Combatting Disease in the Cell

Are oysters more vulnerable now because of ecological changes that have resulted, either directly or indirectly, from human activities?

Why are Dermo and MSX so successful in killing the eastern oyster or, conversely, why has the oyster been so unsuccessful in defending itself? Parasitic organisms like Perkinsus marinus (which causes Dermo disease) and Haplosporidium nelsoni (which causes MSX) are, after all, natural constituents of aquatic ecosystems, and some scientists speculate that Perkinsus itself may have been in the Chesapeake Bay long before it began devastating oyster populations in this last decade.

Are eastern oysters more vulnerable now because of ecological changes that have resulted, either directly or indirectly, from human activities -- for instance, overharvesting, elevated pollution levels and habitat destruction? Have there been natural changes to mid-Atlantic waters that have contributed to the oyster's vulnerability? Or have the parasites themselves improved the weaponry with which they invade, attack and ultimately defeat their host?

Answers to such questions depend on an understanding of how the oyster's immune system acts to defend itself against the biochemical weapons that MSX and Dermo marshal against it, as well as the means by which the parasites are able to evade the oyster's counterattack. For years, scientists have been frustrated by the difficulty of observing a battle which takes place within the very cells of the host, in which the weapons are protein molecules. But recently the tools of biotechnology have given us the means to follow the struggle, and to gain vital insights into the attack strategies of both oyster and parasite. Consistent support from the Oyster Disease Research Program has allowed scientists to initiate the painstaking studies of this cellular war of oyster and parasite, and already this work has led to some remarkable discoveries.

As understanding grows, these new insights hold the key to practical techniques -- from therapeutic agents to genetic manipulation -- that could give the eastern oyster a better-than-fair chance to combat diseases against which it is now helpless.

Cellular Machinations

Unlike vertebrates, molluscs such as the oyster do not possess a sophisticated immune system which produces targeted antibodies against invading disease organisms. The oyster's primary defense against such invaders is phagocytosis, a process in which specialized blood cells called hemocytes recognize a foreign invader, ingest that invader and then internally produce an oxidizing substance such as hydrogen peroxide to destroy it. Scientists have long known that hemocytes, which are analogous to white blood cells in the human body, though far less sophisticated, play a major role in fending off protozoan parasites like Perkinsus. Robert Anderson of the University of Maryland Center for Environmental Science (UMCES), found that when hemocytes are exposed to Dermo cells, the hemocytes engulf them but don't release the toxic compounds -- as a result the parasites survive and proliferate. Why?

Dermo may survive for a number of reasons, Anderson says. It may prevent the the hemocytes from triggering the oxidizing compounds, it may for some reason be able to withstand them, or it may itself produce substances that render the hemocyte's defenses harmless. New molecular tools have been making it possible for Anderson and other scientists such as Gerardo Vasta at the University of Maryland's Center of Marine Biotechnology (COMB) and Muhammad Faisal at the Virginia Institute of Marine Science (VIMS) to better study the chemical armaments that both oysters and Perkinsus marinus deploy in their mutual battle.

Gerardo Vasta (left) and H. Ahmed at the University of Maryland Biotechnology Institute's Center of Marine Biology are purifying virulence factors from the oyster parasite Perkinsus marinus.

Many of these studies depend on large amounts of P. marinus cells, which can now be produced "by the bucketful," says Anderson, thanks to breakthroughs in culturing techniques supported by the Oyster Disease Research Program. Growing P. marinus in the laboratory under controlled conditions makes it possible to study its life cycle and how different environmental conditions (e.g., salinity, temperature, heavy metals, chemicals) affect its growth and behavior. Most importantly, they can begin to tease apart the means by which P. marinus actually causes Dermo disease -- how it invades its host, how it suppresses the response of the oyster's immune system, how it multiplies and, eventually, kills the oyster.

Proteases and Blockers

Researchers soon discovered that, unlike the eastern oyster, the Pacific oyster (Crassostrea gigas) is better able to withstand and fend off the blistering attacks of Dermo and MSX. That's the reason why, says Muhammad Faisal, research in his lab has focused on explaining these different responses. If we can identify the mechanisms that account for the critical difference between these two species, he explains, "it will be the first step towards developing measures to help the eastern oyster better resist these diseases as well."

