Analysis of intestinal epithelial cell responses to Cryptosporidium highlights the temporal effects of IFN-γ on parasite restriction.

The production of IFN-γ is crucial for control of multiple enteric infections, but its impact on intestinal epithelial cells (IEC) is not well understood. Cryptosporidium parasites exclusively infect epithelial cells and the ability of interferons to activate the transcription factor STAT1 in IEC is required for parasite clearance. Here, the use of single cell RNA sequencing to profile IEC during infection revealed an increased proportion of mid-villus enterocytes during infection and induction of IFN-γ-dependent gene signatures that was comparable between uninfected and infected cells. These analyses were complemented by in vivo studies, which demonstrated that IEC expression of the IFN-γ receptor was required for parasite control. Unexpectedly, treatment of Ifng-/- mice with IFN-γ showed the IEC response to this cytokine correlates with a delayed reduction in parasite burden but did not affect parasite development. These data sets provide insight into the impact of IFN-γ on IEC and suggest a model in which IFN-γ signalling to uninfected enterocytes is important for control of Cryptosporidium.

Cryptosporidium rhoptry effector protein ROP1 injected during invasion targets the host cytoskeletal modulator LMO7.

The parasite Cryptosporidium invades and replicates in intestinal epithelial cells and is a leading cause of diarrheal disease and early childhood mortality. The molecular mechanisms that underlie infection and pathogenesis are largely unknown. Here, we delineate the events of host cell invasion and uncover a mechanism unique to Cryptosporidium. We developed a screen to identify parasite effectors, finding the injection of multiple parasite proteins into the host from the rhoptry organelle. These factors are targeted to diverse locations within the host cell and its interface with the parasite. One identified effector, rhoptry protein 1 (ROP1), accumulates in the terminal web of enterocytes through direct interaction with the host protein LIM domain only 7 (LMO7) an organizer of epithelial cell polarity and cell-cell adhesion. Genetic ablation of LMO7 or ROP1 in mice or parasites, respectively, impacts parasite burden in vivo in opposite ways. Taken together, these data provide molecular insight into how Cryptosporidium manipulates its intestinal host niche.

Up-regulation of gasdermin C in mouse small intestine is associated with lytic cell death in enterocytes in worm-induced type 2 immunity.

"Taste-like" tuft cells in the intestine trigger type 2 immunity in response to worm infection. The secretion of interleukin-13 (IL-13) from type 2 innate lymphoid cells (ILC2) represents a key step in the tuft cell-ILC2 cell-intestinal epithelial cell circuit that drives the clearance of worms from the gut via type 2 immune responses. Hallmark features of type 2 responses include tissue remodeling, such as tuft and goblet cell expansion, and villus atrophy, yet it remains unclear if additional molecular changes in the gut epithelium facilitate the clearance of worms from the gut. Using gut organoids, we demonstrated that IL-4 and IL-13, two type 2 cytokines with similar functions, not only induced the classical type 2 responses (e.g., tuft cell expansion) but also drastically up-regulated the expression of gasdermin C genes (Gsdmcs). Using an in vivo worm-induced type 2 immunity model, we confirmed the up-regulation of Gsdmcs in Nippostrongylus brasiliensis-infected wild-type C57BL/6 mice. Consistent with gasdermin family members being principal effectors of pyroptosis, overexpression of Gsdmc2 in human embryonic kidney 293 (HEK293) cells triggered pyroptosis and lytic cell death. Moreover, in intestinal organoids treated with IL-4 or IL-13, or in wild-type mice infected with N. brasiliensis, lytic cell death increased, which may account for villus atrophy observed in worm-infected mice. Thus, we propose that the up-regulated Gsdmc family may be major effectors for type 2 responses in the gut and that Gsdmc-mediated pyroptosis may provide a conduit for the release of antiparasitic factors from enterocytes to facilitate the clearance of worms.

Specific binding of aspartic protease and enterocytes promotes Trichinella spiralis invasion of murine intestinal epithelium cells.

