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1.  Behavioural manipulation in a grasshopper harbouring hairworm: a proteomics approach 
Abstract
The parasitic Nematomorph hairworm, Spinochordodes tellinii (Camerano) develops inside the terrestrial grasshopper, Meconema thalassinum (De Geer) (Orthoptera: Tettigoniidae), changing the insect's responses to water. The resulting aberrant behaviour makes infected insects more likely to jump into an aquatic environment where the adult parasite reproduces. We used proteomics tools (i.e. two-dimensional gel electrophoresis (2-DE), computer assisted comparative analysis of host and parasite protein spots and MALDI-TOF mass spectrometry) to identify these proteins and to explore the mechanisms underlying this subtle behavioural modification. We characterized simultaneously the host (brain) and the parasite proteomes at three stages of the manipulative process, i.e. before, during and after manipulation. For the host, there was a differential proteomic expression in relation to different effects such as the circadian cycle, the parasitic status, the manipulative period itself, and worm emergence. For the parasite, a differential proteomics expression allowed characterization of the parasitic and the free-living stages, the manipulative period and the emergence of the worm from the host. The findings suggest that the adult worm alters the normal functions of the grasshopper's central nervous system (CNS) by producing certain ‘effective’ molecules. In addition, in the brain of manipulated insects, there was found to be a differential expression of proteins specifically linked to neurotransmitter activities. The evidence obtained also suggested that the parasite produces molecules from the family Wnt acting directly on the development of the CNS. These proteins show important similarities with those known in other insects, suggesting a case of molecular mimicry. Finally, we found many proteins in the host's CNS as well as in the parasite for which the function(s) are still unknown in the published literature (www) protein databases. These results support the hypothesis that host behavioural changes are mediated by a mix of direct and indirect chemical manipulation.
doi:10.1098/rspb.2005.3213
PMCID: PMC1559948  PMID: 16191624
extended phenotype; parasite–host systems; parasite manipulation; proteomics
2.  Thioredoxin Glutathione Reductase from Schistosoma mansoni: An Essential Parasite Enzyme and a Key Drug Target 
PLoS Medicine  2007;4(6):e206.
Background
Schistosomiasis—infection with helminth parasites in the genus Schistosoma, including S. mansoni—is a widespread, devastating tropical disease affecting more than 200 million people. No vaccine is available, and praziquantel, the only drug extensively utilized, is currently administered more than 100 million people yearly. Because praziquantel resistance may develop it is essential to identify novel drug targets. Our goal was to investigate the potential of a unique, selenium-containing parasite enzyme thioredoxin glutathione reductase (TGR) as a drug target.
Methods and Findings
Using RNA interference we found that TGR is essential for parasite survival; after silencing of TGR expression, in vitro parasites died within 4 d. We also found that auranofin is an efficient inhibitor of pure TGR (Ki = 10 nM), able to kill parasites rapidly in culture at physiological concentrations (5 μM), and able to partially cure infected mice (worm burden reductions of ~60%). Furthermore, two previously used antischistosomal compounds inhibited TGR activity, suggesting that TGR is a key target during therapy with those compounds.
Conclusions
Collectively, our results indicate that parasite TGR meets all the major criteria to be a key target for antischistosomal chemotherapy. To our knowledge this is the first validation of a Schistosoma drug target using a convergence of both genetic and biochemical approaches.
Using both genetic and biochemical approaches, David Williams and colleagues show that the parasite thioredoxin glutathione reductase meets all the major criteria to be a key target for antischistosomal chemotherapy.
Editors' Summary
Background.
More than 200 million people are infected with schistosomes, a type of parasitic worm. Schistosomes have a complex life cycle that starts with them reproducing in freshwater snails. The snails release free-swimming, infectious parasites that burrow into the skin of people who swim in the contaminated water. Once in the human host, the parasites turn into larvae and migrate to the liver where they become juvenile worms. These mature into 10- to 20-mm-long adult worms and take up long-term residence in the veins draining the gut (Schistosoma mansoni and S. japonicum) or bladder (S. haematobium). Here, the worms mate and release eggs, some of which pass into the feces and so back into water where they hatch and infect fresh snails. Schistosomiasis causes serious health problems (including chronic liver, gut, bladder, and spleen damage) in about 20 million people, making it a disease of great public-health and socioeconomic importance in the developing countries in which it mainly occurs.
Why Was This Study Done?
The only drug available to treat schistosomiasis is praziquantel. Although it is very effective, people regularly get reinfected and need to be retreated once or twice a year. All told, 100 million people are currently being treated with praziquantel. Reliance on a single drug, however, is problematic, as the parasites are likely to develop resistance to the drug over time. The identification of new drug targets in schistosomes is therefore an urgent goal. In this study, the researchers have investigated whether thioredoxin glutathione reductase (TGR), a parasitic enzyme with several functions, might be a key target for antischistosomal chemotherapy. They chose this enzyme because adult worms need to make antioxidants (chemicals that prevent oxygen from damaging cells) to protect themselves against the human immune response. Antioxidant production in these worms depends on TGR; in mammalian cells, two specialized enzymes do its job. The researchers reasoned, therefore, that TGR might be an essential parasite protein and a potentially important drug target.
What Did the Researchers Do and Find?
The researchers made large quantities of pure TGR and tested its activity against various substrates. The enzymatic properties and substrate preferences of TGR, they found, differed somewhat from those of its mammalian counterparts. They then screened different types of compounds for their ability to inhibit TGR. Praziquantel had no effect on TGR activity, but two antischistosomal compounds that are no longer used, potassium antimonyl tartrate and oltipraz, inhibited the enzyme. The most potent inhibitor of TGR, however, was a gold-containing complex called auranofin, low levels of which inhibited TGR in test tubes, completely killed larval, juvenile, and adult parasites living in laboratory dishes within hours, and more than halved the worm burden in infected mice. Finally, the researchers used a technique called RNA silencing to test the importance of TGR for worm survival. Fragments of double-stranded RNA (dsRNA) stop proteins being made from messenger RNA that contains an identical sequence. The addition of TGR dsRNA to larval parasites in a dish greatly reduced TGR enzyme activity and killed nearly all the parasites within days.
What Do These Findings Mean?
These findings suggest TGR as a key target for antischistosomal drug development. Indeed, the discovery that two previously used antischistosomal compounds inhibit TGR suggests that the enzyme has already served as a target protein. The RNA silencing experiment shows that TGR is essential for parasite survival, and the biochemical analyses indicate that TGR and its mammalian counterparts have different substrate specificities. Thus, it should be possible to find compounds that inhibit TGR but have much less effect on the mammalian enzymes. This is certainly true for auranofin, a drug used to treat rheumatoid arthritis. Whether auranofin will be an effective treatment for schistosomiasis remains to be seen—an agent that completely kills schistosomes in animals would be preferable. However, even a 50% reduction in worm burden would decrease the human health problems caused by schistosomiasis, and a combination of auranofin (or another TGR inhibitor) with an agent that works by a different mechanism might be more effective and would also reduce the chances of the parasite developing drug resistance.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040206.
World Health Organization provides information on schistosomiasis, including a fact sheet in English, Spanish, French, Arabic, Chinese, and Russian
US Centers for Disease Control and Prevention provide information for the public and for professionals on schistosomiasis
MedlinePlus encyclopedia includes an entry on schistosomiasis (in English and Spanish)
The Schistosomiasis Control Initiative has information on the disease and its control
Wikipedia has a page on schistosomiasis that is available in several languages (note: Wikipedia is a free online encyclopedia that anyone can edit)
doi:10.1371/journal.pmed.0040206
PMCID: PMC1892040  PMID: 17579510
3.  Aberrant Receptor-Mediated Endocytosis of Schistosoma mansoni Glycoproteins on Host Lipoproteins 
PLoS Medicine  2006;3(8):e253.
Background
Bilharzia is one of the major parasitic infections affecting the public health and socioeconomic circumstances in (sub) tropical areas. Its causative agents are schistosomes. Since these worms remain in their host for decades, they have developed mechanisms to evade or resist the immune system. Like several other parasites, their surface membranes are coated with a protective layer of glycoproteins that are anchored by a lipid modification.
Methods and Findings
We studied the release of glycosyl-phosphatidylinositol (GPI)-anchored proteins of S. mansoni and found them in the circulation associated with host lipoprotein particles. Host cells endocytosed schistosomal GPI-anchored proteins via their lipoprotein receptor pathway, resulting in disturbed lysosome morphology. In patients suffering from chronic schistosomiasis, antibodies attacked the parasite GPI-anchored glycoproteins that were associated with the patients' own lipoprotein particles. These immunocomplexes were endocytosed by cells carrying an immunoglobulin-Fc receptor, leading to clearance of lipoproteins by the immune system. As a consequence, neutral lipids accumulated in neutrophils of infected hamsters and in human neutrophils incubated with patient serum, and this accumulation was associated with apoptosis and reduced neutrophil viability. Also, Trypanosoma brucei, the parasite that causes sleeping sickness, released its major GPI-anchored glycoprotein VSG221 on lipoprotein particles, demonstrating that this process is generalizable to other pathogens/parasites.
