Insight into the host-parasite interplay by proteomic study of host proteins copurified with the human parasite, Schistosoma japonicum

The tegument proteins of schistosome have attracted the most attention in studies of host-parasite interplay, while the host proteins acting at the host-parasite interface remained largely elusive. Here, we undertook a high-throughput proteomic approach to characterize the schistosome-adsorbed host proteins. Fifty five distinct host proteins were confidently identified in S. japonicum samples, including cercaria, schistosomula, adults, eggs, and miracidia, together with tegument and eggshell preparations, of which 23 and 38 host proteins were identified in adult worms and eggs, respectively. Among the schistosome-adsorbed host proteins, host neutrophil elastases were found in the granuloma initiated by schistosome egg deposition, implying that the host innate immune molecules could participate in the granuloma formation for fighting against schistosome invasion, except for the adaptive immune system. In addition, some host proteins, such as proteinase inhibitor and superoxide dismutase, might be utilized by schistosome to counteract or attenuate the host attacks. These parasite-adsorbed host proteins will provide new insights into the host immune responses against schistosome infection, the evasive behavior of the adult worms, and the granuloma formation, which could render an in-depth understanding for the host-parasite interplay.     

The Yeast Saccharomyces cerevisiae: a versatile model system for the identification and characterization of bacterial virulence proteins

Microbial pathogens utilize complex secretion systems to deliver proteins into host cells. These effector proteins target and usurp host cell processes to promote infection and cause disease. While secretion systems are conserved, each pathogen delivers its own unique set of effectors. The identification and characterization of these effector proteins has been difficult, often limited by the lack of detectable signal sequences and functional redundancy. Model systems including yeast, worms, flies, and fish are being used to circumvent these issues. This technical review details the versatility and utility of yeast Saccharomyces cerevisiae as a system to identify and characterize bacterial effectors.     

Enzymatic shaving of the tegument surface of live schistosomes for proteomic analysis: a rational approach to select vaccine candidates

Background

The membrane-associated and membrane-spanning constituents of the Schistosoma mansoni tegument surface, the parasite''s principal interface with the host bloodstream, have recently been characterized using proteomic techniques. Biotinylation of live worms using membrane-impermeant probes revealed that only a small subset of the proteins was accessible to the reagents. Their position within the multilayered architecture of the surface has not been ascertained.

Methodology/Principal Findings

An enzymatic shaving approach on live worms has now been used to release the most accessible components, for analysis by MS/MS. Treatment with trypsin, or phosphatidylinositol-specific phospholipase C (PiPLC), only minimally impaired membrane integrity. PiPLC-enriched proteins were distinguished from those released in parasite vomitus or by handling damage, using isobaric tagging. Trypsin released five membrane proteins, Sm200, Sm25 and three annexins, plus host CD44 and the complement factors C3 and C4. Nutrient transporters and ion channels were absent from the trypsin fraction, suggesting a deeper location in the surface complex; surprisingly, two BAR-domain containing proteins were released. Seven parasite and two host proteins were enriched by PiPLC treatment, the vaccine candidate Sm29 being the most prominent along with two orthologues of human CD59, potentially inhibitors of complement fixation. The enzymes carbonic anhydrase and APD-ribosyl cyclase were also enriched, plus Sm200 and alkaline phosphatase. Host GPI-anchored proteins CD48 and CD90, suggest ‘surface painting’ during worm peregrination in the portal system.

Conclusions/Significance

Our findings suggest that the membranocalyx secreted over the tegument surface is not the inert barrier previously proposed, some tegument proteins being externally accessible to enzymes and thus potentially located within it. Furthermore, the detection of C3 and C4 indicates that the complement cascade is initiated, while two CD59 orthologues suggest a potential mechanism for its inhibition. The detection of several host proteins is a testimonial to the acquisitive properties of the tegument surface. The exposed parasite proteins could represent novel vaccine candidates for combating this neglected disease.     

