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James Chu
SUID: 05406204
ParaSites Project
Feb 25, 2010
Parasites of Parasites: A Review and Possibilities for Future Research
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.
-Jonathan Swift1
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 civilizationsa2. 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 these3.
a
Also referenced slides for Humbio 153 for basic information on these parasites
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Although drugs do exist to treat many diseases, a longstanding fear—especially
with parasites like malaria—is the acquisition of drug resistance4.
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
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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.
History
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 parasite5.
In the 1970s, viral particles were isolated and characterized in Entamoeba
histolytica as well as Leishmania6. 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 parasitic7.
In 1985, a double-stranded RNA (dsRNA) virus of Trichomonas vaginalis
(coined the Trichomonas vaginalis virus or TVV) was formally discovered8. In
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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 kilodaltons9. 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. histolytica10, 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 team11. 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 yeast12. More exciting,
similar viruses were thereafter discovered in several other human parasites such as
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Leishmania braziliensis, the causative agent of mucocutaneous Leishmania13.
Currently, the frontiers of research are on viral infections of amoeba. One of
the most fascinating combinations of parasitism might be found in Acanthamoeba
Figure 1.
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.
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
amoeba14. 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.”
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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 diarrhea15. 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 diseases16. 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.
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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 Russia17, 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 hosts18. 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.
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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) strains19. 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
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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)20. 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
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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 up21.
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 purified22. 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”
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wavelength of the florescence suggested DNA23.
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, matter24. 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
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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 coinfections25. T. vaginalis provides a related
example, as it increases the transmission of AIDS in coinfections26. 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
Taiwan27. 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 virus28.
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 viruses29. Potentially of greater concern, evidence from mycovirus-fungi
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interactions suggests that viruses “encoding toxins lethal to other strains” offer an
advantage to hosts30. 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 evolution31.
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
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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 resistance32.
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 33. 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
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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 genome34), 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.
Methodology
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 strainb35. The cultures will be
grown in a controlled environment based on the work of Tachibana et al. to ensure they
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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are comparable in population and health measures36. A virus inoculum of 104 to 105
infective units based on Diamond’s work37 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.
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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
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already been proven, including the use of epigenetic mechanisms38 and RNAi39. 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
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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 al40.
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.
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Ibid.
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Markell EK John DT, Krotoski WA. p. 147
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Ibid. p. 31
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20
Also see: Diamond LS, Mattern CF, Bartgis IL. Viruses of Entamoeba histolytica. I. Identification
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Figures taken from Mattern et al.
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Ibid.
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40