Conclusions
Transfer of parasite antigens to host cells via host lipoproteins disrupts lipid homeostasis in immune cells, promotes neutrophil apoptosis, may result in aberrant antigen presentation in host cells, and thus cause an inefficient immune response against the pathogen.
The finding that GPI-anchored schistosome proteins are transferred from the parasite surface to human lipoproteins may explain how the parasites interfere with an effective immune response.
Editors' Summary
Background.
More than 200 million people live in a close but uneasy alliance with schistosomes, a type of parasitic worm. Like many parasites, schistosomes have a complicated life cycle. They start life by reproducing in fresh-water snails. The snails release free-swimming, infectious parasites, which burrow into the skin of people who swim in the water. The parasites then migrate to the veins draining the gut and mature into 10–20 mm-long adult worms. The worms mate and lay eggs, some of which pass into the feces and so back into water where they hatch and infect fresh snails. Schistosomiasis does not kill many people but it does cause serious health problems. Most of these are caused by the human immune system responding to eggs that get trapped in the veins of the liver, spleen, and gut. Immune cells recognize proteins on the eggs as foreign and organize a hard shell of immune cells and tough fibres around the egg. Eventually, these fibres block the blood vessels in the liver, spleen, and gut, causing locally raised blood pressure, organ damage, and potentially fatal bleeding.
Why Was This Study Done?
Although the immune system mounts a vigorous attack against schistosome eggs, the parasites themselves somehow evade the immune response—adult worms pull off this feat of “invisibility” for years. The researchers who did this study wanted to find out whether the release of glycoproteins (proteins decorated with sugars) from the surface of the schistosome worms is involved in this immune evasion in some way. These glycoproteins (which are anchored to the parasite's surface by a structure called a GPI-anchor; GPI stands for glycosyl-phosphatidylinositol, a sort of fat or lipid) are the major antigens of schistosomes—the molecules that the immune system normally recognizes on foreign intruders.
What Did the Researchers Do and Find?
The researchers first showed that GPI-anchored schistosomal glycoproteins are released into the circulation of patients and there become attached to human lip oproteinparticles (water-soluble carrier molecules that take fats around the body). Then, using cells grown in the laboratory, the researchers discovered that lipoprotein particles loaded with parasite glycoproteins could enter mammalian cells through an interaction with a protein called the low-density lipoprotein receptor, which normally helps cells absorb the lipids needed to make membranes. Once in the cell, the parasite glycoproteins travelled to cellular regions called lysosomes, which they seemed to disrupt. In addition, the researchers found that the parasite glycoproteins could enter mammalian cells by a second route: This involved the glycoproteins being taken up by neutrophils (a type of immune cells). Many of these neutrophils then died, possibly because of the large amount of lipid they accumulated.
What Does This Mean?
These results provide some tantalising clues to how schistosomes might evade the immune response. First, just binding to lipoprotein particles might change how they are seen by the immune system (possible they are not as clearly recognized as foreign substances) and weaken the immune response against them. On the other hand, the damage done to neutrophils by lipid accumulation might also contribute to how schistosomes hide in the human hosts. Neutrophils are an important type of immune cell, and their destruction could compromise the immune system's response to schistosomes. Furthermore, although the researchers do not investigate this possibility, other cells of the immune system that have might also take up these lipids and be damaged. Finally, even if immune cells are not killed outright by lipid accumulation, disruption of their lysosomes might also affect how well the immune system recognizes schistosomes as foreign. The full details of the complex interplay between schistosomes and their hosts remain a mystery, but these results provide intriguing new avenues to explore that might eventually suggest new treatments for schistosomiasis.
Additional Information
Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030253.
• World Health Organization information on schistosomiasis
• US Centers for Disease Control and Prevention information for the public and for professionals on schistosomiasis
• MedlinePlus encyclopedia entry on schistosomiasis
• Wikipedia page on schistosomiasis (note: Wikipedia is a free online encyclopedia that anyone can edit)
doi:10.1371/journal.pmed.0030253
PMCID: PMC1502155  PMID: 16942390
4.  Schistosomiasis Mansoni: Novel Chemotherapy Using a Cysteine Protease Inhibitor 
PLoS Medicine  2007;4(1):e14.
Background
Schistosomiasis is a chronic, debilitating parasitic disease infecting more than 200 million people and is second only to malaria in terms of public health importance. Due to the lack of a vaccine, patient therapy is heavily reliant on chemotherapy with praziquantel as the World Health Organization–recommended drug, but concerns over drug resistance encourage the search for new drug leads.
Methods and Findings
The efficacy of the vinyl sulfone cysteine protease inhibitor K11777 was tested in the murine model of schistosomiasis mansoni. Disease parameters measured were worm and egg burdens, and organ pathology including hepato- and splenomegaly, presence of parasite egg–induced granulomas in the liver, and levels of circulating alanine aminotransferase activity as a marker of hepatocellular function. K11777 (25 mg/kg twice daily [BID]), administered intraperitoneally at the time of parasite migration through the skin and lungs (days 1–14 postinfection [p.i.]), resulted in parasitologic cure (elimination of parasite eggs) in five of seven cases and a resolution of other disease parameters. K11777 (50 mg/kg BID), administered at the commencement of egg-laying by mature parasites (days 30–37 p.i.), reduced worm and egg burdens, and ameliorated organ pathology. Using protease class-specific substrates and active-site labeling, one molecular target of K11777 was identified as the gut-associated cathepsin B1 cysteine protease, although other cysteine protease targets are not excluded. In rodents, dogs, and primates, K11777 is nonmutagenic with satisfactory safety and pharmacokinetic profiles.
Conclusions
The significant reduction in parasite burden and pathology by this vinyl sulfone cysteine protease inhibitor validates schistosome cysteine proteases as drug targets and offers the potential of a new direction for chemotherapy of human schistosomiasis.
A significant reduction in parasite burden and pathology by a vinyl sulfone cysteine protease inhibitor suggests a new direction for chemotherapy of human schistosomiasis.
Editors' Summary
Background.
Schistosomiasis, a disease caused by a type of parasitic flatworm that lives in the blood, infects around 200 million people worldwide. The disease is a serious problem in sub-Saharan Africa, South America, China, and southeast Asia. Although this disease can kill, it is better known as a lifelong chronic infection with debilitating symptoms mainly due to an immune reaction raised against parasite eggs trapped in the liver, spleen, and gut. The worm's life cycle is complicated and involves a free-swimming form that emerges from certain types of snails that live in lakes and ponds. This can penetrate the skin of people in contact with the water. After a period spent in the skin and around the lungs, the parasites move to veins around the gut, and develop into adult worms that mate and lay eggs. These eggs eventually return to the water through the person's feces or urine. A particular group of proteins called cysteine proteases are thought to be very important in the biology of these worms, especially in their function as digestive enzymes in the parasite's gut. These proteases could represent an exciting opportunity for development of new drugs to treat schistosomiasis. The researchers are looking at whether it is possible to block the activity of cysteine proteases and, as a result, kill the worms or prevent them from developing and thriving.
Why Was This Study Done?
At the moment there is only one drug, praziquantel, in common use for treatment of schistosomiasis; it is cheap and effective. However many organizations are worried about relying on a single drug to treat a serious disease which affects so many people worldwide. The research group here has been looking at molecules that block cysteine protease activity, to see if any of these could be good drug candidates for schistosomiasis. One molecule they have been looking at goes by the name of K11777, which is under evaluation as a drug candidate for another parasitic infection (Chagas' disease). Here, the researchers wanted to find out whether K11777 had any activity against schistosome worms.
What Did the Researchers Do and Find?
In this study, the researchers deliberately infected laboratory mice with the schistosome parasite. These mice were then either injected with K11777 solution twice daily, or with equivalent volumes of water as a comparison. The researchers examined the effects of injecting K11777 either “early” in infection (using a 14 day course, starting 1 day after infection with the parasite) or “late” in the worms' development (using an 8 day treatment course starting 30 days after infection). The outcomes used as measures of success of treatment with K11777 included the number of worms recovered from mice after euthanasia, the number of worm eggs counted in the liver; the extent of the damage to the liver; and finally, the researchers also looked at activity levels of cysteine proteases in the worms themselves, in particular, those proteases associated with the parasite gut.
The results of the early-treatment experiment showed a substantial decrease in worm numbers and egg production. In five of the seven mice treated, eggs were eliminated entirely. Also, there was little measurable liver damage. For the late-treatment experiment, decreased burdens of worms and eggs in the livers of K11777 treated mice were also found, and there was less damage to the livers. Those worms surviving treatment and removed from mice also had much less activity of gut cysteine proteases suggesting that K11777 exerts its effects by targeting worm cysteine proteases.
What Do These Findings Mean?
These experiments show that K11777 is a potent antischistosomal agent in mice. It might therefore be a good ‘candidate' molecule for developing future treatments for human schistosomiasis. However, before that stage can be reached, it would be important to carry out clinical trials to test whether K11777 is both safe and effective in schistosomiasis patients. Full details as to which worm cysteine protease(s) is the critical target of K11777 would also need to be worked out, and more information would be needed as to whether the dosing plan used in this study (twice-daily injections for a week to 14 days) can be decreased.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040014.