A Potential Role for the Interaction of Wolbachia Surface Proteins with the Brugia malayi Glycolytic Enzymes and Cytoskeleton in Maintenance of Endosymbiosis

The human filarial parasite Brugia malayi harbors an endosymbiotic bacterium of the genus Wolbachia. The Wolbachia represent an attractive target for the control of filarial induced disease as elimination of the bacteria affects molting, reproduction and survival of the worms. The molecular basis for the symbiotic relationship between Wolbachia and their filarial hosts has yet to be elucidated. To identify proteins involved in this process, we focused on the Wolbachia surface proteins (WSPs), which are known to be involved in bacteria-host interactions in other bacterial systems. Two WSP-like proteins (wBm0152 and wBm0432) were localized to various host tissues of the B. malayi female adult worms and are present in the excretory/secretory products of the worms. We provide evidence that both of these proteins bind specifically to B. malayi crude protein extracts and to individual filarial proteins to create functional complexes. The wBm0432 interacts with several key enzymes involved in the host glycolytic pathway, including aldolase and enolase. The wBm0152 interacts with the host cytoskeletal proteins actin and tubulin. We also show these interactions in vitro and have verified that wBm0432 and B. malayi aldolase, as well as wBm0152 and B. malayi actin, co-localize to the vacuole surrounding Wolbachia. We propose that both WSP protein complexes interact with each other via the aldolase-actin link and/or via the possible interaction between the host''s enolase and the cytoskeleton, and play a role in Wolbachia distribution during worm growth and embryogenesis.     

Helminthofauna of Opisthonema liberate and Harengula thrissina (Osteichthyes: Clupeidae) from Chamela Bay, Jalisco, Mexico

We collected helminths from the "sardines" Harengula thrissina (N = 61) and Opisthonema libertate (n = 43), from Chamela Bay, Jalisco State, Mexico (12 and six species, respectively). The nematode Pseudoterranova sp., reached the highest values of prevalence and mean abundance in O. libertate (11.6% and 0.13 worms per host, respectively), while in H. thrissina the digenean Parahemiurus merus reached a prevalence of 49.1% and a mean abundance of 1.40 worms per host. Low similarity values (qualitative and quantitative) between helminths of both host species is a result of their opportunistic feeding habits (100% of the parasitic species in H. thrissina and 66% of those from O. libertate infect their host from prey) and differential exposure to helminth larvae.     

Effect of dietary tetrapyrroles on gut pigmentation and perienteric fluid hemoglobin concentration in Ascaris lumbricoides

Ascaris lumbricoides was maintained for 46 days in three pigs fed diets enriched with different metalloporphyrins. Diets, host gut contents, and washed worm guts were differentially extracted for carotenoids, bile pigments, porphyrins and metalloporphyrins, and extracts were compared by spectrophotometric and qualitative chemical tests. Host gut contents from a control diet with no added porphyrin contained carotenoids and bilirubin; worms from this host contained bilirubin and an unidentified metalloporphyrin. An alfalfa-enriched diet yielded phaeophytin A in host gut contents, while worm gut tissue contained an unidentified metalloporphyrin different from the controls. Host gut contents and worms from a blood meal-enriched diet both contained protoheme IX; perienteric fluid concentration in these worms (0·211 mM) was five times that of the other groups. The presence of metalloporphyrins in guts of worms reared on linear tetrapyrroles suggests the ability to convert these compounds to heme.     

Schistosoma bovis: plasminogen binding in adults and the identification of plasminogen-binding proteins from the worm tegument

Schistosoma bovis is a ruminant haematic parasite that lives for years in the mesenteric vessels of the host. The aim of this work was to investigate the ability of adult S. bovis worms to interact with plasminogen, a central component in the host fibrinolytic system. Confocal microscopy analysis revealed that plasminogen bound to the tegument surface of the male-but not female-S. bovis worms and that this binding was strongly dependent on lysine residues. It was also observed that a protein extract of the worm tegument (TG) had the capacity to generate plasmin and to enhance the plasmin generation by the tissue-type plasminogen activator. Proteomic analysis of the TG extract identified 10 plasminogen-binding proteins, among which the major ones were enolase, glyceraldehyde-3-phosphate dehydrogenase and actin. This study represents the first report about the binding of plasminogen to Schistosoma sp. proteins.     

