Feb 25, 2010
Introduction: Blood Sucking Fleas?
So, naturalists observe, a flea
Has smaller fleas that on him prey;
And those have smaller still to bite ‘em;
And so proceed ad infinitum.
The idea of hyperparasites—or parasites of parasites—has been imagined at least since Swift’s observation in the 16th century. While the idea that fleas prey upon fleas is not fully accurate, the idea that an ecosystem can support multiple parasites of each other has certainly been borne out by scientific evidence. More importantly, this concept forms the foundation for an exciting field of inquiry with tangible implications: treatments and new research tools for human parasites.
Parasites such as A. lumbricoides (Ascariasis), L. braziliensis (Leishmania), N. americanus (Hookworm), and E. histolytica (Amebiasis) have indeed plagued humans, sometimes even since the rise of civilizations[a]. According to a study in the New England Journal of Medicine, 56.6 million disability adjusted life years are lost by neglected tropical diseases, the majority caused by parasites such as these. Although drugs do exist to treat many diseases, a longstanding fear—especially with parasites like malaria—is the acquisition of drug resistance.
In this discussion, I will introduce the history of research on hyperparasitism (of relevance to humans), suggest how hyperparasites might contribute to new treatments, highlight a paradigmatic example of hyperparasitism in Entamoeba histolytica, and conclude with a proposal for future research on E. histolytica. I argue that treatments based on current research are limited by ecological intricacies but offer one possible research approach that may begin to elucidate said intricacies.
Definitions: Are Viruses Parasites?
Although the term “parasite” may seem intuitive, to be scientifically rigorous it needs to be operationally defined. Some definitions may exclude viruses as parasites, but I focus much of my attention on viruses. Parasites increase their own reproductive fitness at the cost of the host: by fitness, I refer to the capacity for the organism (or virus) to reproduce its genes to the next generation. For example, P. falciparum is a parasite because by growing and finding nourishment in a human host it has been able to spread its genes effectively, to the particular detriment of children infected with malaria. Similarly, I view the flu virus as a parasite as it develops and reproduces in its host cells to clear detriment. In this discussion I am focusing on so called obligate endoparasites which must live in (“endo”) their host. Facultative parasites, for example, can have an independent life stage. And similarly, ectoparasites such as leeches may gain nourishment from their hosts without living within them.
In the 1960s, researchers began to find what seemed to be viral particles in parasites like Plasmodium. Electron microscopy was one of the only available tools at the time, and there was no ability to molecularly determine whether the particles were actually genetic or replicated at the cost of a host, and as such there was no certainty that they satisfied the definition of being a parasite.
In the 1970s, viral particles were isolated and characterized in Entamoeba histolytica as well as Leishmania. Even over three years of in vitro growth and propagation, the Leishmania promastigotes harbored a similar level of these viral particles, suggesting that viral replication was occurring. Nonetheless, there was no clear detriment to Leishmania, and these particles could easily have been manufactured by the organism rather than being parasitic.
In 1985, a double-stranded RNA (dsRNA) virus of Trichomonas vaginalis (coined the Trichomonas vaginalis virus or TVV) was formally discovered. In addition to using electron microscopy, the scientists adopted a variety of chemical approaches to ascertain the nature of the particles. Using ribonucleases and other cleaving enzymes of nucleic acids, they showed that the particles could indeed hold genetic data. Furthermore, a protein-based coat was discovered that shielded the dsRNA, with this protection annulled when proteinases were added. After analysis, the virus was characterized as having approximately 5.5 kilobase dsRNA and protein structures with a mass of 85 kilodaltons. This data was a clear indication of an obligate virus, and the virus was also unable to infect other protozoa including T. brucei brucei or E. histolytica, suggesting a high level of selectivity. However, there was no clear reduction in host fitness.