World Health Organization pages about schistosomiasis including links to details on further research into the disease
Information from the US Centers for Disease Control for patients and health professionals about schistosomiasis
Wikipedia pages on schistosomiasis (Wikipedia is an internet encyclopedia anyone can edit)
PLoS Neglected Tropical Diseases is a new journal from the Public Library of Science that is devoted to publishing research on the world's most neglected tropical diseases, including schistosomiasis
doi:10.1371/journal.pmed.0040014
PMCID: PMC1764436  PMID: 17214506
5.  Multi-Host Transmission Dynamics of Schistosoma japonicum in Samar Province, the Philippines 
PLoS Medicine  2008;5(1):e18.
Background
Among the 6.7 million people living in areas of the Philippines where infection with Schistosoma japonicum is considered endemic, even within small geographical areas levels of infection vary considerably. In general, the ecological drivers of this variability are not well described. Unlike other schistosomes, S. japonicum is known to infect several mammalian hosts. However, the relative contribution of different hosts to the transmission cycle is not well understood. Here, we characterize the transmission dynamics of S. japonicum using data from an extensive field study and a mathematical transmission model.
Methods and Findings
In this study, stool samples were obtained from 5,623 humans and 5,899 potential nonhuman mammalian hosts in 50 villages in the Province of Samar, the Philippines. These data, with variable numbers of samples per individual, were adjusted for known specificities and sensitivities of the measurement techniques before being used to estimate the parameters of a mathematical transmission model, under the assumption that the dynamic transmission processes of infection and recovery were in a steady state in each village. The model was structured to allow variable rates of transmission from different mammals (humans, dogs, cats, pigs, domesticated water buffalo, and rats) to snails and from snails to mammals. First, we held transmission parameters constant for all villages and found that no combination of mammalian population size and prevalence of infectivity could explain the observed variability in prevalence of infection between villages. We then allowed either the underlying rate of transmission (a) from snails to mammals or (b) from mammals to snails to vary by village. Our data provided substantially more support for model structure (a) than for model structure (b). Fitted values for the village-level transmission intensity from snails to mammals appeared to be strongly spatially correlated, which is consistent with results from descriptive hierarchical analyses.
Conclusions
Our results suggest that the process of acquiring mammalian S. japonicum infection is more important in explaining differences in prevalence of infection between villages than the process of snails becoming infected. Also, the contribution from water buffaloes to human S. japonicum infection in the Philippines is less important than has been recently observed for bovines in China. These findings have implications for the prioritization of mitigating interventions against S. japonicum transmission.
Obtaining schistosome infection data from thousands of humans and mammalian hosts in the Philippines, Steven Riley and colleagues find that mammalian acquisition, rather than transmission to snails, drives prevalence.
Editors' Summary
Background.
Parasitic worms called schistosomes infect about 200 million people worldwide. Different schistosome species are common in different parts of the world. Schistosoma japonicum, for example, is restricted to the Pacific region. In the Philippines, 6.7 million people live in areas where this parasite is endemic (constantly present). S. japonicum has a complex life cycle that starts with the parasite reproducing in freshwater snails. Free-swimming infectious parasites emerge from the snails and which burrow into the skin of people and other mammals (most schistosome species infect only people) when they swim in infected water. The parasites migrate to the veins draining the gut, where they mature into adult worms, mate, and lay eggs, some of which pass into the feces and back into the water where they hatch and infect fresh snails. Infection with S. japonicum does not kill many people but it causes serious health problems, including liver, lung, and gut damage.
Why Was This Study Done?
Schistosome infections can be cured with inexpensive drugs, but people living in endemic countries usually become reinfected. The only way to avoid this problem is to eliminate the parasite in the environment, but to do so the relative contributions of snails, people, and other mammals to the transmission cycle need to be understood. Villages in the Philippines have very different levels of infection with S. japonicum. This variation suggests that the location of snail colonies and water courses and the behavior of the parasite's mammalian hosts (for example, their contact with water and their sanitary habits) affect the efficiency of S. japonicum transmission. Knowing which of these factors are the most important would help public-health officials instigate effective interventions to reduce infection rates. In this study, the researchers devise a mathematical model of S. japonicum transmission and use information on the number of people and other mammals infected in several villages in the Philippines to find out what drives inter-village variability in infection.
What Did the Researchers Do and Find?
The researchers counted schistosome eggs in human and nonhuman mammalian feces collected in 50 villages in the Philippines (about 11,500 samples in total) and used these counts to estimate the parameters (values such as the rate of transmission from snails to people) for an S. japonicum transmission model. They then asked whether the proportion of the human population infected with S. japonicum predicted by the model matched actual data on infection levels in the villages. When they assumed that the transmission parameters were the same in all the villages, the variation in the number of mammalian hosts in each village could not explain the observed variation among villages in human infection levels. The researchers, therefore, tested two slightly more complex models in which the underlying rate of transmission from snails to mammals or from mammals to snails varied between villages to reflect different environmental conditions in each village. The first of these models fitted the available data best and also showed that cats, dogs, pigs, and water buffalo were less susceptible to infection with S. japonicum than people but that rats were more susceptible.
What Do These Findings Mean?
These findings suggest that the snail-to-mammal side of the S. japonicum life cycle affects the inter-village variability in human schistosome infection more than the mammal-to-snail side. The findings also indicate that the contribution of water buffaloes to human S. japonicum infection in the Philippines is not particularly important. This contrasts with a recent study that identified water buffaloes as the major mammalian reservoir for S. japonicum in China. As with all mathematical models, the findings of this one depend on the assumptions made to build the model. Nevertheless, they suggest that interventions to reduce the size of the snail population and the exposure of mammals to parasite-containing water might reduce human infection levels more effectively than interventions that interrupt other parts of the parasite's life cycle. These results also suggest that further studies of the transmission of S. japonicum by water buffalo are needed before efforts are dedicated to treat or vaccinate water buffalo as a control measure against human S. japonicum infection.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0050018.
The MedlinePlus encyclopedia has a page on schistosomiasis (in English and Spanish)
The US Centers for Disease Control and Prevention provides information for the public and professionals on schistosomiasis
The World Health Organization provides information on schistosomiasis and research into its control
doi:10.1371/journal.pmed.0050018
PMCID: PMC2211559  PMID: 18215106
6.  Diverse Host-Seeking Behaviors of Skin-Penetrating Nematodes 
PLoS Pathogens  2014;10(8):e1004305.
Skin-penetrating parasitic nematodes infect approximately one billion people worldwide and are responsible for some of the most common neglected tropical diseases. The infective larvae of skin-penetrating nematodes are thought to search for hosts using sensory cues, yet their host-seeking behavior is poorly understood. We conducted an in-depth analysis of host seeking in the skin-penetrating human parasite Strongyloides stercoralis, and compared its behavior to that of other parasitic nematodes. We found that Str. stercoralis is highly mobile relative to other parasitic nematodes and uses a cruising strategy for finding hosts. Str. stercoralis shows robust attraction to a diverse array of human skin and sweat odorants, most of which are known mosquito attractants. Olfactory preferences of Str. stercoralis vary across life stages, suggesting a mechanism by which host seeking is limited to infective larvae. A comparison of odor-driven behavior in Str. stercoralis and six other nematode species revealed that parasite olfactory preferences reflect host specificity rather than phylogeny, suggesting an important role for olfaction in host selection. Our results may enable the development of new strategies for combating harmful nematode infections.
Author Summary
Parasitic worms are a significant public health problem. Skin-penetrating worms such as hookworms and the human threadworm Strongyloides stercoralis dwell in the soil before infecting their host. However, how they locate and identify appropriate hosts is not understood. Here we investigated the host-seeking behavior of Str. stercoralis. We found that Str. stercoralis moves quickly and actively searches for hosts to infect. We also found that Str. stercoralis is attracted to human skin and sweat odorants, including many that also attract mosquitoes. We then compared olfactory behavior across parasitic worm species and found that parasites with similar hosts respond similarly to odorants even when they are not closely related, suggesting parasitic worms use olfactory cues to select hosts. A better understanding of host seeking in skin-penetrating worms may lead to novel control strategies.
doi:10.1371/journal.ppat.1004305
PMCID: PMC4133384  PMID: 25121736
7.  Differential Spatial Repositioning of Activated Genes in Biomphalaria glabrata Snails Infected with Schistosoma mansoni 
Schistosomiasis is an infectious disease infecting mammals as the definitive host and fresh water snails as the intermediate host. Understanding the molecular and biochemical relationship between the causative schistosome parasite and its hosts will be key to understanding and ultimately treating and/or eradicating the disease. There is increasing evidence that pathogens that have co-evolved with their hosts can manipulate their hosts' behaviour at various levels to augment an infection. Bacteria, for example, can induce beneficial chromatin remodelling of the host genome. We have previously shown in vitro that Biomphalaria glabrata embryonic cells co-cultured with schistosome miracidia display genes changing their nuclear location and becoming up-regulated. This also happens in vivo in live intact snails, where early exposure to miracidia also elicits non-random repositioning of genes. We reveal differences in the nuclear repositioning between the response of parasite susceptible snails as compared to resistant snails and with normal or live, attenuated parasites. Interestingly, the stress response gene heat shock protein (Hsp) 70 is only repositioned and then up-regulated in susceptible snails with the normal parasite. This movement and change in gene expression seems to be controlled by the parasite. Other differences in the behaviour of genes support the view that some genes are responding to tissue damage, for example the ferritin genes move and are up-regulated whether the snails are either susceptible or resistant and upon exposure to either normal or attenuated parasite. This is the first time host genome reorganisation has been seen in a parasitic host and only the second time for any pathogen. We believe that the parasite elicits a spatio-epigenetic reorganisation of the host genome to induce favourable gene expression for itself and this might represent a fundamental mechanism present in the human host infected with schistosome cercariae as well as in other host-pathogen relationships.