Studies on host specificity in Paragonimus westermani: II. Histochemical and cytochemical characterization of metacercariae and worms from rats and dogs

Histochemical tests were done on newly excysted metacercariae and worms recovered from an abnormal host (rat) and the definitive host (dog) for some oxidoreductases, phosphatases and glycosidases. The results demonstrate that rat worms have enzymatic distribution and intensities more similar to those of metacercariae than to adult worms from dogs. Ultracytochemical examination of acid and alkaline phosphatase and Mg-ATPase activity was also carried out. Acid phosphatase activity occurred exceptionally in the excretory bladder and caeca of dog worms. No activity was observed in rat worms except for lysosomal granules in the tegument. Alkaline phosphatase activity was exhibited in the excretory bladder in both dog and rat worms. Mg-ATPase activity occurred in the tegument and parenchymal cells in dog worms and in the excretory bladder in rat worms. In metacercariae, little or no reaction for these enzymes was present except for Mg-ATPase activity on the excretory ducts. These observations, together with the histochemical results, indicate that metabolic activity in rat worms is higher than in metacercariae although it is strongly reduced compared with dog worms.     

Schistosome immunomodulators

Schistosomes are long lived, intravascular parasitic platyhelminths that infect >200 million people globally. The molecular mechanisms used by these blood flukes to dampen host immune responses are described in this review. Adult worms express a collection of host-interactive tegumental ectoenzymes that can cleave host signaling molecules such as the “alarmin” ATP (cleaved by SmATPDase1), the platelet activator ADP (SmATPDase1, SmNPP5), and can convert AMP into the anti-inflammatory mediator adenosine (SmAP). SmAP can additionally cleave the lipid immunomodulator sphingosine-1-phosphate and the proinflammatory anionic polymer, polyP. In addition, the worms release a barrage of proteins (e.g., SmCB1, SjHSP70, cyclophilin A) that can impinge on immune cell function. Parasite eggs also release their own immunoregulatory proteins (e.g., IPSE/α1, omega1, SmCKBP) as do invasive cercariae (e.g., Sm16, Sj16). Some schistosome glycans (e.g., LNFPIII, LNnT) and lipids (e.g., Lyso-PS, LPC), produced by several life stages, likewise affect immune cell responses. The parasites not only produce eicosanoids (e.g., PGE2, PGD2—that can be anti-inflammatory) but can also induce host cells to release these metabolites. Finally, the worms release extracellular vesicles (EVs) containing microRNAs, and these too have been shown to skew host cell metabolism. Thus, schistosomes employ an array of biomolecules—protein, lipid, glycan, nucleic acid, and more, to bend host biochemistry to their liking. Many of the listed molecules have been individually shown capable of inducing aspects of the polarized Th2 response seen following infection (with the generation of regulatory T cells (Tregs), regulatory B cells (Bregs) and anti-inflammatory, alternatively activated (M2) macrophages). Precisely how host cells integrate the impact of these myriad parasite products following natural infection is not known. Several of the schistosome immunomodulators described here are in development as novel therapeutics against autoimmune, inflammatory, and other, nonparasitic, diseases.     

Excretory/Secretory Proteome of the Adult Developmental Stage of Human Blood Fluke, Schistosoma japonicum