Trichinella spiralis is an important foodborne zoonotic parasite and it is necessary to develop vaccine to prevent T. spiralis infection in food animals. T. spiralis aspartic protease-2 (TsASP2) has been demonstrated to play a crucial role in larval invasion of intestinal epithelium cells (IECs). The purpose of this study was to assess the interaction between TsASP2 and IECs and to investigate the immune protection elicited by vaccination with rTsASP2. The results showed that the enzymatic activity of native aspartic protease was detected in crude proteins of all T. spiralis development stages other than NBL stage, the highest activity was observed in the IIL stage. The results of Western blot showed that TsASP2 protein was expressed at ML, IIL and AW but not NBL, and the TsASP2 expression level at IIL stage was significantly higher than those of other three worm stages (P < 0.05). The specific binding between rTsASP2 and IECs was observed by immunofluorescence test (IFT) and confocal microscopy, and the binding site was localized at the IEC membrane and this binding ability was inhibited by aspartic protease specific inhibitor pepstain A. The results of ELISA showed that the binding ability was protein dose-dependent. Vaccination with rTsASP2 triggered a mixed Th1/Th2 humoral and mucosal immune responses, as demonstrated by the elevation levels of Th1/Th2 cytokines (IFN-γ and IL-4) secreted by the spleen and mesenteric lymph nodes (MLNs) of immunized mice. The mice vaccinated with rTsASP2 exhibited a 54.17% reduction in enteral adult worms and a 54.58% reduction in muscle larvae after T. spiralis challenge. The results demonstrated that TsASP2 might be a potential molecular target for anti-Trichinella vaccines.

Interaction of a Trichinella spiralis cathepsin B with enterocytes promotes the larval intrusion into the cells.

Cathepsin B is one member of cysteine protease family and widely distributed in organisms, it plays an important function in parasite penetrating, migrating, molting and immune escaping. The aim of this work was to investigate whether exist interaction between a Trichinella spiralis cathepsin B (TsCB) and mouse intestinal epithelium cells (IECs), and its influence in the process of larva cell invasion. The results of ELISA, indirect immunofluorescence assay (IIFA), confocal microscopy and Far western blotting showed that there was a strong specific binding of rTsCB and IEC proteins, and the binding positions were located in cytoplasm and nuclei of IECs. The results of the in vitro larva penetration test revealed that rTsCB facilitated the larva invasion of IECs, whereas anti-rTsCB antibodies impeded partially the larva intrusion of enterocytes, this promotive or inhibitory roles were dose-dependent of rTsCB or anti-rTsCB antibodies. Silencing TsCB by siRNA mediated RNA interference reduced the TsCB expression in T. spiralis larvae, and markedly inhibited the larva penetration of enterocytes. The results indicated that TsCB binding to IECs promoted larva penetration of host's enteral epithelia, and it is a promising molecular target against intestinal invasive stages of T. spiralis.

Cryptosporidium parvum alters glucose transport mechanisms in infected enterocytes.

The parasite Cryptosporidium parvum Tyzzer 1912 destroys parts of the intestinal brush border membrane which is important for the uptake of nutrients like glucose. In this study, glucose transport mechanisms of the host cells (IPEC-J2 cells) infected by C. parvum were investigated. The mRNA expression levels of glucose transporters (GLUT) 1 and 2 and Na+-coupled glucose transporter (SGLT) 1 were compared in infected and uninfected cells over an infection time of 24-96 h by RT-qPCR. Furthermore, the protein expression of SGLT 1 and GLUT 2 was quantified in western blot studies. While the protein expression of SGLT 1 was not altered in infected cells, mRNA expression of SGLT 1 and GLUT 1 was significantly increased 24 h p. i. and decreased 96 h p. i. The mRNA expression of GLUT 2 was significantly decreased 24 h, 72 h, and 96 h p. i. and also correlated significantly with the infection dose at 72 h p. i. In contrast to that, the protein expression of GLUT 2 was significantly increased 48 h p. i., associated with a significantly higher intracellular glucose level in infected cells compared with control cells at that time point of infection. This points to an adaptation of the host cells' glucose uptake taking place in the acute phase of the infection. A better understanding of these molecular mechanisms following a C. parvum infection may probably lead to an improvement of therapy strategies in the future.