Author Summary
Bilharzia is a parasitic disease endemic in many parts of the world. The schistosoma parasite that causes Bilharzia infects humans but uses a fresh water snail as a secondary host. These two organisms have co-evolved together, and as such the parasite will have mechanisms to overcome the host defences. Understanding this delicately balanced relationship is fundamental to controlling or eradicating the disease. We have studied how this parasite can influence how the DNA within the snail behaves. We have shown snail genes have specific locations within the cell nuclei. Further, we have revealed that specific snail genes related to a schistosome infection change to a new non-random nuclear location as they are turned on or up-regulated. We have snail strains that are susceptible or resistant to the infection of parasites and we can also take live parasites and make them unable to complete an infection by irradiating them. In this unique study, we have shown a gene that is involved in stress pathways moves to a new nuclear location and becomes turned on, but only in susceptible snails, infected with fully functional parasite. Our data suggest that this gene is regulated by the parasite, which has control over the host's DNA, so that the gene is moved to an area where it can be actively expressed. We have uncovered a novel mechanism whereby the spatial organization of a host organism is interfered with by a pathogen. This type of control is probably found in other host-pathogen relationships.
doi:10.1371/journal.pntd.0003013
PMCID: PMC4161332  PMID: 25211244
8.  Immunoprotection of Mice against Schistosomiasis Mansoni Using Solubilized Membrane Antigens 
Background
Schistosomiasis continues to be one of the most prevalent parasitic diseases in the world. Despite the existence of a highly effective antischistosome drug, the disease is spreading into new areas, and national control programs do not arrive to complete their tasks particularly in low endemic areas. The availability of a vaccine could represent an additional component to chemotherapy. Experimental vaccination studies are however necessary to identify parasite molecules that would serve as vaccine candidates. In the present work, C57BL/6 female mice were subcutaneously immunized with an n-butanol extract of the adult worm particulate membranous fraction (AWBE) and its protective effect against a S. mansoni challenge infection was evaluated.
Methodology and Findings
Water-saturated n-butanol release into the aqueous phase a set of membrane-associated (glyco)proteins that are variably recognized by antibodies in schistosome-infected patients; among the previously identified AWBE antigens there is Alkaline Phosphatase (SmAP) which has been associated with resistance to the infection in mice. As compared to control, a significantly lower number of perfuse parasites was obtained in the immunized/challenged mouse group (P<0.05, t test); and consequently, a lower number of eggs and granulomas (with reduced sizes), overall decreasing pathology. Immunized mice produced high levels of sera anti-AWBE IgG recognizing antigens of ∼190-, 130-, 98-, 47-, 28-23, 14-, and 9-kDa. The ∼130-kDa band (the AP dimer) exhibited in situ SmAP activity after addition of AP substrate and the activity was not apparently inhibited by host antibodies. A preliminary proteomic analysis of the 25-, 27-, and 28-kDa bands in the immunodominant 28–23 kDa region suggested that they are composed of actin.
Conclusions
Immunization with AWBE induced the production of specific antibodies to various adult worm membrane molecules (including AP) and a partial (43%) protection against a challenging S. mansoni infection by mechanism(s) that still has to be elucidated.
Author Summary
Schistosomiasis is a neglected disease affecting more than 200 million people globally, especially in sub-Saharan Africa. The mainstay of control of schistosomiasis is Praziquantel, but the mass administration of this drug is unsustainable due to the high rates of re-infection after treatment. These high rates of re-infection point towards the potential emergence of schistosoma drug resistance, making the anti-schistosome vaccine an essential component for the future control of schistosomiasis, as an adjunct to chemotherapy. Multiple strategies have been used to develop an anti-schistosome vaccine with different levels of success. These studies found that the tegument is the most important source of protective antigens; a logical assumption considering this structure represents the surface where the parasite and host interact. In our laboratory, we have isolated a (glyco)protein extract (AWBE) from the whole membrane fraction of adult worms, which is enriched by enzymatic and somatic antigens. Some of these antigens are recognized by infected patients and by mice immunized with irradiated cercariae. Given this context, we tested the possible protective effect of AWBE in mice. The results showed that immunization with AWBE induced a strong humoral response (IgG) with 43% protection against a challenge infection. The AWBE-vaccinated mice showed specific recognition of epitopes in identified proteins, such as schistosome phosphatase and probably actin, pointing to a possible association of these antigens with immunoprotection. These antigens may join the gallery of candidate proteins for vaccination against the infection by schistosomes.
doi:10.1371/journal.pntd.0002254
PMCID: PMC3688544  PMID: 23818994
9.  Induced Release of a Plant-Defense Volatile ‘Deceptively’ Attracts Insect Vectors to Plants Infected with a Bacterial Pathogen 
PLoS Pathogens  2012;8(3):e1002610.
Transmission of plant pathogens by insect vectors is a complex biological process involving interactions between the plant, insect, and pathogen. Pathogen-induced plant responses can include changes in volatile and nonvolatile secondary metabolites as well as major plant nutrients. Experiments were conducted to understand how a plant pathogenic bacterium, Candidatus Liberibacter asiaticus (Las), affects host preference behavior of its psyllid (Diaphorina citri Kuwayama) vector. D. citri were attracted to volatiles from pathogen-infected plants more than to those from non-infected counterparts. Las-infected plants were more attractive to D. citri adults than non-infected plants initially; however after feeding, psyllids subsequently dispersed to non-infected rather than infected plants as their preferred settling point. Experiments with Las-infected and non-infected plants under complete darkness yielded similar results to those recorded under light. The behavior of psyllids in response to infected versus non-infected plants was not influenced by whether or not they were carriers of the pathogen. Quantification of volatile release from non-infected and infected plants supported the hypothesis that odorants mediate psyllid preference. Significantly more methyl salicylate, yet less methyl anthranilate and D-limonene, was released by infected than non-infected plants. Methyl salicylate was attractive to psyllids, while methyl anthranilate did not affect their behavior. Feeding on citrus by D. citri adults also induced release of methyl salicylate, suggesting that it may be a cue revealing location of conspecifics on host plants. Infected plants were characterized by lower levels of nitrogen, phosphorus, sulfur, zinc, and iron, as well as, higher levels of potassium and boron than non-infected plants. Collectively, our results suggest that host selection behavior of D. citri may be modified by bacterial infection of plants, which alters release of specific headspace volatiles and plant nutritional contents. Furthermore, we show in a laboratory setting that this apparent pathogen-mediated manipulation of vector behavior may facilitate pathogen spread.
Author Summary
In this investigation, we experimentally demonstrate specific mechanisms through which a bacterial plant pathogen induces plant responses that modify behavior of its insect vector. Candidatus Liberibacter asiaticus, a fastidious, phloem-limited bacterium responsible for causing huanglongbing disease of citrus, induced release of a specific volatile chemical, methyl salicylate, which increased attractiveness of infected plants to its insect vector, Diaphorina citri, and caused vectors to initially prefer infected plants. However, the insect vectors subsequently dispersed to non-infected plants as their preferred location of prolonged settling because of likely sub-optimal nutritional content of infected plants. The duration of initial feeding on infected plants was sufficiently long for the vectors to acquire the pathogen before they dispersed to non-infected plants, suggesting that the bacterial pathogen manipulates behavior of its insect vector to promote its own proliferation. The behavior of psyllids in response to infected versus non-infected plants was not influenced by whether or not they were carriers of the pathogen and was similar under both light and dark conditions. Feeding on citrus by D. citri adults also induced the release of methyl salicylate, suggesting that it may be a cue revealing location of conspecifics on host plants.
doi:10.1371/journal.ppat.1002610
PMCID: PMC3310815  PMID: 22457628
10.  Identification of Leishmania Proteins Preferentially Released in Infected Cells Using Change Mediated Antigen Technology (CMAT) 
Although Leishmania parasites have been shown to modulate their host cell's responses to multiple stimuli, there is limited evidence that parasite molecules are released into infected cells. In this study, we present an implementation of the change mediated antigen technology (CMAT) to identify parasite molecules that are preferentially expressed in infected cells. Sera from mice immunized with cell lysates prepared from L. donovani or L. pifanoi-infected macrophages were adsorbed with lysates of axenically grown amastigotes of L. donovani or L. pifanoi, respectively, as well as uninfected macrophages. The sera were then used to screen inducible parasite expression libraries constructed with genomic DNA. Eleven clones from the L. pifanoi and the L. donovani screen were selected to evaluate the characteristics of the molecules identified by this approach. The CMAT screen identified genes whose homologs encode molecules with unknown function as well as genes that had previously been shown to be preferentially expressed in the amastigote form of the parasite. In addition a variant of Tryparedoxin peroxidase that is preferentially expressed within infected cells was identified. Antisera that were then raised to recombinant products of the clones were used to validate that the endogenous molecules are preferentially expressed in infected cells. Evaluation of the distribution of the endogenous molecules in infected cells showed that some of these molecules are secreted into parasitophorous vacuoles (PVs) and that they then traffic out of PVs in vesicles with distinct morphologies. This study is a proof of concept study that the CMAT approach can be applied to identify putative Leishmania parasite effectors molecules that are preferentially expressed in infected cells. In addition we provide evidence that Leishmania molecules traffic out of the PV into the host cell cytosol and nucleus.