Schistosomes are the causative agents of schistosomiasis, one of the most prevalent and serious of the parasitic diseases that currently infects ∼200 million people worldwide. Schistosome excretory/secretory (ES) proteins have been shown to play important roles in modulating mammalian host immune systems. In our current study, we performed a global proteomics identification of the ES proteins from adult worms of Schistosoma japonicum, one of the three major schistosome species. Our results unambiguously identified 101 proteins, including 53 putatively secreted proteins. By quantitative analysis, we revealed fatty acid-binding protein as a major constituent of the in vitro ES proteome. Strikingly the heat shock proteins HSP70s, HSP90, and HSP97 constituted the largest protein family in the ES proteome, implying a central role for these proteins in immunomodulation in the host-parasite relationship. Other important S. japonicum ES proteins included actins, 14-3-3, aminopeptidase, enolase, and glyceraldehyde-3-phosphate dehydrogenase, some of which have been considered as viable vaccine candidates and therapeutic targets. A comparison with previous studies suggests that 48.5% of S. japonicum ES proteins are common to other parasite ES products, indicating that the molecular mechanisms involved in evading the host immune response may be conserved across different parasites. Interestingly seven host proteins, including antimicrobial protein CAP18, immunoglobulins, and a complement component, were identified among in vitro S. japonicum ES products likely originating from the schistosome tegument or gut, indicating that host innate and acquired immune systems could defend against schistosome invasion. Our present study represents the first attempt at profiling S. japonicum ES proteins, provides an insight into host-parasite interactions, and establishes a resource for the development of diagnostic agents and vaccines for the control of schistosomiasis.Schistosomes, or blood flukes, are water-borne parasites that are the causative agents of schistosomiasis. An estimated 200 million people worldwide are infected with schistosomes with an additional 650 million people at risk of infection (1). One of the major species of schistosomes, Schistosoma japonicum, is a mammalian parasite endemic in East Asia, especially in China and the Philippines. Schistosomes have complex life cycles. Larval schistosome worms (cercariae) are released by freshwater snails and subsequently invade their definite hosts, human or other mammals, via skin penetration. Once in a host animal, cercariae develop into schistosomula and adult worms, which reside in the portal mesenteric system of the host. When the females lay eggs, some eggs leave the host body and hatch in bodies of water as miracidia. The miracidia seek out and penetrate intermediate host snails, completing the schistosome life cycle.Schistosome strategies for evasion of the host immune system, which permit extended survival in mammalian hosts, are not well understood. These dominant evasion strategies have been described as a system of mimicry capable of producing antigens that are similar to endogenous host components (2–4), antigen disguise through acquisition of host molecules to cover the outer worm surface (5, 6), and immunological modulation through interference with host immune systems (7–10). Among these strategies, schistosome excretory/secretory (ES)¹ products have been shown to elicit host immunological modulation functions (7, 11). Schistosome ES proteins are released or secreted from epithelial surfaces of the gut and/or tegument as well as other specialized ES organs throughout almost all life stages. Schistosoma mansoni primary sporocysts have been reported to synthesize and secrete a wide variety of glycoproteins when cultured in vitro (12, 13). These glycoproteins were shown to have antioxidant activities against potential oxidative killing by mollusk defense systems (14). Similarly ES molecules from schistosome cercariae were also reported to down-regulate host immune responses (7). The anti-inflammatory activity of S. mansoni schistosomula ES products (ESPs) was found to be dominantly associated with Sm16.8 protein (15). In addition, ES proteins from S. mansoni adult worms (16), eggs (17), and miracidia (18) have also been investigated.Identification of all ES complex components is important for understanding how schistosomes regulate host immune systems to establish chronic infections and also other aspects of parasite-host interaction. Importantly this information can be expected to facilitate the discovery of vaccines and new therapeutic drug targets as well as new diagnostic reagents for schistosomiasis control. Proteomics approaches encompass the most efficient and powerful tools for identification of protein complexes and have been widely used to decipher the ES components of the filarial parasite Brugia malayi (19), Leishmania (Trypanosomatidae) (20), nematodes (21–26), and Trematoda (27–37). For the genus Schistosoma, the ES compositions of S. mansoni have been identified in many developmental life stages, including sporocyst (34), cercaria (35, 36), and egg (37) but have not been characterized in the adult worm.Characterization of the S. japonicum ES proteome has not been reported. S. japonicum is significantly different from S. mansoni and Schistosoma hematobium in skin invasion, skin migration, and its developmental patterns of swift migration and maturation (38–40). As such, S. japonicum represents a distinct and valuable model for the study of blood fluke immune evasion strategies. Our research group recently generated and reported a large number of S. japonicum protein-coding genes and expressed sequence tags (ESTs) (41, 42). This preliminary work provides important translated protein sequence data resources for mass spectrum data searching. The present study characterized the in vitro ES proteome of adult worms of S. japonicum (43) using a high throughput LC-MS/MS screening. This life stage spans the longest time frame of parasitic interaction with the host that is distinct from previous reports on proteomics identification of ES compositions in other species. Finally the present study confidently identified 101 S. japonicum ES proteins. This information represents substantial progress toward deciphering the worm ES proteome. These new data provide the basis for further investigations into the molecular basis of schistosome modulation of host immunity, increase the possibility of identifying vaccine candidates and new drug targets, and may aid the development of protein probes for selective and sensitive diagnosis of schistosomiasis.     