P-Glycoprotein-Mediated Efflux Reduces the In Vivo Efficacy of a Therapeutic Targeting the Gastrointestinal Parasite Cryptosporidium.

Recent studies have illustrated the burden Cryptosporidium infection places on the lives of malnourished children and immunocompromised individuals. Treatment options remain limited, and efforts to develop a new therapeutic are currently underway. However, there are unresolved questions about the ideal pharmacokinetic characteristics of new anti-Cryptosporidium therapeutics. Specifically, should drug developers optimize therapeutics and formulations to increase drug exposure in the gastrointestinal lumen, enterocytes, or systemic circulation? Furthermore, how should researchers interpret data suggesting their therapeutic is a drug efflux transporter substrate? In vivo drug transporter-mediated alterations in efficacy are well recognized in multiple disease areas, but the impact of intestinal transporters on therapeutic efficacy against enteric diseases has not been established. Using multiple in vitro models and a mouse model of Cryptosporidium infection, we characterized the effect of P-glycoprotein efflux on bumped kinase inhibitor pharmacokinetics and efficacy. Our results demonstrated P-glycoprotein decreases bumped kinase inhibitor enterocyte exposure, resulting in reduced in vivo efficacy against Cryptosporidium. Furthermore, a hollow fiber model of Cryptosporidium infection replicated the in vivo impact of P-glycoprotein on anti-Cryptosporidium efficacy. In conclusion, when optimizing drug candidates targeting the gastrointestinal epithelium or gastrointestinal epithelial infections, drug developers should consider the adverse impact of active efflux transporters on efficacy.

Intestinal NF-κB and STAT signalling is important for uptake and clearance in a Drosophila-Herpetomonas interaction model.

Dipteran insects transmit serious diseases to humans, often in the form of trypanosomatid parasites. To accelerate research in more difficult contexts of dipteran-parasite relationships, we studied the interaction of the model dipteran Drosophila melanogaster and its natural trypanosomatid Herpetomonas muscarum. Parasite infection reduced fecundity but not lifespan in NF-κB/Relish-deficient flies. Gene expression analysis implicated the two NF-κB pathways Toll and Imd as well as STAT signalling. Tissue specific knock-down of key components of these pathways in enterocytes (ECs) and intestinal stem cells (ISCs) influenced initial numbers, infection dynamics and time of clearance. Herpetomonas triggered STAT activation and proliferation of ISCs. Loss of Relish suppressed ISCs, resulting in increased parasite numbers and delayed clearance. Conversely, overexpression of Relish increased ISCs and reduced uptake. Finally, loss of Toll signalling decreased EC numbers and enabled parasite persistence. This network of signalling may represent a general mechanism with which dipteran respond to trypanosomatids.

Gametogony of Eimeria macusaniensis Guerrero, Hernandez, Bazalar and Alva, 1971 in llama (Lama glama).

Camelids (llama, alpaca, vicunãs, guanacos) are important for the economy of South America and Eimeria infections are an important cause of mortality in camelids. Of the six species of Eimeria in camelids, Eimeria macusaniensis, considered the most pathogenic, is distinctive; its oocysts are the largest among all Eimeria species in animals, its prepatent period is more than 1 month, and its oocysts have been found in mummies from prehistoric times. Although, E. macusaniensis gametogonic stages are found associated with enteritis in naturally infected camelids, the schizogonic stages are unknown and clinical disease has been reported in some camelids with no oocysts in feces. Described herein are morphological details of gametogonic development and oocyst formation of E. macusaniensis in a naturally infected llama (Lama lama), solely infected with this parasite. Microgamonts, macrogamonts and oocysts were located in large (up to 300 µm diameter) parasitophorous vacuoles of enterocytes in the ileum. Schizonts were not found. Review of previous reports suggests that multinucleated microgamonts have been mistaken for schizonts. Gametogonic development described in the present study can serve as a guide for differential diagnosis of Eimeria species in the histological sections of intestines.

