Eric S. Loker

Invertebrate immune systems – not homogeneous, not simple, not well understood

Summary:  The approximate 30 extant invertebrate phyla have diversified along separate evolutionary trajectories for hundreds of millions of years. Although recent work understandably has emphasized the commonalities of innate defenses, there is also ample evidence, as from completed genome studies, to suggest that even members of the same invertebrate order have taken significantly different approaches to internal defense. These data suggest that novel immune capabilities will be found among the different phyla. Many invertebrates have intimate associations with symbionts that may play more of a role in internal defense than generally appreciated. Some invertebrates that are either long lived or have colonial body plans may diversify components of their defense systems via somatic mutation. Somatic diversification following pathogen exposure, as seen in plants, has been investigated little in invertebrates. Recent molecular studies of sponges, cnidarians, shrimp, mollusks, sea urchins, tunicates, and lancelets have found surprisingly diversified immune molecules, and a model is presented that supports the adaptive value of diversified non‐self recognition molecules in invertebrates. Interactions between invertebrates and viruses also remain poorly understood. As we are in the midst of alarming losses of coral reefs, increased pathogen challenge to invertebrate aquaculture, and rampant invertebrate‐transmitted parasites of humans and domestic animals, we need a better understanding of invertebrate immunology.

Reduced Susceptibility to Praziquantel among Naturally Occurring Kenyan Isolates of Schistosoma mansoni

Background The near exclusive use of praziquantel (PZQ) for treatment of human schistosomiasis has raised concerns about the possible emergence of drug-resistant schistosomes. Methodology/Principal Findings We measured susceptibility to PZQ of isolates of Schistosoma mansoni obtained from patients from Kisumu, Kenya continuously exposed to infection as a consequence of their occupations as car washers or sand harvesters. We used a) an in vitro assay with miracidia, b) an in vivo assay targeting adult worms in mice and c) an in vitro assay targeting adult schistosomes perfused from mice. In the miracidia assay, in which miracidia from human patients were exposed to PZQ in vitro, reduced susceptibility was associated with previous treatment of the patient with PZQ. One isolate (“KCW”) that was less susceptible to PZQ and had been derived from a patient who had never fully cured despite multiple treatments was studied further. In an in vivo assay of adult worms, the KCW isolate was significantly less susceptible to PZQ than two other isolates from natural infections in Kenya and two lab-reared strains of S. mansoni. The in vitro adult assay, based on measuring length changes of adults following exposure to and recovery from PZQ, confirmed that the KCW isolate was less susceptible to PZQ than the other isolates tested. A sub-isolate of KCW maintained separately and tested after three years was susceptible to PZQ, indicative that the trait of reduced sensitivity could be lost if selection was not maintained. Conclusions/Significance Isolates of S. mansoni from some patients in Kisumu have lower susceptibility to PZQ, including one from a patient who was never fully cured after repeated rounds of treatment administered over several years. As use of PZQ continues, continued selection for worms with diminished susceptibility is possible, and the probability of emergence of resistance will increase as large reservoirs of untreated worms diminish. The potential for rapid emergence of resistance should be an important consideration of treatment programs.

A comparative study of the life-histories of mammalian schistosomes

Available data in the literature pertaining to the life-history characteristics of all known species of mammalian schistosomes have been gathered, and correlations between such variables as length of pre-patent period, adult worm size, rate of progeny production and progeny size have been explored. Accommodation of the schistosome life-cycle to the constraints imposed by certain host characteristics such as life-expectancy and size is discussed. Of the 23 known species of mammalian schistosomes, 20 species apparently rely to a major extent on relatively large-bodied and long-lived mammals such as primates, ungulates and proboscideans for their transmission. Only 1 species, Schistosomatium douthitti, is exclusively dependent on rodents for its transmission. S. douthitti attains maturity within its definitive host faster than any other mammalian schistosome, and is the only species known to be capable of producing viable eggs by facultative parthenogenesis. For all species of mammalian schistosomes, adult worm size, as estimated by female length, is positively correlated with the number of uterine eggs contained within the female (r = 0.682). For the 7 species for which data exist, rate of egg production/worm pair/day is positively correlated with uterine egg counts (r = 0.873) and inversely correlated with egg length (r = -0.787) and miracidium length (r = -0.953). Length of the pre-patent period is positively correlated with egg length (r = 0.503). With respect to the molluscan host, the number of cercariae produced by snails is positively correlated with the shell size of the snail (r = 0.657). For the 5 species for which data exist, the rate of egg production is inversely correlated with shell size of the intermediate host (r = -0.955) and the common logarithm of the number of cercariae produced (r = -0.893). Comparisons between species suggest that exceptionally low rates of cercariae production in the intermediate host may be compensated for by rapid rates of egg production in the definitive host, implying a degree of integration in the schistosome life-cycle not previously appreciated. Most species of mammalian schistosomes have long-lived definitive hosts, and snail hosts capable of producing many cercariae; compensatory relationships are therefore less obvious in such species. Additional quantitative data on all aspects of schistosome life-histories, particularly rate and duration of egg production, are needed to confirm or refute the relationships discussed above.