A few years after this discovery, a similar finding of a virus infecting Giardia was discovered and characterized by the same team. Here, the genetic material was directly analyzed and compared with existing viruses of yeast. Some similarities including the shape of these viruses (isometric), diameter (around 33 nanometers), lack of lipids, and configuration of one strand of dsRNA enclosed in a major protein complex suggested the evolutionary link between protozoan viruses and yeast viruses, which often code for toxins that damage yeast. More exciting, similar viruses were thereafter discovered in several other human parasites such as Leishmania braziliensis, the causative agent of mucocutaneous Leishmania.
An electron micrograph of A. polyphaga, showing the mimivirus (MVF) and the arrows pointing at the sputnik virus clusters.
Sputnik cannot replicate when A. polyphaga is not infected by MVF.
Currently, the frontiers of research are on viral infections of amoeba. One of the most fascinating combinations of parasitism might be found in Acanthamoeba polyphaga, which is frequently infected with the so-called mimivirus. Its 1185-kilobase dsRNA (compare to the 5.5 kb dsRNA of TVV) codes more genetic data than many bacteria. In a twist, Mimivirus is also frequently infected with a newly-discovered virus affectionately coined “sputnik.” Sputnik is a class of new “virophages” being discovered and characterized with exciting implications: the virophage can alter the genetic material of the virus, which in turn will affect the amoeba. Such findings suggest multiple levels of parasitism can be sustained in a given ecology. With Acanthamoeba occasionally a parasite in its own right, Swift’s imagined fleas pale in comparison to these “parasites of parasites of parasites.”
New Treatments and Possibilities
Although current research has characterized several different viruses that infect human parasites, there is less known about exactly how and whether the parasites are impacted. A direct treatment suggested by these studies may be phage therapy, well characterized and even currently implemented for certain veterinary issues like E. coli caused diarrhea. One idea with promise is to harness the viruses already present in the ecology of parasites like T. vaginalis or L. braziliensis. Phages that will lyse or otherwise reduce the fitness of human parasites could be developed (see figure 2), and current literature suggests promising results in phage therapy for bacterial diseases. If developed, such viruses might even be harnessed to lyse P. falciparum merozoites, co-evolving with the parasite to challenge its drug resistance.
Figure 2. A schematic of how bacteriophages function and the two types—lysogenic and lytic phages. The bound spaces are bacteria with basic geometric forms to describe DNA and protein production.
Although theoretically sound, these possibilities may not necessarily be borne out in reality. Whether viruses truly harm the parasites is mediated by a number of different factors, including resource availability, immune responses of the parasites, and viral load. Before moving to understanding whether and how hyperparasites may shed light on new treatment possibilities, I wish to consider the paradigmatic example of Entamoeba histolytica.
Entamoeba histolytica: A Paradigm Case
E. histolytica, first described in 1875 in the Northern extremes of Russia, inhabits in the large intestine and may feed upon red blood cells. “Invading the mucosal crypts,” it may lead to ulceration and dysentery or so called amebiasis. In rare cases, it may wind up in the lungs, liver, or even the brain and lead to abscesses. On average 20 micrometers in diameter, this strain is closely related to a family of other lumen-dwelling amoeba like E. dispar and E. gingivalis, and shares general similarities with tissue-dwelling and formerly mentioned Acanthamoeba. However, amongst other amoeba, it is one of the most virulent species, especially for immune-suppressed or otherwise susceptible hosts. Given both its connection to existing research and its comparative virulence, this species serves as a well-suited paradigm model to consider the role of hyperparasites.
The majority of research on viruses of E. histolytica stem from a series of papers published by Diamond et al. from 1972 to 1977. In 1972, they hypothesized two separate polyhedral and filamentous viral strains within E. histolytica that caused cell lysis. I now turn to present their data, which was the basis for constructing the first full-scale model of a viral infection of protozoa.
Six E. histolytica strains were acquired (from infected hosts in the United States, Korea, and existing strains) and first axenized (isolated) by isolating them with antibiotics (e.g. streptomycin and Penicillin) and a series of additional cultures to ensure purity. Perhaps the most novel observation was that two kinds of viral strains existed, and that within one type of amoeba (dubbed HB-301) the polyhedral strain had no detrimental effect but led to cell lysis in other (dubbed HK-9) strains. Vice versa, filamentous viral particles were found in another strain led to cell lysis when introduced to other amoeba. These findings suggest that at least two viral strains exist that are native to certain amoeba strains but are highly infectious to others.