Re-evaluation of the life cycle of Eimeria maxima Tyzzer, 1929 in chickens (Gallus domesticus).

A time-course study was conducted to resolve discrepancies in the literature and better define aspects of the Eimeria maxima life cycle such, as sites of development and both morphology and number of asexual stages. Broiler chickens were inoculated orally with five million E. maxima oocysts (APU1), and were necropsied at regular intervals from 12 to 120 h p.i. Small intestine tissue sections and smears were examined for developmental stages. The jejunum contained the highest numbers of developmental stages. At 12 h p.i., sporozoites were observed inside a parasitophorous vacuole (PV) in the epithelial villi and the lamina propria. By 24 h, sporozoites enclosed by a PV were observed in enterocytes of the glands of Lieberkühn. At 48 h p.i., sporozoites, elongated immature and mature schizonts, were all seen in the glands with merozoites budding off from a residual body. By 60 h, second-generation, sausage-shaped schizonts containing up to 12 merozoites were observed around a residual body in the villar tip of invaded enterocytes. At 72 and 96 h, profuse schizogony associated with third- and fourth-generation schizonts was observed throughout the villus. At 120 h, another generation (fifth) of schizonts were seen in villar tips as well as in subepithelium where gamonts and oocysts were also present; a few gamonts were in epithelium. Our finding of maximum parasitization of E. maxima in jejunum is important because this region is critical for nutrient absorption and weight gain.

Toxoplasma gondii infection causes structural changes in the jejunum of rats infected with different inoculum doses.

AIM: To evaluate the mucosal tunic and submucosal plexus of the jejunum of rats infected with different inoculum doses of Toxoplasma gondii. MAIN METHODS: Rats were infected with different inoculum doses (50, 500, 1000 and 5000 oocysts) of the T. gondii for 30days, while a control group (CG) received saline solution. Blood and feces were collected before euthanasia for analysis of blood and fecal leukocytes (LEs). Histological analysis of the mucosa, submucosa, villi, crypts and enterocytes were performed. Goblet cells, intraepithelial lymphocytes (IELs) and Paneth cells were quantified. Immunohistochemistry was used to assess enteroendocrine serotonergic (5HT-IR) cells, proliferative cells (PCNA+) and mast cells. Whole mounts were obtained to determine the total submucosal neurons by Giemsa staining and metabolically active neurons (NADH-d+), nitrergic neurons (NADPH-d+) and glial cells (S100). KEY FINDINGS: An increase in blood LEs was observed 30days post-infection (dpi). Fecal LEs were more abundant in the feces in all infected groups at 21 dpi when compared to the CG. The number of IELs, sulfomucin-producing goblet cells, Paneth cells, PCNA+ cells and mast cells increased, whereas the number of 5HT-IR cells decreased. The jejunal architecture was altered, with atrophy of the mucosa, submucosa, villi and crypts. The number of total submucosal neurons decreased, but the NADPH-d+ subpopulation increased. SIGNIFICANCE: The results show how chronic toxoplasmic infection affects the tissue and cellular composition of the rat jejunum. These structural changes tend to intensify with the inoculum dose, demonstrating the importance of the parasitic load on intestinal alterations.

Asexual expansion of Toxoplasma gondii merozoites is distinct from tachyzoites and entails expression of non-overlapping gene families to attach, invade, and replicate within feline enterocytes.