Author Summary
Leishmania are intracellular parasites that reside within parasitophorous vacuoles (PV) in phagocytes. From within these compartments parasites control the host cell's responses to multiple stimuli. There is limited knowledge of the molecules that Leishmania parasites elaborate in the host cell to target processes therein. Furthermore, the mechanism by which such molecules would access their targets beyond the PV is not known. In the study presented here, we implemented the change mediated antigen technology (CMAT) to identify parasite molecules that are preferentially expressed inside infected cells. The approach was based on the reasoning that parasites express ‘new’ or antigenically modified molecules in the intracellular environment; therefore antiserum that is reactive to infected cells would contain immunoglobulins that are specific to these ‘new’ molecules. After adsorption of the antiserum with axenically cultured parasites, the antiserum was used to screen a parasite genomic expression library to identify genes encoding the molecules that are preferentially expressed in infected cells. We present for the first time evidence that some of these CMAT molecules accumulate in the PV and then traffic into the host cell in vesicles of distinct morphologies. Furthermore, several of these parasite molecules become localized in discrete compartments within the host cell.
doi:10.1371/journal.pntd.0000842
PMCID: PMC2950143  PMID: 20957202
11.  Erythrocyte G Protein as a Novel Target for Malarial Chemotherapy 
PLoS Medicine  2006;3(12):e528.
Background
Malaria remains a serious health problem because resistance develops to all currently used drugs when their parasite targets mutate. Novel antimalarial drug targets are urgently needed to reduce global morbidity and mortality. Our prior results suggested that inhibiting erythrocyte Gs signaling blocked invasion by the human malaria parasite Plasmodium falciparum.
Methods and Findings
We investigated the erythrocyte guanine nucleotide regulatory protein Gs as a novel antimalarial target. Erythrocyte “ghosts” loaded with a Gs peptide designed to block Gs interaction with its receptors, were blocked in β-adrenergic agonist-induced signaling. This finding directly demonstrates that erythrocyte Gs is functional and that propranolol, an antagonist of G protein–coupled β-adrenergic receptors, dampens Gs activity in erythrocytes. We subsequently used the ghost system to directly link inhibition of host Gs to parasite entry. In addition, we discovered that ghosts loaded with the peptide were inhibited in intracellular parasite maturation. Propranolol also inhibited blood-stage parasite growth, as did other β2-antagonists. β-blocker growth inhibition appeared to be due to delay in the terminal schizont stage. When used in combination with existing antimalarials in cell culture, propranolol reduced the 50% and 90% inhibitory concentrations for existing drugs against P. falciparum by 5- to 10-fold and was also effective in reducing drug dose in animal models of infection.
Conclusions
Together these data establish that, in addition to invasion, erythrocyte G protein signaling is needed for intracellular parasite proliferation and thus may present a novel antimalarial target. The results provide proof of the concept that erythrocyte Gs antagonism offers a novel strategy to fight infection and that it has potential to be used to develop combination therapies with existing antimalarials.
Erythrocyte G protein signaling is needed for intracellular malarial parasite proliferation and thus may present a novel antimalarial target.
Editors' Summary
Background.
New drugs for treatment of malaria are urgently needed, because the malaria parasite has evolved resistance against virtually all types of commonly used drugs. When a person is bitten by a malaria-infected mosquito, the parasite first infects the person's liver cells before going on to infect red blood cells, where the parasites multiply and develop into a parasite stage called a schizont. The red blood cells then burst and release more schizonts into the bloodstream; it is this “blood stage” of infection in humans that causes the symptoms of disease. Therefore efforts to develop new drugs against malaria often focus on this “blood stage” of infection. One strategy for developing new drugs is termed the “host-targeted” approach. This means that rather than trying to block processes occurring within the parasite itself, a drug can be developed which blocks processes within the person's red blood cells, and which would otherwise be needed for the parasite to complete its life cycle. It will be difficult for malaria parasites to evolve resistance to such a drug, because changes in a person's red blood cells occur much more slowly than in the parasites themselves.
Why Was This Study Done?
This research group has been studying a set of molecular processes within human red blood cells which seemed to be required for entry of malaria parasites into the cells. They wanted to get a better understanding of those processes and, specifically, to find out whether it would be possible to use particular molecules to block those processes, and by doing so to prevent malaria parasites from entering and multiplying within red blood cells. In particular, when the malaria parasites invade the red blood cell, they form membranes around the red blood cell, containing lipids and proteins “hijacked” from the red blood cell membrane. These researchers already knew that two particular proteins were hijacked in this way; the β2-adrenergic receptor (β2-AR) and heterotrimeric G protein (Gs). These two proteins act together to pass messages across the surface of the membrane to inside the cell. Small molecules could be used to block signaling through β2-AR and Gs, and therefore potentially to provide a new way of preventing malaria parasites from entering red blood cells and multiplying within them.
What Did the Researchers Do and Find?
Firstly, the researchers made red blood cell “ghosts” in which to study these molecular processes. This meant that they took fresh red blood cells from healthy human volunteers, burst them to remove half the contents and loaded them with markers and other cargoes before resealing the membranes of the cell. These resealed markers and cargoes allowed them to see what was happening inside the cells. Malaria parasites were able to invade these ghosts normally and multiply within them. When the researchers introduced a specific peptide (a molecule consisting of a short series of amino acids), they found that it blocked Gs signaling within the ghosts. This peptide also prevented malaria parasites from developing inside the ghosts. Therefore, they concluded that Gs signaling inside the red blood cell was important for the parasite life cycle. The researchers then examined a drug called propranolol which is already known to act on Gs signaling and which is commonly prescribed for high blood pressure. This drug also blocked development of malaria parasites inside the ghosts when used at a particular concentration. Finally, the researchers studied the effect of giving propranolol, along with other antimalarial drugs, to human malaria parasites in a culture dish and to mice injected with a malaria parasite that infects rodents. In these experiments, adding propranolol reduced the amount of other “parasite-targeted” drugs that were needed to effectively treat malarial infection in tissue culture and in mice.
What Do These Findings Mean?
Showing that the Gs signaling pathway is important for the malaria parasite's life cycle opens up new possibilities for drug development. Specifically, propranolol (which is already approved for treatment of high blood pressure and other conditions) might itself provide a new candidate therapy, either alone or in combination with existing drugs. These combinations would first, however, need to be tested in human clinical trials, perhaps by seeing whether they have antimalarial activity in people who have not responded to existing antimalarial drugs. Since it acts to lower blood pressure, which can already be low in some people with malaria, there are some concerns that propranolol might not be a suitable drug candidate for use, especially with existing antimalarial drugs that also reduce blood pressure. However, other molecules which block Gs signaling could be tested for activity against malaria should propranolol prove not to be an ideal drug candidate.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030528.
The World Health Organization publishes a minisite containing links to information about all aspects of malaria worldwide, including treatment, prevention, and current programs for malaria control
Medicines for Malaria Venture is a collaboration between public and private organizations (including the pharmaceutical industry) that aims to fund and manage the development of new drugs for treatment and prevention of malaria
Wikipedia entries for drug discovery and drug development (Wikipedia is an internet encyclopedia that anyone can edit)
doi:10.1371/journal.pmed.0030528
PMCID: PMC1716186  PMID: 17194200
12.  The Neurotropic Parasite Toxoplasma Gondii Increases Dopamine Metabolism 
PLoS ONE  2011;6(9):e23866.