Schistosoma mansoni and Biomphalaria: past history and future trends

Schistosoma mansoni is one of the most abundant infectious agents of humankind. Its widespread distribution is permitted by the broad geographic range of susceptible species of the freshwater snail genus Biomphalaria that serve as obligatory hosts for its larval stages. Molecular phylogenetic studies suggest that Schistosoma originated in Asia, and that a pulmonate-transmitted progenitor colonized Africa and gave rise to both terminal-spined and lateral-spined egg species groups, the latter containing S. mansoni. Schistosoma mansoni likely appeared only after the trans-Atlantic dispersal of Biomphalaria from the Neotropics to Africa, an event that, based on the present African fossil record, occurred only 2-5 million years ago. This parasite became abundant in tropical Africa and then entered the New World with the slave trade. It prospered in the Neotropics because a remarkably susceptible and productive host, B. glabrata, was widely distributed there. Indeed, a snail similar to B. glabrata may have given rise to the African species of Biomphalaria. Schistosoma mansoni has since spread into other Neotropical Biomphalaria species and mammalian hosts. The distribution of S. mansoni is in a state of flux. In Egypt, S. mansoni has nearly completely replaced S. haematobium in the Nile Delta, and has spread to other regions of the country. A susceptible host snail, B. straminea, has been introduced into Asia and there is evidence of S. mansoni transmission in Nepal. Dam and barrage construction has lead to an epidemic of S. mansoni in Senegal, and the parasite continues its spread in Brazil. Because of competition with introduced aquatic species and environmental changes, B. glabrata and consequently S. mansoni have become less abundant on the Caribbean islands. Control of S. mansoni using praziquantel and oxamniquine has reduced global prevalence but control is difficult to sustain, and S. mansoni can develop tolerance/resistance to praziquantel, raising concerns about its future efficacy. Because of legitimate environmental concerns, snail control is unlikely to be an option in future control efforts. Global warming will impact the distribution of Biomphalaria and S. mansoni, but the magnitude and nature of the effects are poorly understood.

Origin and diversification of the human parasite Schistosoma mansoni

Schistosoma mansoni is the most widespread of the human‐infecting schistosomes, present in 54 countries, predominantly in Africa, but also in Madagascar, the Arabian Peninsula, and the Neotropics. Adult‐stage parasites that infect humans are also occasionally recovered from baboons, rodents, and other mammals. Larval stages of the parasite are dependent upon certain species of freshwater snails in the genus Biomphalaria, which largely determine the parasites geographical range. How S. mansoni genetic diversity is distributed geographically and among isolates using different hosts has never been examined with DNA sequence data. Here we describe the global phylogeography of S. mansoni using more than 2500 bp of mitochondrial DNA (mtDNA) from 143 parasites collected in 53 geographically widespread localities. Considerable within‐species mtDNA diversity was found, with 85 unique haplotypes grouping into five distinct lineages. Geographical separation, and not host use, appears to be the most important factor in the diversification of the parasite. East African specimens showed a remarkable amount of variation, comprising three clades and basal members of a fourth, strongly suggesting an East African origin for the parasite 0.30–0.43 million years ago, a time frame that follows the arrival of its snail host. Less but still substantial variation was found in the rest of Africa. A recent colonization of the New World is supported by finding only seven closely related New World haplotypes which have West African affinities. All Brazilian isolates have nearly identical mtDNA haplotypes, suggesting a founder effect from the establishment and spread of the parasite in this large country.

Ultrastructure of encapsulation of Schistosoma mansoni mother sporocysts by hemocytes of juveniles of the 10-R2 strain of Biomphalaria glabrata.

Dynamic aspects of the cellular responses of juvenile (2-3 mm shell diameter) 10-R2 strain Biomphalaria glabrata to newly penetrated Schistosoma mansoni (NIH-Sm-PR-1 strain) were studied at the ultrastructural level. As early as 3 hr postexposure (PE), host hemocytes had contacted the parasites surface and by 7.5 hr, had phagocytosed sporocyst microvilli and small pieces of underlying tegument. Most sporocysts observed at 24 hr PE lacked tegumental cytoplasm, and germinal cells and other internal structures showed extensive pathological changes. By 48 hr, capsules were filled with hemocytes containing numerous, large phagosomes, and only scattered remnants of sporocyst material remained. Capsules were difficult to find at 4 days PE, suggesting that hemocytes participating in the encapsulation response had dispersed. Hemocytes responsible for the rapid and consistent destruction of S. mansoni sporocysts in the head-foot region typically formed extensive pseudopodia and contained large numbers of lysosomelike bodies, characteristics associated with granulocytes of B. glabrata. No evidence was found to suggest that hemocytes were lysed, or formed multinuclear syncytia, during the encapsulation response.