Rhythmic behaviour of intestinal helminths in rodents

Intestinal helminths exhibit two types of circadian rhythms, either involving the migration of adults worms ( Hymenolepis and Syphacia ) within the gut or periodic release of eggs by female worms ( Aspiculuris and Heligmosomoides ) to the exterior of the host to maximize transmission efficiency. By modifying the feeding regimes of rodents, there is clear evidence that these rhythmic processes are directly linked with the digestive and defaecating activities of the host with neurohormonal control mechanisms likely to be involved at least in the migratory behaviour of adult worms. Furthermore, in Heligmosomoides polygyrus rhythmic egg output appears to be dependent upon parasite age and the population density of the host and parasite. A brief reference is also made to studies on long-term rhythms of egg output by intestinal nematodes and their effect on patterns of host faecal production.     

Host resistance and tolerance of parasitic gut worms depend on resource availability

Resource availability can significantly alter host–parasite dynamics. Abundant food can provide more resources for hosts to resist infections, but also increase host tolerance of infections by reducing competition between hosts and parasites for food. Whether abundant food favors host resistance or tolerance (or both) might depend on the type of resource that the parasite exploits (e.g., host tissue vs. food), which can vary based on the stage of infection. In our study, we evaluated how low and high resource diets affect Cuban tree frog (Osteopilus septentrionalis) resistance and tolerance of a skin-penetrating, gut nematode Aplectana sp. at each stage of the infection. Compared to a low resource diet, a high resource diet enhanced frog resistance to worm penetration and tolerance while worms traveled to the gut. In contrast, a low resource diet increased resistance to establishment of the infection. After the infection established and worms could access food resources in the gut, a high resource diet enhanced host tolerance of parasites. On a high resource diet, parasitized frogs consumed significantly more food than non-parasitized frogs; when food was then restricted, mass of non-parasitized frogs did not change, whereas mass of parasitized frogs decreased significantly. Thus, a high resource diet increased frog tolerance of established worms because frogs could fully compensate for energy lost to the parasites. Our study shows that host–parasite dynamics are influenced by the effect of resource availability on host resistance and tolerance, which depends on when parasites have access to food and the stage of infection.     

Intestinal distribution of worms and host ingesta in Nippostrongylus brasiliensis.

The gastrointestinal nematode Nippostrongylus brasiliensis is thought to feed on host ingesta, and it is generally thought that the presence of ingesta determines the distribution of this parasite within the host intestine. However, these assertions have not been supported by direct evidence. The purpose of this study was to test the hypothesis that N. brasiliensis worms are preferentially found in regions of the host small intestine containing ingesta. The relationship between worm and ingesta distribution was investigated using mice infected with N. brasiliensis and killed on day 8 postinfection at 0130, 0730, 1330, or 1930 hr. There was an inverse relationship between worm and ingesta distributions, and the worms were distributed significantly more anteriad in the intestine than host ingesta, at all times during the 24 hr. To determine what the worms fed on, host ingesta, tissue, and blood were differentially labeled with the fluorescent dyes rhodamine B and Fluoresbrite. The results of this study suggest that N. brasiliensis feeds on the host's intestinal wall, and that habitat distribution of this parasite within the small intestine is not directly related to the presence of luminal ingesta.     