The highly prevalent parasite Toxoplasma gondii manipulates its host's behavior. In infected rodents, the behavioral changes increase the likelihood that the parasite will be transmitted back to its definitive cat host, an essential step in completion of the parasite's life cycle. The mechanism(s) responsible for behavioral changes in the host is unknown but two lines of published evidence suggest that the parasite alters neurotransmitter signal transduction: the disruption of the parasite-induced behavioral changes with medications used to treat psychiatric disease (specifically dopamine antagonists) and identification of a tyrosine hydroxylase encoded in the parasite genome. In this study, infection of mammalian dopaminergic cells with T. gondii enhanced the levels of K+-induced release of dopamine several-fold, with a direct correlation between the number of infected cells and the quantity of dopamine released. Immunostaining brain sections of infected mice with dopamine antibody showed intense staining of encysted parasites. Based on these analyses, T. gondii orchestrates a significant increase in dopamine metabolism in neural cells. Tyrosine hydroxylase, the rate-limiting enzyme for dopamine synthesis, was also found in intracellular tissue cysts in brain tissue with antibodies specific for the parasite-encoded tyrosine hydroxylase. These observations provide a mechanism for parasite-induced behavioral changes. The observed effects on dopamine metabolism could also be relevant in interpreting reports of psychobehavioral changes in toxoplasmosis-infected humans.
doi:10.1371/journal.pone.0023866
PMCID: PMC3177840  PMID: 21957440
13.  Parasite virulence when the infection reduces the host immune response 
Parasite infections often induce a reduction in host immune response either because of a direct manipulation of the immune system by the parasite or because of energy depletion. Although infection-induced immunodepression can favour the establishment of the parasite within the host, a too severe immunodepression may increase the risk of infection with opportunistic pathogens, stopping the period over which the parasite can be transmitted to other hosts. Here, we explore how the risk of contracting opportunistic diseases affects the survival of the amphipod Gammarus pulex infected by the acanthocephalan Pomphorhynchus laevis. Previous work with this system has shown that upon infection, G. pulex has a substantially reduced immune response. Non-infected and P. laevis-infected hosts were maintained either in control or in micro-organism-enriched water, so as to vary the risk of encountering opportunistic pathogens. As predicted, we found that host mortality was exacerbated when infected gammarids were maintained in micro-organism-enriched water compared with clean, control water; whereas for non-infected gammarids, living in micro-organism-enriched water only moderately increased the risk of mortality. These results show that the virulence of parasites that reduce the host immune response is an environmentally sensitive trait that depends on the concomitant risk for the host of contracting opportunistic diseases. This extra source of host mortality probably represents a cost for P. laevis, and we tentatively predict that the optimal level of parasite exploitation should depend on environmental conditions.
doi:10.1098/rspb.2010.0138
PMCID: PMC2871884  PMID: 20200031
disease ecology; Pomphorhynchus laevis; immunodepression; opportunistic pathogens; virulence
14.  An Analysis of Genetic Diversity and Inbreeding in Wuchereria bancrofti: Implications for the Spread and Detection of Drug Resistance 
Estimates of genetic diversity in helminth infections of humans often have to rely on genotyping (immature) parasite transmission stages instead of adult worms. Here we analyse the results of one such study investigating a single polymorphic locus (a change at position 200 of the β-tubulin gene) in microfilariae of the lymphatic filarial parasite Wuchereria bancrofti. The presence of this genetic change has been implicated in benzimidazole resistance in parasitic nematodes of farmed ruminants. Microfilariae were obtained from patients of three West African villages, two of which were sampled prior to the introduction of mass drug administration. An individual-based stochastic model was developed showing that a wide range of allele frequencies in the adult worm populations could have generated the observed microfilarial genetic diversity. This suggests that appropriate theoretical null models are required in order to interpret studies that genotype transmission stages. Wright's hierarchical F-statistic was used to investigate the population structure in W. bancrofti microfilariae and showed significant deficiency of heterozygotes compared to the Hardy-Weinberg equilibrium; this may be partially caused by a high degree of parasite genetic differentiation between hosts. Studies seeking to quantify accurately the genetic diversity of helminth populations by analysing transmission stages should increase their sample size to account for the variability in allele frequency between different parasite life-stages. Helminth genetic differentiation between hosts and non-random mating will also increase the number of hosts (and the number of samples per host) that need to be genotyped, and could enhance the rate of spread of anthelmintic resistance.
Author Summary
During the last decade, there has been a substantial increase in the use of mass drug administration to reduce the disease caused by parasitic worms. With so many people regularly receiving treatment, there is a risk that drug resistance may develop. As a result, the number of studies looking for genetic markers of drug resistance has increased noticeably. In this paper we analyse the results of one such study that investigated the presence of genes associated with drug resistance in parasites responsible for elephantiasis. This study, like many other studies of human parasitic infections, relies on analysing parasite immature stages (such as eggs) because the adult worms are often inaccessible within the human body. Using computer models we show how the gene frequency in the immature stages may vary from that in the adult worm population. Parasites with these markers for drug resistance might also be unevenly distributed across the host population even prior to treatment. This may increase the spread of drug resistance and make it harder to detect. We suggest that studies conducted only on parasite immature stages should be interpreted with caution and should carefully consider the number of people and the number of parasites they analyse.
doi:10.1371/journal.pntd.0000211
PMCID: PMC2275205  PMID: 18382607
15.  Experimental demonstration of a parasite-induced immune response in wild birds: Darwin's finches and introduced nest flies 
Ecology and Evolution  2013;3(8):2514-2523.
Abstract
Ecological immunology aims to explain variation among hosts in the strength and efficacy of immunological defenses. However, a shortcoming has been the failure to link host immune responses to actual parasites under natural conditions. Here, we present one of the first experimental demonstrations of a parasite-induced immune response in a wild bird population. The recently introduced ectoparasitic nest fly Philornis downsi severely impacts the fitness of Darwin's finches and other land birds in the Galápagos Islands. An earlier study showed that female medium ground finches (Geospiza fortis) had P. downsi-binding antibodies correlating with presumed variation in fly exposure over time. In the current study, we experimentally manipulated fly abundance to test whether the fly does, in fact, cause changes in antibody levels. We manipulated P. downsi abundance in nests and quantified P. downsi-binding antibody levels of medium ground finch mothers, fathers, and nestlings. We also quantified host behaviors, such as preening, which can integrate with antibody-mediated defenses against ectoparasites. Philornis downsi-binding antibody levels were significantly higher among mothers at parasitized nests, compared to mothers at (fumigated) nonparasitized nests. Mothers with higher antibody levels tended to have fewer parasites in their nests, suggesting that antibodies play a role in defense against parasites. Mothers showed no behavioral changes that would enhance the effectiveness of the immune response. Neither adult males, nor nestlings, had P. downsi-induced immunological or behavioral responses that would enhance defense against flies. None of the parasitized nests fledged any offspring, despite the immune response by mothers. Thus, this study shows that, while the immune response of mothers appeared to be defensive, it was not sufficient to rescue current reproductive fitness. This study further shows the importance of testing the fitness consequences of immune defenses, rather than assuming that such responses increase host fitness.
Host immune responses can protect against the negative fitness consequences of parasitism; however, the strength and effectiveness of these responses vary among hosts. Strong host immune responses are often assumed to correlate with greater host fitness. This study investigates the relationship between host immune response, parasite load, and host fitness using Darwin's finches and an invasive nest parasite. We found that while the immune response of mothers appeared defensive, it did not rescue current reproductive fitness.
doi:10.1002/ece3.651
PMCID: PMC3930052  PMID: 24567824
Antibody; defense; ecoimmunology; Geospiza fortis; invasive species; Philornis downsi
16.  Conflict between parasites with different transmission strategies infecting an amphipod host 
Competition between parasites within a host can influence the evolution of parasite virulence and host resistance, but few studies examine the effects of unrelated parasites with conflicting transmission strategies infecting the same host. Vertically transmitted (VT) parasites, transmitted from mother to offspring, are in conflict with virulent, horizontally transmitted (HT) parasites, because healthy hosts are necessary to maximize VT parasite fitness. Resolution of the conflict between these parasites should lead to the evolution of one of two strategies: avoidance, or sabotage of HT parasite virulence by the VT parasite. We investigated two co-infecting parasites in the amphipod host, Gammarus roeseli: VT microsporidia have little effect on host fitness, but acanthocephala modify host behaviour, increasing the probability that the amphipod is predated by the acanthocephalan's definitive host. We found evidence for sabotage: the behavioural manipulation induced by the Acanthocephala Polymorphus minutus was weaker in hosts also infected by the microsporidia Dictyocoela sp. (roeselum) compared to hosts infected by P. minutus alone. Such conflicts may explain a significant portion of the variation generally observed in behavioural measures, and since VT parasites are ubiquitous in invertebrates, often passing undetected, conflict via transmission may be of great importance in the study of host–parasite relationships.
doi:10.1098/rspb.2005.3244
PMCID: PMC1599785  PMID: 16271976
Acanthocephala; microsporidia; Gammarus roeseli; conflict; parasite transmission; behavioural manipulation
17.  Do Parasitic Trematode Cercariae Demonstrate a Preference for Susceptible Host Species? 
PLoS ONE  2012;7(12):e51012.
Many parasites are motile and exhibit behavioural preferences for certain host species. Because hosts can vary in their susceptibility to infections, parasites might benefit from preferentially detecting and infecting the most susceptible host, but this mechanistic hypothesis for host-choice has rarely been tested. We evaluated whether cercariae (larval trematode parasites) prefer the most susceptible host species by simultaneously presenting cercariae with four species of tadpole hosts. Cercariae consistently preferred hosts in the following order: Anaxyrus ( = Bufo) terrestris (southern toad), Hyla squirella (squirrel tree frog), Lithobates ( = Rana) sphenocephala (southern leopard frog), and Osteopilus septentrionalis (Cuban tree frog). These host species varied in susceptibility to cercariae in an order similar to their attractiveness with a correlation that approached significance. Host attractiveness to parasites also varied consistently and significantly among individuals within a host species. If heritable, this individual-level host variation would represent the raw material upon which selection could act, which could promote a Red Queen “arms race” between host cues and parasite detection of those cues. If, in general, motile parasites prefer to infect the most susceptible host species, this phenomenon could explain aggregated distributions of parasites among hosts and contribute to parasite transmission rates and the evolution of virulence. Parasite preferences for hosts belie the common assumption of disease models that parasites seek and infect hosts at random.
doi:10.1371/journal.pone.0051012
PMCID: PMC3525650  PMID: 23272084
18.  Empirical Support for Optimal Virulence in a Castrating Parasite 
PLoS Biology  2006;4(7):e197.