In laboratory studies, Faisal found that Pacific oysters infected with Dermo showed an increase in the production of hemocytes and blood plasma proteins. But just as in the eastern oyster, Dermo cells were capable of surviving phagocytosis by C. gigas hemocytes. Researchers in his lab found that P. marinus readily grows and divides in hemocytes of infected Pacific oysters, apparently suppressing its hemocyte functions as effectively as it does those of the eastern oyster. This finding, Faisal says, strongly suggests that "factors other than hemocytes may be important in the [Pacific] oyster's resistance." These factors are probably proteins in the oyster's blood (more properly called hemolymph) which in some manner inhibit the ability of Dermo to invade host cells or to multiply.

As part of their attack, notes Faisal, parasites such as Dermo cells release enzymes called proteases that are capable of digesting proteins, thus weakening the host's tissues as well as undoing its molecular defense mechanisms. For example, Faisal and his co-workers have found that P. marinus proteases suppress activity of oyster hemocytes. In a counterattack, the oyster's hemolymph produces protease inhibitors, enzymes which in turn break down the proteases fired off by P. marinus.

Muhammad Faisal has been pursuing the role that proteases, enzymes released by Perkinsus marinus , play in suppressing the oyster's defense mechanisms; his aim is to develop a therapeutant, a protease "blocker" that could help the oyster better defend itself.

The evidence is mounting, says Faisal, "that protease inhibitors play a vital role in the Pacific oyster's defensive arsenal." This finding is especially striking when contrasted with the eastern oyster: the sharp increases in plasma protein concentrations following exposure to Dermo cells, are seen only in Pacific oysters. For instance, in one set of experiments, he found the inhibitory activity of Pacific oysters against P. marinus proteases to be three times greater than the eastern oysters.

If further research confirms these initial findings, Faisal is hoping that it may be possible to develop "protease blockers" that would act something like antibiot-ics in fighting the parasite, at least under controlled conditions. These materials could be delivered to the oyster in its food, or in tiny microcapsules called liposomes. Various practical approaches are now being investigated in Faisal's lab- oratory.

Also being tested is whether eastern oysters which show the most protease inhibitory activity in the lab (albeit lower than that of C. gigas) will prove less vulnerable to Dermo disease in the field. This could provide a means of predicting disease resistance, and contribute to the eventual development of resistant genetic strains.

An Arsenal of Weapon

The arsenal of a complex parasite like Perkinsus marinus is not limited to a single weapon. Scientists are finding that it has many ways of evading or defusing the defense mechanisms of its oyster host. They have also discovered that there are several strains (or types) of P. marinus, which differ in virulence and possibly in their modus operandi. The general strategy, explains Dr. Gerardo Vasta, of the University of Maryland's Center of Marine Biotechnology, appears to be that "the best defense is a good offense." The parasite produces a wide variety of proteins with different actions, all of which contribute to its successful survival within the oyster by disrupting the normal functioning of the oyster's phagocytes. The diversity of these compounds, says Vasta, suggest that they are directed towards challenging the hemocytes "up front" and putting them at an immediate disadvantage, instead of simply responding to the hemocyte's initial attack.

Vasta is on the trail of these "virulence factors." He and his coworkers are investigating what determines the ability of a parasite to combat the oyster's defenses, and how quickly it can respond to challenges from the oyster's immune system. Even more basically, exactly how does the parasite invade? Why are some P. marinus strains (such as those from the Gulf coast) apparently less damaging than those from the Chesapeake Bay -- is it the parasite which differs, are Gulf oysters more resistant, or does the very different Gulf Coast environment reduce the parasite's virulence?

In studies on the cellular functioning of the immune system, a researcher extracts circulatory fluid from the oyster's adductor mussel

Surprisingly, it appears as if the oyster hemocytes themselves may be the key to the initial infection. Current thinking is that infectious P. marinus cells in the surrounding water are taken up by the oyster as it feeds. In the oyster's gut, the parasite crosses the epithelium of the stomach or the intestine into the body of the oyster. It is not known whether this invasion is entirely passive (through phagocytosis) or whether the parasite can also actively penetrate the oyster's cells. The first scenario is highly likely. Unlike the case in many other parasitic diseases (where the parasites "hide" within the host), the invading P. marinus is apparently readily recognized by the oyster's hemocytes. In fact, it almost appears as if the parasite "wants" to be recognized and engulfed by the hemocytes, says Gerardo Vasta.

The parasite possesses cell surface features (such as sugar molecules) which attract the hemocyte, and enhance phagocytosis. Once inside the hemocyte, P. marinus evades or (more probably) inhibits the cellular defense mechanisms, multiplies, and spreads throughout the oyster's tissues. The invader excretes various enzymes which break down the oyster's proteins, providing food for the multiplying parasite. As the disease progresses, hemocytes, oyster tissue and parasite cells are sloughed into the oyster's gut, and released into the water column, thereby spreading the infection.