The fecundity of Schistosoma mansoni in chronic nonhuman primate infections and after transplantation into naive hosts

Adult Schistosoma mansoni worms were transplanted from 8 nonhuman primates with chronic infections into 8 naive recipients, in an effort to test the hypothesis that worm fecundity reduction in chronic infections is the result of host immunity or some other host effect. Techniques for perfusing living donors without the added use of anti-schistosomal drugs and for reducing the likelihood of post-operative bacterial endotoxemia and septic shock are described. Fecundity values in terms of eggs per day per female worm were obtained for the worms in their original and in their new hosts and compared. In 3 experiments, perfusions were incomplete and the donors were saved, enabling direct comparisons of fecundity to be made in subpopulations of worms in both their original and new hosts, after equal life spans. In only 1 of the 8 transplantations was there a clear increase in fecundity after surgical introduction into a naive host. Therefore, these experiments fail to support the hypothesis that reduced fecundity of S. mansoni worms in permissive nonhuman primate hosts is a reversible result of host immunity or some other host-derived factor. Despite this negation, further evidence for reduced worm fecundity in older infections was obtained. In the absence of in vivo evidence for immune-mediated antifecundity, worm senescence is the most likely explanation for this finding, with irreversible immune damage to the worms being a less attractive alternative hypothesis.     

The effect of diet and live host presence on the growth and reproduction of Terebrasabella heterouncinata (Polychaeta: Sabellidae)

Summary

The effect of diet type and the presence or absence of a live host on the growth and production of eggs and larvae by Terebrasabella heterouncinata were quantified. Diet was shown not to have a significant effect on the time in which the worms on live hosts reached their maximum size. Diet did, however, influence the maximum size and consequently the growth rate of worms, which were larger and grew faster on kelp-fed abalone than on those fed a commercial pellet diet. Despite diet having no effect on fecundity and offspring size, kelp-fed worms matured earlier. The maximum size of kelp-fed worms was unaffected by the absence of a live host, suggesting that the worms do not rely on the host for food. The absence of a live host reduced the growth and sexual maturation rates of worms. There was no difference in the size of offspring in the two treatments, but fewer worms matured sexually and fewer broods were produced on “shells only” than on live hosts. The lower growth rate and reproductive output of worms on “shells only” may be due to the diversion of energy from growth and reproduction to burrow expansion.     

The ability of single sex infections of Schistosoma japonicum to induce resistance to reinfection in mice

In four experiments, mice harbouring an average 50, 76 or over 200 Schistosoma japonicum female worms were not resistant when challenged six to eight weeks after infection. The female worms from these single sex infections were stunted and immature (average length 4.6 mm) and induced no overt pathology in the host. Male worm burdens of 60, 135 or 140 also induced little or no resistance to challenge in the host. The males from these single sex infections were fully grown for their age (average length 9 mm) and burdens of 135 or 140 induced distended hepatic portal veins and marked deposition of pigment in the livers of infected mice.     

Inequalities in size and intensity-dependent growth in a mermithid nematode parasitic in beach hoppers

Inequality in body sizes is a common feature in populations of helminth parasites, with potential consequences for egg production and population genetics. Inequalities in body lengths and the effects of intraspecific competition on worm length were studied in a species of mermithid nematode parasitic in the crustacean Talorchestia quoyana (Amphipoda: Talitridae). The majority of the 753 worms recovered were relatively small, and an analysis using a Lorenz curve and Gini coefficient suggested that there were no marked inequalities in body lengths among the worms. Total worm length in the 356 infected amphipods (i.e. the sum of the lengths of all the worms in a host) increased steadily as a function of the number of worms per amphipod, whereas the length of the longest worm per amphipod peaked in amphipods harbouring intermediate numbers of worms. This last result was not significantly accounted for by the observed increase in host size with increasing intensity of infection, but resulted from a correlation between worm length and host size. As the number of worms per amphipod increased, the relative sizes of the second-, third-, and fourth-longest worms per host increased markedly. This means that relative inequalities in sizes become less pronounced, i.e. subordinate worms get closer in size to the longest worm, as the number of worms per host increases. The main consequence of this phenomenon is that worm sizes in the mermithid population are more homogeneous than they would be if intraspecific competition had stronger effects on worm growth.     

Failure of in vitro-cultured schistosomes to produce eggs: how does the parasite meet its needs for host-derived cytokines such as TGF-β?