The trade-off hypothesis for the evolution of virulence predicts that parasite transmission stage production and host exploitation are balanced such that lifetime transmission success (LTS) is maximised. However, the experimental evidence for this prediction is weak, mainly because LTS, which indicates parasite fitness, has been difficult to measure. For castrating parasites, this simple model has been modified to take into account that parasites convert host reproductive resources into transmission stages. Parasites that kill the host too early will hardly benefit from these resources, while postponing the killing of the host results in diminished returns. As predicted from optimality models, a parasite inducing castration should therefore castrate early, but show intermediate levels of virulence, where virulence is measured as time to host killing. We studied virulence in an experimental system where a bacterial parasite castrates its host and produces spores that are not released until after host death. This permits estimating the LTS of the parasite, which can then be related to its virulence. We exposed replicate individual Daphnia magna (Crustacea) of one host clone to the same amount of bacterial spores and followed individuals until their death. We found that the parasite shows strong variation in the time to kill its host and that transmission stage production peaks at an intermediate level of virulence. A further experiment tested for the genetic basis of variation in virulence by comparing survival curves of daphniids infected with parasite spores obtained from early killing versus late killing infections. Hosts infected with early killer spores had a significantly higher death rate as compared to those infected with late killers, indicating that variation in time to death was at least in part caused by genetic differences among parasites. We speculate that the clear peak in lifetime reproductive success at intermediate killing times may be caused by the exceptionally strong physiological trade-off between host and parasite reproduction. This is the first experimental study to demonstrate that the production of propagules is highest at intermediate levels of virulence and that parasite genetic variability is available to drive the evolution of virulence in this system.
Exposing replicate Daphnia hosts to the same amount of bacterial spores from the castrating bacterium Pasteuria ramose provides experimental evidence that parasite fitness is maximized at intermediate levels of virulence.
doi:10.1371/journal.pbio.0040197
PMCID: PMC1470460  PMID: 16719563
19.  TGF-β Signaling Controls Embryo Development in the Parasitic Flatworm Schistosoma mansoni 
PLoS Pathogens  2007;3(4):e52.
Over 200 million people have, and another 600 million are at risk of contracting, schistosomiasis, one of the major neglected tropical diseases. Transmission of this infection, which is caused by helminth parasites of the genus Schistosoma, depends upon the release of parasite eggs from the human host. However, approximately 50% of eggs produced by schistosomes fail to reach the external environment, but instead become trapped in host tissues where pathological changes caused by the immune responses to secreted egg antigens precipitate disease. Despite the central importance of egg production in transmission and disease, relatively little is understood of the molecular processes underlying the development of this key life stage in schistosomes. Here, we describe a novel parasite-encoded TGF-β superfamily member, Schistosoma mansoni Inhibin/Activin (SmInAct), which is key to this process. In situ hybridization localizes SmInAct expression to the reproductive tissues of the adult female, and real-time RT-PCR analyses indicate that SmInAct is abundantly expressed in ovipositing females and the eggs they produce. Based on real-time RT-PCR analyses, SmInAct transcription continues, albeit at a reduced level, both in adult worms isolated from single-sex infections, where reproduction is absent, and in parasites from IL-7R−/− mice, in which viable egg production is severely compromised. Nevertheless, Western analyses demonstrate that SmInAct protein is undetectable in parasites from single-sex infections and from infections of IL-7R−/− mice, suggesting that SmInAct expression is tightly linked to the reproductive potential of the worms. A crucial role for SmInAct in successful embryogenesis is indicated by the finding that RNA interference–mediated knockdown of SmInAct expression in eggs aborts their development. Our results demonstrate that TGF-β signaling plays a major role in the embryogenesis of a metazoan parasite, and have implications for the development of new strategies for the treatment and prevention of an important and neglected human disease.
Author Summary
Schistosomes are parasitic worms that infect hundreds of millions of people in developing countries. They cause disease by virtue of the fact that the eggs that they produce, which are intended for release from the host in order to allow transmission of infection, can become trapped in target organs such as the liver, where they induce damaging inflammation. Egg production by female schistosomes is critically dependent on the presence of male parasites, without which females never fully develop, and (counterintuitively) on the contribution of signals from the host's immune system. Very little is understood about the molecular basis of these interactions. Here, we describe a newly discovered schistosome gene, which is expressed in the reproductive tract of the female parasite and in parasite eggs. The protein encoded by this gene is made only when females are paired with males in an immunologically competent setting. Using recently developed tools that allow gene function to be inhibited in schistosomes, we show that the product of this gene plays a crucial role in egg development. Examining how the expression of this gene is controlled has the potential to provide insight into the molecular nature of the interactions between male and female parasites and their hosts. Moreover, the pivotal role of this gene in the egg makes it a potential target for blocking transmission and disease development.
doi:10.1371/journal.ppat.0030052
PMCID: PMC1847691  PMID: 17411340
20.  Toxoplasma gondii Rhoptry Kinase ROP16 Activates STAT3 and STAT6 Resulting in Cytokine Inhibition and Arginase-1-Dependent Growth Control 
PLoS Pathogens  2011;7(9):e1002236.
The ROP16 kinase of Toxoplasma gondii is injected into the host cell cytosol where it activates signal transducer and activator of transcription (STAT)-3 and STAT6. Here, we generated a ROP16 deletion mutant on a Type I parasite strain background, as well as a control complementation mutant with restored ROP16 expression. We investigated the biological role of the ROP16 molecule during T. gondii infection. Infection of mouse bone marrow-derived macrophages with rop16-deleted (ΔROP16) parasites resulted in increased amounts of IL-12p40 production relative to the ROP16-positive RH parental strain. High level IL-12p40 production in ΔROP16 infection was dependent on the host cell adaptor molecule MyD88, but surprisingly was independent of any previously recognized T. gondii triggered pathway linking to MyD88 (TLR2, TLR4, TLR9, TLR11, IL-1ß and IL-18). In addition, ROP16 was found to mediate the suppressive effects of Toxoplasma on LPS-induced cytokine synthesis in macrophages and on IFN-γ-induced nitric oxide production by astrocytes and microglial cells. Furthermore, ROP16 triggered synthesis of host cell arginase-1 in a STAT6-dependent manner. In fibroblasts and macrophages, failure to induce arginase-1 by ΔROP16 tachyzoites resulted in resistance to starvation conditions of limiting arginine, an essential amino acid for replication and virulence of this parasite. ΔROP16 tachyzoites that failed to induce host cell arginase-1 displayed increased replication and dissemination during in vivo infection. We conclude that encounter between Toxoplasma ROP16 and the host cell STAT signaling cascade has pleiotropic downstream effects that act in multiple and complex ways to direct the course of infection.
Author Summary
Toxoplasma gondii is an extremely widespread intracellular protozoan parasite that establishes long-lasting infection in humans and animals. Because Toxoplasma infection is most often asymptomatic, it is evident that this parasite has developed sophisticated ways to manipulate host immunity. Recently, the parasite ROP16 kinase was identified as an important determinant of host cell signaling. During cell invasion, ROP16 is injected into the host cell cytoplasm and subsequently localizes to the nucleus. Here, we report the generation of ROP16 knockout parasites (ΔROP16) as well as ΔROP16 complementation mutants (ΔROP16:1) and we describe the biological effects of deleting and re-inserting this molecule. We find that ROP16 controls the ability to activate multiple host cell signaling pathways and simultaneously suppress macrophage proinflammatory responses. Deletion of ROP16 increases parasite ability to replicate and disseminate during in vivo infection. This increased growth response may arise from ROP16-dependent activation of host arginase-1. Induction of arginase-1 limits availability of arginine, an amino acid that is required for parasite growth and host-inducible nitric oxide production. Our results provide new insight into the complex interactions between an intracellular eukaryotic pathogen and its host cell.
doi:10.1371/journal.ppat.1002236
PMCID: PMC3169547  PMID: 21931552
21.  Serine Protease(s) Secreted by the Nematode Trichuris muris Degrade the Mucus Barrier 
The polymeric mucin component of the intestinal mucus barrier changes during nematode infection to provide not only physical protection but also to directly affect pathogenic nematodes and aid expulsion. Despite this, the direct interaction of the nematodes with the mucins and the mucus barrier has not previously been addressed. We used the well-established Trichuris muris nematode model to investigate the effect on mucins of the complex mixture of immunogenic proteins secreted by the nematode called excretory/secretory products (ESPs). Different regimes of T. muris infection were used to simulate chronic (low dose) or acute (high dose) infection. Mucus/mucins isolated from mice and from the human intestinal cell line, LS174T, were treated with ESPs. We demonstrate that serine protease(s) secreted by the nematode have the ability to change the properties of the mucus barrier, making it more porous by degrading the mucin component of the mucus gel. Specifically, the serine protease(s) acted on the N-terminal polymerising domain of the major intestinal mucin Muc2, resulting in depolymerisation of Muc2 polymers. Importantly, the respiratory/gastric mucin Muc5ac, which is induced in the intestine and is critical for worm expulsion, was protected from the depolymerising effect exerted by ESPs. Furthermore, serine protease inhibitors (Serpins) which may protect the mucins, in particular Muc2, from depolymerisation, were highly expressed in mice resistant to chronic infection. Thus, we demonstrate that nematodes secrete serine protease(s) to degrade mucins within the mucus barrier, which may modify the niche of the parasite to prevent clearance from the host or facilitate efficient mating and egg laying from the posterior end of the parasite that is in intimate contact with the mucus barrier. However, during a TH2-mediated worm expulsion response, serpins, Muc5ac and increased levels of Muc2 protect the barrier from degradation by the nematode secreted protease(s).