BACKGROUND: The apicomplexan parasite Toxoplasma gondii is cosmopolitan in nature, largely as a result of its highly flexible life cycle. Felids are its only definitive hosts and a wide range of mammals and birds serve as intermediate hosts. The latent bradyzoite stage is orally infectious in all warm-blooded vertebrates and establishes chronic, transmissible infections. When bradyzoites are ingested by felids, they transform into merozoites in enterocytes and expand asexually as part of their coccidian life cycle. In all other intermediate hosts, however, bradyzoites differentiate exclusively to tachyzoites, and disseminate extraintestinally to many cell types. Both merozoites and tachyzoites undergo rapid asexual population expansion, yet possess different effector fates with respect to the cells and tissues they develop in and the subsequent stages they differentiate into. RESULTS: To determine whether merozoites utilize distinct suites of genes to attach, invade, and replicate within feline enterocytes, we performed comparative transcriptional profiling on purified tachyzoites and merozoites. We used high-throughput RNA-Seq to compare the merozoite and tachyzoite transcriptomes. 8323 genes were annotated with sequence reads across the two asexually replicating stages of the parasite life cycle. Metabolism was similar between the two replicating stages. However, significant stage-specific expression differences were measured, with 312 transcripts exclusive to merozoites versus 453 exclusive to tachyzoites. Genes coding for 177 predicted secreted proteins and 64 membrane- associated proteins were annotated as merozoite-specific. The vast majority of known dense-granule (GRA), microneme (MIC), and rhoptry (ROP) genes were not expressed in merozoites. In contrast, a large set of surface proteins (SRS) was expressed exclusively in merozoites. CONCLUSIONS: The distinct expression profiles of merozoites and tachyzoites reveal significant additional complexity within the T. gondii life cycle, demonstrating that merozoites are distinct asexual dividing stages which are uniquely adapted to their niche and biological purpose.

Life Cycle of Hammondia hammondi (Apicomplexa: Sarcocystidae) in Cats.

Hammondia hammondi and Toxoplasma gondii are feline coccidians that are morphologically, antigenically, and phylogenitically related. Both parasites multiply asexually and sexually in feline intestinal enterocytes, but H. hammondi remains confined to enterocytes whereas T. gondii also parasitizes extra-intestinal tissues of the cat. Here, we studied multiplication of H. hammondi in feline intestine and compared with T. gondii cycle. Five parasite-free cats were inoculated orally with tissue cysts and free bradyzoites from skeletal muscles of gamma interferon gene knockout mice and killed at 1, 3, 4, 6, and 7 d later. At 1 and 3 d post inoculation (DPI), numerous individual intracellular bradyzoites were detected in histological sections of small intestine. At 4 DPI only schizonts were found and they were located in enterocyte cytoplasm above the host cell nucleus. At 6 and 7 DPI both schizonts and gamonts were seen and they were located in enterocytes. Ultrastucturally, schizogonic and gametogonic development of H. hammondi was similar to T. gondii. However, in H. hammondi merozoites rhoptries were longer, and coiled and contained more micronemes than in T. gondii. Ultrastructural development is illustrated in detail.

Investigation of effects of Giardia duodenalis on transcellular and paracellular transport in enterocytes using in vitro Ussing chamber experiments.

The mechanisms by which different genotypes of Giardia duodenalis result in different symptoms remain unresolved. In particular, we lack detailed knowledge on which transport mechanisms (transcellular or paracellular) are affected by different Giardia isolates. Using horse radish peroxidase (HRP) and creatinine as transcellular and paracellular probes, respectively, we developed a robust assay that can be used with an Ussing chamber to investigate epithelial transport, as well as short-circuit current as an indicator of net ion transport. We investigated 2 Giardia isolates, both Assemblage A, one a lab-adapted strain and the other a field isolate. Results indicate that products from sonicated Giardia trophozoites increase both transcellular and paracellular transport. A non-significant increase in transepithelial electrical resistance (TEER) and short-circuit current were also noted. The paracellular transport was increased significantly more in the field isolate than in the lab-adapted strain. Our results indicate that while both transcellular and paracellular transport mechanisms may be increased following exposure of cells to Giardia trophozoite sonicate, perhaps by inducing non-specific increases in cellular traffic, it is important that in vitro studies of Giardia pathophysiology are conducted with different Giardia isolates, not just lab-attenuated strains.

Parasite distribution and associated immune response during the acute phase of Toxoplasma gondii infection in sheep.

