Pfiesteria piscicida is a type of plankton found from Florida to Deleware that is famous for its association with fish kills,
including massive ones that have killed thousands if not millions of fish in North Carolina and in the Chesapeake Bay. Nonetheless,
some questions about the species' true toxicity, or lack thereof, remain and Belas is one of many researchers working to answer
remaining questions about the organism, in his case through examining its association with bacteria.
Understanding the kills, and the role of
P. piscicida, has been severely hampered by the organism's complexity.
P. piscicida is a
dinoflagelate, which are typically classified as algae, but may also have animal-like characteristics such as consuming food rather
than just photosynthesizing. The more confusing aspect of
P. piscicida, though, is its life cycle, which scientists believe may
include over 20 potential different stages, a few of which may be toxic.
Understanding of what might cause toxic outbreaks of
P. piscicida remains cloudy, in fact some researchers have found evidence
suggesting that either a fungus or another species of Pfiesteria may be responsible for the fish kills. But many scientists believe
pollution and the presence of significant numbers of fish are involved in triggering
P. piscicida to enter a toxic stage, which in
turn can lead to the production of toxins that cause lesions that can eventually kill fish.
P. piscicida has also been linked to
toxins blamed for human health problems such as memory loss, respiratory and skin problems for those exposed to tainted waters.
Belas became involved in Pfiesteria research after fish kills began impacting the Chesapeake Bay in 1997. He and his team explore
the interactions between Pfiesteria and the myriad organisms, especially bacteria, with which they live in association in coastal
and marine environments. This work has led to the discovery of a bacterium called TM1040 (named after Todd Miller, who discovered it
while a graduate student in the Belas lab), that interacts beneficially with
P. piscicida to keep it alive.
Studies of the impacts of various organisms such as TM1040 on
P. piscicida are facilitated by the fact that one of the species's
life cycles is a dormant and tough cyst phase. During this phase the organism is resistant to chemicals that kill other associated
species. So, the group adds bleach to cultures of cysts killing everything else off and producing a pure culture to which single
bacterial species can be added to observe interactions.
The group has determined that TM1040 has over 20 unique receptor proteins that respond to information from outside world and
transmit that information into the cell. By comparison,
E. coli has only about 4 such proteins, suggesting that TM1040 is very
responsive to its environment. These proteins seem to give TM1040 the ability to respond to the production of certain metabolic
products by dinoflagellates with the production of proteins that allow it to use dinoflagellate products for their own needs. Also,
the group has found that TM1040 actively moves toward
P. piscicida and hovers around it, and may even have fibers that allow it to
attach to the dinoflagellate.
Belas believes the study of such interactions may one day lead to important discoveries about roles the bacteria may play in
facilitating or enabling dinoflagellate toxin production. However, at this point the question remains open as to whether the
production of toxins responsible for the fish kills was mediated by bacteria, as indeed does the question of what produced the
toxins to begin with.
- VIDEO CLIP 2: "Mysterious Chesapeake Bay Fishkills: Is Pfiesteria piscicida the real culprit?"
- VIDEO CLIP 3: "Ineractions between Pfiesteria and the bacterial associate Silicibacter species TM1040"
Assembly Disruption
One very hot area of research in both marine and general microbiology, to which Belas has dedicated years of his work, is the formation of biofilms
by bacteria and possible ways to prevent it. Biofilms are thin layers of bacterial and other growth that can be found everywhere from teeth to
intestines to the bottoms of ships, causing tooth decay, disease biofouling, and more in the process.
One of the most commonly used model bacteria species for biofilm work is
Proteus mirabili. Though it is at times found in coastal waters,
Proteus is
by and large a terrestrial species. It remains nonetheless an excellent model for marine and other research because it has been widely studied and
has many similarities to marine species.
The focus of much biofilm formation research has been on determining just how simple bacteria, which are generally the instigators of biofilms, can
even know when they are on a surface on which to aggregate.
Like many marine bacteria,
Proteus produces a unique cell-type when it is on a surface that is distinct from its free-swimming form, and this
characteristic has simplified the search for the surface trigger in bacteria. The process is not yet fully understood but it appears that bacteria
are able to sense a surface by monitoring the rotation of their flagella. Flagella rotate at a constant speed, so if movement of a cell's flagellum
is slowed or inhibited, it generally indicates close proximity to some kind of surface, on which the cell can "land".
Information about flagellar rotation is conveyed into the cell through a complex motor known as the flagellar basal body, which contains a set of
three proteins. Belas and his colleagues are studying these proteins to determine how they work at a molecular level to transmit information from the
flagella to the organism's DNA, where the real interaction takes place, as researchers have identified over 50 genes that are turned on in response
to sensed surfaces.
Though the research remains at the basic level, the ultimate goal is to increase understanding of bacterial surface sensing and then find ways to
inhibit the changes that allow bacteria to settle and form biofilms. One possibility the Belas group is exploring is to target one of those proteins
integral to the flagellar motion sensing system to prevent its activity and thus prevent surface sensing and the resulting settlement.
Controlling the sensing could have a variety of important applications. Appropriate additives might be used in boat and ship bottom paints to prevent
biofouling, or the effects of diseases such as cystic fibrosis might be diminished or eliminated.
"That certainly is right now a utopian vision," says Belas, of the search for a magic bullet to block biofilms, "but it's something we're looking
for."
- VIDEO CLIP 4: "Out of the Sea and Into Our Gut: the bacterium Proteus mirabilis and biofilm formation"
Creating New Biosensors
A standard plan for developing a biosensor based on genetics is to attach a gene that codes for something easy to spot that you don't need to detect,
to a gene that codes for something you want to detect that is not easy to spot. For instance, if you're trying to detect a toxic chemical and
discover a bacterium that happens to produce some protein in response to that chemical, you might attach to that gene a gene for, say, bacterial
luciferase, which glows once expressed. That would cause the bacterium to glow in the presence of the toxin, an easily detectable response.
The Belas group has developed expertise at making these luciferase attachments to explore potential toxin sensors. The group is also looking at
similar paths to creating sensors that detect surfaces or viscosity by attaching a luminescence gene to proteins produced in response to sensed
surfaces, which can be the same as those produced in response to increasing viscosity.
"You give me the toxic material or the condition and we should be able to figure out how to detect it," says Belas.
At this point, Belas is most interested in exploring possibilities for sensors, whether a commercial use is immediately apparent or not. All the work
is still in the early stages, with no products yet on the market from the lab.
- VIDEO CLIP 5: "Putting the Technology in Marine Biotechnology"
Educational Background
One promising potential application for belas's chaperone research is in increasing the stability of vaccines to make them easier to deliver
to remote parts of the world. Many vaccines contain weak versions of the disease-causing agent they are used to inoculate against, and these
live organisms can be rather delicate, requiring refrigeration. Though they have not yet stabilized a particular vaccine, belas's team has
experimental results already that suggest it may be possible to add chaperones to vaccines that would stabilize them enough to avoid or
reduce the need for refrigeration, which could significantly increase the areas in undeveloped countries that could be reached. Chaperones
may be used to either prevent the breakdown of proteins in the living cells found in certain vaccines, or of the proteins in vaccine
additives known as adjuvants that are necessary for making a vaccine effective. Two examples of vaccines that might be improved through such
research are those for typhoid and cholera.
- VIDEO CLIP 6: "How I Became a Marine Microbiologist"
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