Author Summary
Gastrointestinal parasitic worm infections cause significant morbidity, affecting up to a third of the world's populationand their domestic pets and livestock. Mucus, the gel-like material that blankets the surface of the intestine, forms a protective barrier that is an important part of our innate immune system. The whipworm Trichuris is closely associated with the intestinal mucus barrier. The major structural component of this barrier, large glycoproteins known as mucins play a significant role in the expulsion of these worms in a mouse model. Using mice that get longterm chronic infections and others able to expel the worms from the intestine, we uncover a novel role for products secreted by the worms. Enzymes secreted by whipworms can disrupt the mucin network that gives mucus its viscous properties. Moreover, we unravel that worm products are unable to degrade forms of mucins present in the mucus barrier during worm expulsion, suggesting that these enzymes may be released by the worm as part of its regime to improve its niche and survival in the host. However, the host is capable of producing mucins and other protective molecules that protect the mucus barrier from degradation and are detrimental to the viability of the worm.
doi:10.1371/journal.pntd.0001856
PMCID: PMC3469553  PMID: 23071854
22.  Parasite-induced aggression and impaired contest ability in a fish host 
Parasites & Vectors  2010;3:17.
Background
Success of trophically transmitted parasites depends to a great extent on their ability to manipulate their intermediate hosts in a way that makes them easier prey for target hosts. Parasite-induced behavioural changes are the most spectacular and diverse examples of manipulation. Most of the studies have been focused on individual behaviour of hosts including fish. We suggest that agonistic interactions and territoriality in fish hosts may affect their vulnerability to predators and thus the transmission efficiency of trophically transmitted parasites. The parasite Diplostomum spathaceum (Trematoda) and juvenile rainbow trout, Oncorhynchus mykiss, were used to study whether infection can alter aggression rates and territorial behaviour of intermediate fish hosts.
Results
The changes in behaviour of rainbow trout, Oncorhynchus mykiss, infected with an eye fluke Diplostomum spathaceum (Trematoda), was monitored over the course of an experimental infection for 1.5 months. At the beginning of their development, not yet infective D. spathaceum metacercariae decreased the aggressiveness of rainbow trout. By the time that metacercariae were fully infective to their definitive hosts, the aggressiveness increased and exceeded that of control fish. Despite the increased aggressiveness, the experimentally infected fish lost contests for a territory (dark parts of the bottom) against the control fish.
Conclusions
The results obtained indicate that the parasitized fish pay the cost of aggressiveness without the benefit of acquiring a territory that would provide them with better protection against predators. This behaviour should increase transmission of the parasite as expected by the parasite manipulation hypothesis.
doi:10.1186/1756-3305-3-17
PMCID: PMC2845576  PMID: 20226098
23.  Trypanosoma brucei Modifies the Tsetse Salivary Composition, Altering the Fly Feeding Behavior That Favors Parasite Transmission 
PLoS Pathogens  2010;6(6):e1000926.
Tsetse flies are the notorious transmitters of African trypanosomiasis, a disease caused by the Trypanosoma parasite that affects humans and livestock on the African continent. Metacyclic infection rates in natural tsetse populations with Trypanosoma brucei, including the two human-pathogenic subspecies, are very low, even in epidemic situations. Therefore, the infected fly/host contact frequency is a key determinant of the transmission dynamics. As an obligate blood feeder, tsetse flies rely on their complex salivary potion to inhibit host haemostatic reactions ensuring an efficient feeding. The results of this experimental study suggest that the parasite might promote its transmission through manipulation of the tsetse feeding behavior by modifying the saliva composition. Indeed, salivary gland Trypanosoma brucei-infected flies display a significantly prolonged feeding time, thereby enhancing the likelihood of infecting multiple hosts during the process of a single blood meal cycle. Comparison of the two major anti-haemostatic activities i.e. anti-platelet aggregation and anti-coagulation activity in these flies versus non-infected tsetse flies demonstrates a significant suppression of these activities as a result of the trypanosome-infection status. This effect was mainly related to the parasite-induced reduction in salivary gland gene transcription, resulting in a strong decrease in protein content and related biological activities. Additionally, the anti-thrombin activity and inhibition of thrombin-induced coagulation was even more severely hampered as a result of the trypanosome infection. Indeed, while naive tsetse saliva strongly inhibited human thrombin activity and thrombin-induced blood coagulation, saliva from T. brucei-infected flies showed a significantly enhanced thrombinase activity resulting in a far less potent anti-coagulation activity. These data clearly provide evidence for a trypanosome-mediated modification of the tsetse salivary composition that results in a drastically reduced anti-haemostatic potential and a hampered feeding performance which could lead to an increase of the vector/host contact and parasite transmission in field conditions.
Author Summary
Human African Trypanosomiasis, or sleeping sickness, is a devastating parasitic disease that is fatal if left untreated. Infections are acquired via the bite of an obligate blood feeding fly, the tsetse fly, that is exclusively present on the African continent. In this insect vector, the trypanosome parasite has a complex development ending in the salivary glands. In this experimental study we demonstrate that the Trypanosoma brucei parasites change the composition of the tsetse fly saliva making it less efficient to keep the blood fluid at the biting site in the mammalian host. This results in a more difficult blood feeding process and favors the fly biting activity on multiple hosts, thereby promoting the survival and circulation of the parasite within the natural host population. These findings give us a better understanding of how trypanosome infections in the human population can be maintained given the fact that only very few tsetse flies are actually carrying the parasite.
doi:10.1371/journal.ppat.1000926
PMCID: PMC2880569  PMID: 20532213
24.  Eosinophil deficiency compromises parasite survival in chronic nematode infection 
Immune responses elicited by parasitic worms share many features with those of chronic allergy. Eosinophils contribute to the inflammation that occurs in both types of disease and helminths can be damaged or killed by toxic products released by eosinophils in vitro. Such observations inform the widely held view that eosinophils protect the host against parasitic worms. The mouse is a natural host for Trichinella spiralis, a worm that establishes chronic infection in skeletal muscle. We tested the influence of eosinophils on T. spiralis infection in two mouse strains in which the eosinophil lineage is ablated. Eosinophils were prominent in infiltrates surrounding infected muscle cells of wild-type mice; however, in the absence of eosinophils T. spiralis muscle larvae died in large numbers. Parasite death correlated with enhanced IFN-γ and decreased IL-4 production. Larval survival improved when mice were treated with inhibitors of inducible nitric oxide synthase, implicating the nitric oxide pathway in parasite clearance. Thus, the long-standing paradigm of eosinophil toxicity in nematode infection requires reevaluation, as our results suggest that eosinophils may influence the immune response in a manner that would sustain chronic infection and insure worm survival in the host population. Such a mechanism may be deployed by other parasitic worms that depend upon chronic infection for survival.
PMCID: PMC3923382  PMID: 19155506
Eosinophils; T cells; parasitic infections
25.  An unlikely partnership: parasites, concomitant immunity and host defence. 
Concomitant immunity (CI) against macroparasites describes a state of effective anti-larval immunity coupled with persistent adult infection. Experimental studies indicate that immunologically concealed adult worms might promote anti-larval immunity via the release of cross-reactive antigens, thus creating a barrier against continual infection and restricting burden size within the host. CI offers an important potential benefit to established worms by preventing overcrowding within the host. Thus, CI may be interpreted as akin to vaccination; relatively long-lived adult worms 'vaccinate' their host with larval surface antigens and so benefit from reduced conspecific competition. The shared responsibility for host vaccination among adult worms leads to a problem of collective action. Here, we build on earlier analytical findings about the evolutionary forces that shape cooperation among parasites in order to produce a stochastic simulation model of macroparasite social evolution. First, we theoretically investigate a parasite adaptation hypothesis of CI and demonstrate its plausibility under defined conditions, despite the possibility of evolutionary 'cheats'. Then we derive a set of predictions for testing the hypothesis that CI is partly a host-manipulative parasite adaptation. Evidence in support of this model would present an unusual case of adaptive population regulation.
doi:10.1098/rspb.2001.1821
PMCID: PMC1088913  PMID: 11749708

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