Compared with a healthy control, in the first 24 hours, infected cells were still motile and spread across the slide as usual (see figure 3). However, by the 48th hour many infected cells lysed or clumped together in multinucleate masses. Significantly, supernatant (after centrifuging) from strains that showed lysis were used to inoculate
Figure 3. LEFT: a healthy, axenized community of E. histolytica showing a healthy “sheet” of amoeba. RIGHT: after being infected with the virus for 48 hours, amoeba debris is evidence to cell lysis, and multinucleated clusters were occasionally found (see arrow)
identical strains that also showed signs of cell lysis, thus demonstrating that the virus could infect other strains. Moreover, infected cells were frozen-thawed (which killed the amoeba without destroying the virus) and reintroduced to healthy cultures. The result was that healthy cells developed identical infections—either polyhedral or filamentous particles (see figure 4 and 5). Although the existence of two different phenotypes is not conclusive evidence for different viruses, at the very least these experiments demonstrated an infectious agent that led to the direct demise of amoeba compared with controls.
In follow up studies, these researchers were able to isolate the virus (with exception to amoeba membrane debris). More importantly, these purified viruses were not infectious to other organisms like E. coli or T. cruzi, even though these organisms were originally growing alongside the amoeba. Although hardly conclusive, this finding does suggest the possibility that the virus was evolved with amoeba rather than being introduced by other species nearby only recently (in evolutionary terms).
Figure 4. One virus forms beaded and filamentous forms within the amoeba. Note the beaded form within the close up.
Figure 5. By contrast to the filamentous and beaded forms, polyhedral viruses were also found that were selectively infectious to certain amoeba strains.
Unfortunately the genetic information in the viruses was unable to be sequenced. The viruses have particular affinity to amoeba membrane debris and, as mentioned, could not be completely purified. However, in a staining experiment, the research team discovered evidence suggesting that the viruses contains double-stranded DNA: Chemicals that interfered with the synthesis of DNA (but not RNA) also inhibited the virus. When the genetic material was stained with acridine orange (a florescent dye), the telltale “yellow-green” wavelength of the florescence suggested DNA.
Overall, these data suggest the likely existence of at least two highly selective virus strains that target specific E. histolytica strains. When infected amoeba were frozen and thawed, the dead amoeba could still cause pathology in healthy amoeba, clearly suggesting that the infectious agent was viral in nature. Indeed, these viruses are likely double-stranded DNA, and lead to cell lysis and abnormal clumping within 48 hours.
A Verdict: Treatments Are Still Elusive
Given such selectivity and infectivity of these viruses, one might consider engineering treatment using these viruses to curtail amoeba growth in human intestinal crypts. Surely current successes in veterinary phage therapy suggests a possibility.
However, I argue that treatments are elusive given the still undeciphered complexities of ecological interactions between Entamoeba and these viruses. More seriously, before such complexities are sufficiently understood, harnessing such viruses to treat disease may lead to particularly nefarious outcomes. The ecology surrounding hyperparasites and their parasite hosts, in short, matter. More often than not, interactions between parasites may accentuate damage to human hosts.
For example, coinfections of TB and HIV are well-characterized and established as being particularly dangerous. Not only does the HIV virus modify the “clinical presentation of TB,” certain medical treatments are countermanded, curtailing the arsenal available to combat HIV/TB coinfections. T. vaginalis provides a related example, as it increases the transmission of AIDS in coinfections. Turning specifically again to our paradigm case, the HIV virus and Entamoeba histolytica may just well be a mutualistic interaction as well. AIDS accentuates the damage and pathogenicity of E. histolytica in a study of men who have sex with men in Taiwan. The favor might be repaid, however, as AIDS “hitch-hikes” with amoeba when cells infected with AIDS are consumed by Entamoeba histolytica. Infective HIV remains viable within the amoeba, although fortunately so far there has been no proof of human reinfection from amoeba carrying this virus.