When adult schistosome worm pairs are transferred from experimental hosts to in vitro culture they cease producing viable eggs within a few days. Female worms in unisexual infections fail to mature, and when mature adult females are separated from male partners they regress sexually. Worms cultured from the larval stage are also permanently reproductively defective. The cytokine transforming growth factor beta derived from the mammalian host is considered important in stimulating schistosome female worm maturation and maintenance of fecundity. The means by which schistosomes acquire TGF-β have not been elucidated, but direct uptake in vivo seems unlikely as the concentration of free, biologically active cytokine in host blood is very low. Here we review the complexities of schistosome development and male–female interactions, and we speculate about two possibilities on how worms obtain the TGF-β they are assumed to need: (i) worms may have mechanisms to free active cytokine from the latency-inducing complex of proteins in which it is associated, and/or (ii) they may obtain the cytokine from alpha 2-macroglobulin, a blood-borne protease inhibitor to which TGF-β can bind. These ideas are experimentally testable.     

Trichinella spiralis: behavior, structure, and biochemistry of larvae following exposure to components of the host enteric environment

Four layers are present on the surface of infective larvae of Trichinella spiralis isolated from host muscle in pepsin-HCl. Trypsin treatment of pepsin-HCl isolated worms caused partial degradation and removal of large patches of the two outer surface layers. Following exposure to bile, only traces of the outer layers remained on the worms surface. These changes in the worm surface were accompanied by a shift from Type I behavior, typical of pepsin-HCl isolated larvae, to Type II behavior, (snakelike) following exposure to either trypsin or bile. Worm behavior was also temperature dependent. Type I behavior was typical of worms maintained at room temperature regardless of treatment, while Type II behavior displayed by worms held at 37 C was treatment dependent. The absorption of in vitro glucose or beta-methyl-D-glucoside was lowest in pepsin-HCl isolated first stage infective larvae, significantly higher in trypsin treated worms and greatest in worms following exposure to bile. Sugar uptake by worms isolated from the host small intestine after 1 hr of enteral infection was similar to that seen in worms isolated from host muscle in pepsin-HCl. Sugar uptake in vitro in worms 2 hr following enteral infection was similar to worms following exposure to bile. The highest levels of sugar absorption in vitro occurred in worms which had resided in the small intestine for 3 hr. The lowest rates of incorporation of label into worm tissues was seen in 1 hr enteral and pepsin-HCl isolated worms. Infective larvae treated with trypsin or bile incorporated significantly greater amounts of label than the two former groups. The highest levels of incorporation of label into worm tissues was seen in 3 hr enteral worms. These findings support the view that trypsin, bile, and temperature serve as environmental cues which lead to alteration of the parasite's behavioral and nutritional status.     

Trans-cellular introduction of HIV-1 protein Nef induces pathogenic response in Caenorhabditis elegans

Background

Caenorhabditis elegans has emerged as a very powerful model for studying the host pathogen interactions. Despite the absence of a naturally occurring viral infection for C. elegans, the model is now being exploited experimentally to study the basic aspects of virus-host interplay. The data generated from recent studies suggests that the virus that infects mammalian cells does infect, replicate and accumulate in C. elegans.

Methodology/Principal Findings

We took advantage of the easy-to-achieve protein introduction in C. elegans and employing the methodology, we administered HIV-1 protein Nef into live worms. Nef is known to be an important protein for exacerbating HIV-1 pathogenesis in host by enhancing viral replication. The deletion of nef from the viral genome has been reported to inhibit its replication in the host, thereby leading to delayed pathogenesis. Our studies, employing Nef introduction into C. elegans, led to creation of an in-vivo model that allowed us to study, whether or not, the protein induces effect in the whole organism. We observed a marked lipodystrophy, effect on neuromuscular function, impaired fertility and reduced longevity in the worms exposed to Nef. The observed effects resemble to those observed in Nef transgenic mice and most interestingly the effects also relate to some of the pathogenic aspects exhibited by human AIDS patients.

Conclusions/Significance

Our studies underline the importance of this in vivo model for studying the interactions of Nef with host proteins, which could further be used for identifying possible inhibitors of such interactions.