapshot in time, not a video of the GFP moving through a live cell. Like any photograph, the image of the GFP in the tubes is open to some interpretation. Is the GFP sitting in the tube actually moving towards the other cell? Are there molecular barriers inside the tubes we cannot see in these snapshots? These questions remain open; however, the observation of GFP inside these tubes strengthens the authors’ argument that GFP can pass through these intercellular nanotubes, even if it does not prove it outright.

Lastly, both GFP transfer and nanotube formation occurred between not only two populations of Bacillus subtilis bacteria, but also between Bacillus subtilis and methicillin-drug resistant Staphylococcus aureus (MRSA). MRSA is a strain of pathogenic (disease-causing) bacteria that is resistant to many common antibiotics and poses an especially serious problem for those in hospitals and other healthcare settings [5]. The finding that nanotubes can form between two extremely different species of bacteria poses the tantalizing idea that this could be a more universal strategy for cross-species chatting.

Biological role of membrane nanotubes

So, what is the biological purpose of these nanotubes? One idea is that they help spread molecular tools that improve the ability of a group of bacteria to survive and reproduce. In addition to their experiments with GFP, Dubey and Ben-Yehuda showed evidence suggesting that nanotubes could also transport proteins that protect cells against antibiotics. Several other experiments suggested that DNA plasmids carrying antibiotic resistance genes could also travel from one bacterium to another through these nanotubes. Acquiring DNA, rather than protein, would provide the advantage of being able to pass on the protection to future generations. One caveat of these experiments is that Bacillus subtilis bacteria are naturally able to enter a state called competence under certain conditions, in which they can directly uptake DNA molecules from their surroundings. Thus, it’s possible that the DNA plasmids for antibiotic resistance could have been passed among cells using a means other than nanotubes.

This study presents exciting new ideas for how bacteria may interact with each other. Plants use an extensive system of membrane tubes called plasmodesmata to exchange proteins, lipids, and various nutrients among its cells [3]. Animals have long been known to use protein-based connections called gap junctions to communicate with adjacent cells; in recent years, membrane tubes called tunneling nanotubes have also been documented in some animal cells to facilitate long-distance exchanges [2]. What similarities do bacterial nanotubes have in common with their analogs in plants and animals? Could bacterial populations in the wild use nanotubes to spread resistance genes or infective traits? Or, do they use them to better coordinate group behavior? These and other questions will be of great interest in the future, as we start to understand more about the nature and biological function of membrane nanotubes, particularly in pathogenic bacteria.

Tina Liu is a graduate student at Harvard Medical School.

References:

[1] Bassler B.L., Losick R. Bacterially speaking. Cell. Apr 21, 2006. 125: pp. 237-246.

[2] Gerdes H., Bukoreshtliev N.V., Barroso J.F.V.. Tunneling nanotubes: A new route for the exchange of components between animal cells. FEBS Letters. Apr 4, 2007. 581: pp. 2194–2201

[3] Gallagher K.L., Benfey P.N. Not just another hole in the wall: understanding intercellular protein trafficking. Genes Dev. 2005. 19: pp. 189-195

[4] Dubey G.P., Ben-Yehuda S. Intercellular nanotubes mediate bacterial communication. Cell. Feb 18, 2011. 144(4). pp. 590-600.

[5] Center for Disease Control. Staphylococcus aureus (MRSA) infections. http://www.cdc.gov/mrsa/index.html

Other news coverage:

Edyta Zielinska. “Trading resistance via nanotubes?” http://www.the-scientist.com/news/display/57991/