As such, even if the virus seems to target amoeba, there is no guarantee that attempting to use the virus on a widespread basis will lead to a beneficial effect. But most importantly, in addition to serving no benefit, there is also no guarantee that the virus will not exacerbate disease. First, Mattern et al. clearly noticed the same implications when they attempted to find parallels between viruses and bacteriophages. However, they find no significant changes in Entamoeba histolytica virulence when infected by viruses. Potentially of greater concern, evidence from mycovirus-fungi interactions suggests that viruses “encoding toxins lethal to other strains” offer an advantage to hosts. Without sufficient ecological understanding of the Entamoeba viruses, the possibilities of particularly virulent strains of E. histolytica being promoted a particular reason for concern. Indeed, amoebas are identified as particularly well suited grounds for viral evolution.
Ultimately, a more nuanced understanding of how the virus interacts with the amoeba must be obtained, and care must be taken to use existing knowledge of hyperparasitic viruses to avoid additional harm.
A Possible Experiment
Instead of only arguing for caution when adopting such viruses for novel treatments, I outline a possible experimental design that could take one constructive step toward understanding with greater precision specifically how Entamoeba viruses interact with their host. This research design might be replicated with other virus-parasite pairs, but I focus on the paradigm example for clarity.
I start with the observation that in the majority of cases a parasite’s pathogenicity is directly related to its ability to evade the human immune system. Immunocompromised patients, for example, are far more susceptible to opportunistic infections, including the amoeba. In the same line of thought, I advocate for better understanding of the amoeba’s own response—specifically its immune system, however basic it may be—against the viruses. Specifically, given that the E. histolytica viruses currently studied are selectively pathogenic, one of the first questions to consider is whether and why the polyhedral virus seems to not harm the HB-301 strain.
Indeed, as Diamond’s team suggests,
the persistence of virus in normal, axenically cultivated HB-301 E. histolytica without any discernible [lysis…] is suggestive of either a carrier culture or a lysogenic system. A carrier culture, as defined by Walker (15), is a mixed population of cells and virus in a more or less stable equilibrium. The equilibrium is usually maintained by some antiviral substance, antiserum or interferon, or some inherent host cell resistance.
Understanding host cell resistance and/or tolerance is therefore a productive first step to identifying how the existing viruses might be utilized for clinical treatment. There is, for example, currently some evidence that certain cells of social amoeba exhibit selective phagocytic activity of bacteria, suggesting innate immune responses in amoeba. Does such innate immunity occur in E. histolytica? The following is a research protocol that might take the first step toward this question:
Research Aim and Hypothesis
E. histolytica strain HB-301 is not negatively influenced by infection of polyhedral virus strain (V301) but strain HK-9 has been qualitatively noted to face severe cell lysis when infected. As such, I hypothesize that an innate immune factor is maintaining strain HB-301’s carrier state. To test this hypothesis, I will track three amoeba health measures as well as viral load: amoeba count, clustering, and comparative survival to controls (based on Diamond). I will test three fully axenized strains HK-9, HM-1: IMSS (a strain chose because it has a fully sequenced genome), and HB-301. Although when infected, health measures are naturally highest for strain HB-301 and lowest for strain HK-9, I propose that inoculating HK-9 with the cytoplasm of HB-301 will abolish the pathogenicity of V301 in HK-9. When repeated in comparison to strain HM-1: IMSS, I expect the same findings, which would suggest the existence of a genetically expressed immune factor in the cytoplasm of strain HB-301 against V301.
STEP A. After axenizing strains HB-301, HM-1: IMSS, and HK-9, take baseline (t=0) health measures of at least 10 comparable cultures of each strain[b]. The cultures will be grown in a controlled environment based on the work of Tachibana et al. to ensure they are comparable in population and health measures. A virus inoculum of 104 to 105 infective units based on Diamond’s work will be used to inoculate 5 cultures of each strain, while 5 cultures will be inoculated with the same solution with heat-killed viral particles (sham). Health measures will be taken at additional times t=1, 2, 12, 24, 36, and 48 hours as referencing Diamond et al.