In research supported by the Oyster Disease Research Program, both Robert Anderson and Fu-Lin Chu, of the Virginia Institute of Marine Science, observed that while oyster hemocytes usually produced bursts of superoxides when engulfing foreign organisms – typically killing the invaders – they did not do so when ingesting P. marinus cells. Vasta's research has shown that the parasite has the ability to produce several enzymes which inhibit the hemocyte's "oxidative burst." His laboratory is now investigating the genetic basis for the production and regulation of these and other factors which contribute to the virulence of Dermo. Which of the many "weapons" employed by P. marinus are the most important in controlling the disease process? Does this vary among the different strains of the parasite? What about the effects of environmental variables? In the future, this information may allow development of biotechnological applications for control of P. marinus, as well as clarify some still-puzzling aspects of its life cycle.

One interesting finding is that many other common Chesapeake Bay bivalves, such as the hard clam Mercenaria and the Baltic clam Macoma, are also hosts to genetically distinct forms of Perkinsus. Whether these animals can also serve as a "reservoir" for oyster Dermo disease is, however, unknown. With support from the Oyster Disease Research Program, this lead is now being investigated in the laboratory and field by Vasta and Greg Ruiz of the Smithsonian's Environmental Research Center.

Environment and Disease

One provocative hypothesis which has developed from ODRP and related research is that environmental degradation of the Chesapeake Bay has exacerbated the spread of Dermo disease. For example, Fu-Lin Chu and her co-workers exposed oysters to water containing contaminants extracted from sediments of the Elizabeth River in Virginia. She observed that these oysters contracted Dermo disease earlier, and had more extensive infections, than oysters exposed to clean water.

Similarly, Anderson observed that exposure to various contaminants (all of which are found in waters or sediments of the Chesapeake) inhibit the oyster's immune responses, in particular the hemocyte's ability to generate a parasite-killing "oxidative burst."

Nutrient overenrichment may also be affecting the oyster's immune system, in a subtle manner. Some of the parasite's most effective weapons are a suite of enzymes which essentially "shut down" the production of the killing superoxide burst by the oyster hemocytes. Gerardo Vasta notes that one essential element in the production of these enzymes is iron – each enzyme molecule contains a single atom of iron.

In fact, since iron is also essential to the production of many important oyster proteins, the parasite must compete with its host for this important element -- a lack of iron would slow proliferation of P. marinus, as well as affect its ability to evade the oyster's immune response. In the saline waters of Chesapeake Bay, under well-oxygenated conditions, free iron concentrations are low, and most iron is bound into very stable iron-phosphate complexes in the Bay sediments. However, in warm months much of the deeper waters of the Chesapeake are extremely low in oxygen (due to nutrient enrichment and decay of resulting algal blooms). Under these conditions, the iron-phosphate compounds break down, releasing free iron into the water column. This ready availability of free iron may help stimulate the proliferation of Dermo disease in Chesapeake Bay. Certainly there is a seasonal component to Dermo virulence which might reflect the greater iron availability (and uptake of the element by oysters) found in summer months.

Results from earlier field studies which show a higher rate of Dermo infection in oysters in deeper bars are also suggestive of environmental influence on Dermo disease. A number of researchers have found that oysters infected with Perkinsus have significantly more acid blood, a condition called acidosis. There is evidence that acidosis enhances the action of some of the parasite's potent weapons (e.g., protease), and speeds the rate of infection. Ken Paynter, of the University of Maryland College Park, points out that environmental factors can also cause acidosis, which would exacerbate disease. Among these factors are high salinity, high temperature and low levels of dissolved oxygen -- all characteristic of the Chesapeake Bay in summer.

These hypotheses are now being tested in a controlled manner, with support from the Oyster Disease Research Program, by Vasta, Anderson and Chu in the laboratory, and by Paynter and others in the field.

For example, in the laboratory Vasta and his co-workers are studying the mechanism by which Perkinsus acquires iron from its host (or the environment), and how iron controls the production of enzymes needed for parasite survival and growth. One goal is to develop a means for blocking infection or parasite proliferation (possibly through use of iron chelators), which could be employed in aquaculture. Initial tests are promising: in culture, iron chelators were shown to inhibit the multiplication of parasite cells, whereas the addition of soluble iron enhanced their growth, supporting the initial premise.

The most intriguing aspect of these hypotheses is that current efforts to reduce nutrient and toxicant pollution of the Chesapeake Bay and other estuaries, and to restore a well-oxygenated water column throughout the year, may have the unexpected side benefit of reducing the incidence of Dermo disease as well.