BACKGROUND: In many countries, Toxoplasma gondii (T. gondii) is a major cause of reproductive disorders and abortions in the sheep industry, and therefore responsible for important financial and economic losses. In addition, undercooked infected lamb is an important risk factor for human toxoplasmosis. In the present study, the initial phase of the infection was investigated: the parasite's entry site, the subsequent distribution of the parasite and the host-immune response. RESULTS: Parasite DNA was already detected in the cranial small intestinal mucosa the first days after oral infection with T. gondii tissue cysts. Simultaneously, high IFN-gamma and IL-12 responses were induced mainly in the mesenteric lymph nodes. The emergence of IgG1 (at 8dpi), and IgG2 (at 11 dpi) was accompanied by a decrease or even disappearance of the IFN-gamma and IL-12 response in the Peyers' patches (PP), PBMC's and popliteal LN's. Meanwhile the parasite DNA could be recovered from most mucosal and systemic tissues to become undetectable in the small intestine, popliteal LN, PBMC and spleen 3 weeks pi. CONCLUSIONS: Our results indicate that parasites enter the cranial small intestine the first days after infection and that after an increase the first two weeks after infection, the parasite DNA levels in the intestine drop below the detection limit three weeks after infection. This coincides with an increase in parastic-specific serum IgG1 and IgG2 and a decrease of the antigen-specific IFN-gamma response in PP, PBMC and popliteal LN. We suggest a role for IFN-gamma and IL-12 in controlling the infection.

Strain-dependent induction of human enterocyte apoptosis by blastocystis disrupts epithelial barrier and ZO-1 organization in a caspase 3- and 9-dependent manner.

Blastocystis is an emerging protistan parasite colonizing the human intestine. It is frequently reported to cause general intestinal symptoms of vomiting, diarrhea, and abdominal pain. We recently demonstrated that Blastocystis rearranged cytoskeletal proteins and induced intestinal epithelial barrier compromise. The effect of Blastocystis on enterocyte apoptosis is unknown, and a possible link between microbially induced enterocyte apoptosis and increased epithelial permeability has yet to be determined. The aim of this study is to assess if Blastocystis induces human enterocyte apoptosis and whether this effect influences human intestinal epithelial barrier function. Monolayers of polarized human colonic epithelial cell-line Caco-2 were incubated with Blastocystis subtype 7 and subtype 4. Assays for both early and late markers of apoptosis, phosphatidylserine externalization, and nuclear fragmentation, respectively, showed that Blastocystis ST-7, but not ST-4, significantly increased apoptosis in enterocytes, suggesting that Blastocystis exhibits host specificity and strain-to-strain variation in pathogenicity. ST-7 also activated Caco-2 caspases 3 and 9 but not 8. ST-7 induced changes in epithelial resistance, permeability, and tight junction (ZO-1) localization. Pretreatment of Caco-2 monolayers with a pan-caspase inhibitor z-VAD-fmk significantly inhibited these changes. This suggests a role for enterocyte apoptosis in Blastocystis-mediated epithelial barrier compromise in the human intestine.

Role of macrophages in the altered epithelial function during a type 2 immune response induced by enteric nematode infection.

Parasitic enteric nematodes induce a type 2 immune response characterized by increased production of Th2 cytokines, IL-4 and IL-13, and recruitment of alternatively activated macrophages (M2) to the site of infection. Nematode infection is associated with changes in epithelial permeability and inhibition of sodium-linked glucose absorption, but the role of M2 in these effects is unknown. Clodronate-containing liposomes were administered prior to and during nematode infection to deplete macrophages and prevent the development of M2 in response to infection with Nippostrongylus brasiliensis. The inhibition of epithelial glucose absorption that is associated with nematode infection involved a macrophage-dependent reduction in SGLT1 activity, with no change in receptor expression, and a macrophage-independent down-regulation of GLUT2 expression. The reduced transport of glucose into the enterocyte is compensated partially by an up-regulation of the constitutive GLUT1 transporter consistent with stress-induced activation of HIF-1α. Thus, nematode infection results in a "lean" epithelial phenotype that features decreased SGLT1 activity, decreased expression of GLUT2 and an emergent dependence on GLUT1 for glucose uptake into the enterocyte. Macrophages do not play a role in enteric nematode infection-induced changes in epithelial barrier function. There is a greater contribution, however, of paracellular absorption of glucose to supply the energy demands of host resistance. These data provide further evidence of the ability of macrophages to alter glucose metabolism of neighboring cells.