STEP B. Identical to the methods of step A, with the following changes: after inoculating each strain with the virus, transfer 2 microliters of healthy HB-301 cytoplasm to the other strains (and shams) by micropipette.
STEP C. Repeating step B, heat kill noninfected HB-301 before transferring cytoplasm to other strains. This step should negate any protective effects of the cytoplasm transfer by denaturing any protective (protein) factors and thus serve as a check of methodology.
STEP D. Repeating step B, transfer 24-hour infected HB-301 cytoplasm to other strains. This step double-checks for the possibility that HB-301 begins to translate protective proteins or factors (e.g. RNAi) after infection with the virus.
Significance and Conclusion
If such an experiment finds that the cytoplasm of HB-301 contains protective factors that at least partially explain its carrier state, further experimentation might be done to identify gene targets. Abilities to silence gene expression in E. histolytica has already been proven, including the use of epigenetic mechanisms and RNAi. Such tools can test whether certain gene targets are necessary in maintaining HB-301’s carrier state by knocking out those genes with these silencing mechanisms.
More importantly, this direction of research elucidates whether and subsequently how the HB-301 E. histolytica strain avoids being lysed by its hyperparasite V300. Such findings could have direct clinical application: a medicine that, for example, is able to knock out such mechanisms of innate immune protection will allow the virus to overrun its host. This approach would avoid the complexities of having to introduce a virus to a new amoeba species and with a high degree of unknowns. Furthermore, because most E. histolytica strains are already known to carry viral strains, if this pathway is conserved across strains, such a medical treatment could seriously curtail the fitness of pathogenic amoeba without affecting the other flora in the intestine.
While I have not focused on other possibilities in this discussion, I wish to underline the significance and potential richness of such research by briefly raising a further example. Viruses have fostered evolution in bacteria by the process of gene transduction. In the same way, viruses may possibly be able to insert desired genes into parasites, creating experimental conditions currently not found naturally. For example, a virus may be able to knock out a certain gene in P. falciparum by inserting a disruptive transposon, and an experiment with a wild type control would illuminate the role of that gene. Such work is already being explored by Que et al.
I have now illustrated the possibilities that exist in studying viruses of human parasites. I have introduced in particular the paradigm model E. histolytica and its two (or more) characterized viruses. While suggesting a high number of uncertainties and doubts regarding the current use of such viruses as clinical or research tools, I have demonstrated a plausible research design that could lead to the eventual discovery of a viable drug against E. histolytica that capitalizes on the viruses that is carries.
Through this discussion, I ultimately hope to have suggested that the possibilities for combating parasites may not necessarily lie only in discovering an arsenal of drugs but rather in probing the ecological framework in which the parasite itself is a part. By understanding, for example, that Entamoeba histolytica carries viruses suggested the possibilities of treatment outlined in this discussion. Indeed, Swift’s albeit inaccurate observation suggests that parasites are equally prone to being hosts. Perhaps in moving forward, a deeper examination of the ecological framework surrounding far more pathogenic and significant parasites like Plasmodium falciparum could yield exciting possibilities as well. While cliché, human lives are indeed at stake.
[a] Also referenced slides for Humbio 153 for basic information on these parasites
[b] In greater detail, “E. histolytica trophozoites are maintained under axenic conditions through successive passages from culture tubes placed horizontally in an incubator at 37 °C until ∼90% confluence (∼1 × 106 trophozoites/tube). Once confluence is reached, culture media supernatant is carefully decanted and adherent cells are rinsed gently with 2 mL per tube of TYI-S-33 medium previously incubated at 37 °C. Tubes are immediately refilled with 5 mL of fresh medium, and trophozoites are detached by striking the bottom of tubes three times against the workbench surface. The cellular suspension that is obtained is used immediately to prepare new culture tubes.”
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 Figures taken from Mattern et al.
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