Towards an understanding of the mechanism of action of praziquantel.

Although praziquantel (PZQ) has been used to treat schistosomiasis for over 20 years its mechanism of action remains unknown. We have developed an assay based on the transcriptional response of Schistosoma mansoni PR-1 to heat shock to confirm that while 6-week post-infection (p.i.) schistosomes are sensitive to PZQ, 4-week p.i. schistosomes are not. Further, we have used this assay to demonstrate that in mice this sensitivity develops between days 37 and 40 p.i. When PZQ is linked to the fluorophore BODIPY to aid microscopic visualization, it appears to enter the cells of intact 4 and 6-week p.i. schistosomes as well as mammalian NIH 3T3 cells with ease suggesting that the differential effects of PZQ is not based on cell exclusion. A transcriptomal analysis of gene expression between 4 and 6 weeks p.i. revealed 607 up-regulated candidate genes whose products are potential PZQ targets. A comparison of this gene list with that of genes expressed by PZQ sensitive miracidia reduced this target list to 247 genes, including a number involved in aerobic metabolism and cytosolic calcium regulation. Finally, we also report the effect of an in vitro sub-lethal exposure of PZQ on the transcriptome of S. mansoni PR-1. Annotation of genes differentially regulated by PZQ exposure suggests that schistosomes may undergo a transcriptomic response similar to that observed during oxidative stress.

Evolutionary relationships among the schistosomatidae (Platyhelminthes: Digenea) and an Asian origin for Schistosoma

Schistosome blood flukes parasitize birds, mammals, and crocodilians and are responsible for causing one of the great neglected diseases of humanity, schistosomiasis. A phylogenetic study of 10 schistosome genera using approximately 1,100 bases of the large subunit of the nuclear ribosomal gene complex revealed 2 major clades. One clade is entirely mammalian and includes the genera Schistosoma and Orientobilharzia. A close examination of relationships in this group suggests that the medically important Schistosoma arose in Asia and not in Africa as generally presumed and is paraphyletic. The second clade is primarily avian, consisting of 6 genera of exclusively avian parasites and 2 genera of North American mammal flukes. These results indicate a secondary host capture of mammals on the North American continent. This study provides little evidence concerning the ancestral molluscan or vertebrate schistosome host but does demonstrate that host switching has been an important feature of schistosome evolution. Evidence also indicates that the reduced sexual dimorphism characteristic of some avian schistosomes is derived evolutionarily.

Role for a somatically diversified lectin in resistance of an invertebrate to parasite infection

Invertebrates lack adaptive immune systems homologous to those of vertebrates, yet it is becoming increasingly clear that they can produce diversified antigen recognition molecules. We have previously noted that the snail Biomphalaria glabrata produces a secreted lectin, fibrinogen-related protein 3 (FREP3), unusual among invertebrate defense molecules because it is somatically diversified by gene conversion and point mutation. Here we implicate FREP3 in playing a central role in resistance to a major group of snail pathogens, digenetic trematodes. FREP3 is up-regulated in three models of resistance of B. glabrata to infection with Schistosoma mansoni or Echinostoma paraensei, and functions as an opsonin favoring phagocytosis by hemocytes. Knock-down of FREP3 in resistant snails using siRNA-mediated interference resulted in increased susceptibility to E. paraensei, providing a direct link between a gastropod immune molecule and resistance to trematodes. FREP3 up-regulation is also associated with heightened responsiveness following priming with attenuated digenetic trematodes (acquired resistance) in this model invertebrate immune system.

On being a parasite in an invertebrate host: a short survival course.

Many parasites develop in invertebrate hosts that possess internal defense systems (IDS) that vigorously defined self-integrity. Invertebrates apparently do not produce a large diversity of finely tuned immunorecognition molecules but rather rely on recognition of patterns. As a consequence, requirements for immune evasion are likely to be fundamentally different in such hosts. Although parasites of invertebrates certainly employ diverse tactics to evade host IDS, this review focuses on parasite-mediated interference with the structural and functional integrity of host hemocytes and argues that this is a common strategy of immune evasion. Parasites mediating such effects on host hemocytes are termed suppressors. In some cases, interference is mediated by mutualistic symbionts carried by the suppressors. Hemocytes from infected hosts exhibit diminished adherence to substrates, impaired spreading ability, and reduced ability to participate in phagocytosis or encapsulation reactions. As a result of the action of suppressors, the hosts vulnerability to opportunistic parasites is increased, a phenomenon termed acquired susceptibility. A strategy of interference is therefore risky, particularly for suppressors with relatively long development times. As a result, suppressors may provoke either a partial generalized interference or a selective interference with host IDS function, actively contribute to protection of the host to discourage growth of opportunists (termed parasite-mediated internal defense), or induce compensatory host responses that protect the host but that do not jeopardize their own development. Some parasites consistently colonize previously infected hosts and seem to be specialized opportunists.