Construction and use of a Trichinella spiralis phage display library to identify the interactions between parasite and host enterocytes.

Although it has been known for many years that Trichinella spiralis initiates infection by penetrating the columnar epithelium of the small intestine, the mechanisms by which T. spiralis infective larvae recognize and invade the intestinal epithelial cells (IECs) are unknown. It is speculated that the molecular interactions between the parasite and host enterocytes may mediate the recognition and invasion of IECs by T. spiralis. However, no Trichinella proteins that interact with the enterocytes have been identified previously. The aim of this study was to identify Trichinella proteins that bind to IECs by screening a T7 phage display cDNA library constructed using messenger RNA from T. spiralis intestinal infective larvae. Following five rounds of biopanning, sequencing, and bioinformatics analysis, ten T. spiralis proteins (Tsp1-Tsp10) with significant binding to normal mouse IECs were identified. The results of the protein classification showed that six proteins (Tsp1, calcium-transporting ATPase 2 protein; Tsp4, ovochymase-1; Tsp6, T-complex protein 1 subunit eta; Tsp7, glycosyl hydrolase family 47; Tsp8, DNA replication licensing factor MCM3; and Tsp10, nudix hydrolase) of these T. spiralis proteins were annotated with putative molecular functions. Out of the six proteins, five have catalytic activity, four have binding activity, and one has transporter activity. Anti-Tsp10 antibodies prevented the in vitro partial invasion of IECs by infective larvae and the mice immunized with the recombinant phage T7-Tsp10 showed a 62.8 % reduction in adult worms following challenge with T. spiralis muscle larvae. Although their biological functions are not yet fully known, these proteins might be related to the larval invasion of host enterocytes. Future experiments will be necessary to ascertain whether these proteins play important roles in the recognition and invasion of host enterocytes. The construction and biopanning of Trichinella phage display libraries provide a novel approach for searching for candidate genes that are related to invasion and for identifying protein interactions between parasite and host.

Cryptosporidium parvum scavenges LDL-derived cholesterol and micellar cholesterol internalized into enterocytes.

Cryptosporidium spp. are responsible for devastating diarrhoea in immunodeficient individuals. In the intestinal tract, the developmental stages of the parasite are confined to the apical surfaces of epithelial cells. Upon invasion, Cryptosporidium incorporates the microvillous membrane of the enterocyte to form the parasitophorous vacuole (PV) and sequesters itself from the host cytoplasm by rearranging the host cytoskeleton. Cryptosporidium parvum has minimal anabolic capabilities and relies on transporters and salvage pathways to meet its basic metabolic requirements. The cholesterol salvage pathway is crucial for the development of protozoan parasites. In this study, we have examined the sources of cholesterol from C. parvum infecting enterocytes. We illustrated that the intracellular stages of Cryptosporidium as well as the oocysts shed by the host, contain cholesterol. Incubation of infected enterocytes in lipoprotein-free medium impairs parasite development and results in substantial decrease in cholesterol content associated with the PV. Among lipoproteins, LDL constitutes an important source of cholesterol for Cryptosporidium. Dietary cholesterol incorporated into micelles is internalized into enterocytes by the NPC1L1 transporter. We showed that C. parvum also obtains cholesterol from micelles in enterocytes.Pharmacological blockade of NPC1L1 function by ezetimibe or moderate downregulation of NPC1L1 expression decreases parasite infectivity. These observations indicate that, despite its dual sequestration from the intestinal lumen and the host cytoplasm, C. parvum can, in fact, obtain cholesterol both from the gut's lumen and the host cell. This study highlights the evolutionary advantages for epicellular pathogens to access to nutrients from the outside and inside of the host cell.