The Interaction of Classical Complement Component C1 with Parasite and Host Calreticulin Mediates Trypanosoma cruzi Infection of Human Placenta

Background

9 million people are infected with Trypanosoma cruzi in Latin America, plus more than 300,000 in the United States, Canada, Europe, Australia, and Japan. Approximately 30% of infected individuals develop circulatory or digestive pathology. While in underdeveloped countries transmission is mainly through hematophagous arthropods, transplacental infection prevails in developed ones.

Methodology/Principal Findings

During infection, T. cruzi calreticulin (TcCRT) translocates from the endoplasmic reticulum to the area of flagellum emergence. There, TcCRT acts as virulence factor since it binds maternal classical complement component C1q that recognizes human calreticulin (HuCRT) in placenta, with increased parasite infectivity. As measured ex vivo by quantitative PCR in human placenta chorionic villi explants (HPCVE) (the closest available correlate of human congenital T. cruzi infection), C1q mediated up to a 3–5-fold increase in parasite load. Because anti-TcCRT and anti-HuCRT F(ab′)₂ antibody fragments are devoid of their Fc-dependent capacity to recruit C1q, they reverted the C1q-mediated increase in parasite load by respectively preventing its interaction with cell-bound CRTs from both parasite and HPCVE origins. The use of competing fluid-phase recombinant HuCRT and F(ab′)₂ antibody fragments anti-TcCRT corroborated this. These results are consistent with a high expression of fetal CRT on placental free chorionic villi. Increased C1q-mediated infection is paralleled by placental tissue damage, as evidenced by histopathology, a damage that is ameliorated by anti-TcCRT F(ab′)₂ antibody fragments or fluid-phase HuCRT.

Conclusions/Significance

T. cruzi infection of HPCVE is importantly mediated by human and parasite CRTs and C1q. Most likely, C1q bridges CRT on the parasite surface with its receptor orthologue on human placental cells, thus facilitating the first encounter between the parasite and the fetal derived placental tissue. The results presented here have several potential translational medicine aspects, specifically related with the capacity of antibody fragments to inhibit the C1q/CRT interactions and thus T. cruzi infectivity.     

Bioluminescent imaging of Trypanosoma cruzi infection

Chagas disease, caused by infection with the protozoan parasite Trypanosoma cruzi, is a major public health problem in Central and South America. The pathogenesis of Chagas disease is complex and the natural course of infection is not completely understood. The recent development of bioluminescence imaging technology has facilitated studies of a number of infectious and non-infectious diseases. We developed luminescent T. cruzi to facilitate similar studies of Chagas disease pathogenesis. Luminescent T. cruzi trypomastigotes and amastigotes were imaged in infections of rat myoblast cultures, which demonstrated a clear correlation of photon emission signal strength to the number of parasites used. This was also observed in mice infected with different numbers of luminescent parasites, where a stringent correlation of photon emission to parasite number was observed early at the site of inoculation, followed by dissemination of parasites to different sites over the course of a 25-day infection. Whole animal imaging from ventral, dorsal and lateral perspectives provided clear evidence of parasite dissemination. The tissue distribution of T. cruzi was further determined by imaging heart, spleen, skeletal muscle, lungs, kidneys, liver and intestines ex vivo. These results illustrate the natural dissemination of T. cruzi during infection and unveil a new tool for studying a number of aspects of Chagas disease, including rapid in vitro screening of potential therapeutical agents, roles of parasite and host factors in the outcome of infection, and analysis of differential tissue tropism in various parasite-host strain combinations.     

Local association of Trypanosoma cruzi chronic infection foci and enteric neuropathic lesions at the tissue micro-domain scale

Digestive Chagas disease (DCD) is an enteric neuropathy caused by Trypanosoma cruzi infection. The mechanism of pathogenesis is poorly understood and the lack of a robust, predictive animal model has held back research. We screened a series of mouse models using gastrointestinal tracer assays and in vivo infection imaging systems to discover a subset exhibiting chronic digestive transit dysfunction and significant retention of faeces in both sated and fasted conditions. The colon was a specific site of both tissue parasite persistence, delayed transit and dramatic loss of myenteric neurons as revealed by whole-mount immunofluorescence analysis. DCD mice therefore recapitulated key clinical manifestations of human disease. We also exploited dual reporter transgenic parasites to home in on locations of rare chronic infection foci in the colon by ex vivo bioluminescence imaging and then used fluorescence imaging in tissue microdomains to reveal co-localisation of infection and enteric nervous system lesions. This indicates that long-term T. cruzi-host interactions in the colon drive DCD pathogenesis, suggesting that the efficacy of anti-parasitic chemotherapy against chronic disease progression warrants further pre-clinical investigation.     

Bioluminescence imaging of chronic Trypanosoma cruzi infections reveals tissue‐specific parasite dynamics and heart disease in the absence of locally persistent infection

Chronic Trypanosoma cruzi infections lead to cardiomyopathy in 20–30% of cases. A causal link between cardiac infection and pathology has been difficult to establish because of a lack of robust methods to detect scarce, focally distributed parasites within tissues. We developed a highly sensitive bioluminescence imaging system based on T. cruzi expressing a novel luciferase that emits tissue‐penetrating orange‐red light. This enabled long‐term serial evaluation of parasite burdens in individual mice with an in vivo limit of detection of significantly less than 1000 parasites. Parasite distributions during chronic infections were highly focal and spatiotemporally dynamic, but did not localize to the heart. End‐point ex vivo bioluminescence imaging allowed tissue‐specific quantification of parasite loads with minimal sampling bias. During chronic infections, the gastro‐intestinal tract, specifically the colon and stomach, was the only site where T. cruzi infection was consistently observed. Quantitative PCR‐inferred parasite loads correlated with ex vivo bioluminescence and confirmed the gut as the parasite reservoir. Chronically infected mice developed myocarditis and cardiac fibrosis, despite the absence of locally persistent parasites. These data identify the gut as a permissive niche for long‐term T. cruzi infection and show that canonical features of Chagas disease can occur without continual myocardium‐specific infection.     

Transplacental Zika virus transmission in ex vivo perfused human placentas

A Zika virus (ZIKV) infection during pregnancy can result in severe birth defects such as microcephaly. To date, it is incompletely understood how ZIKV can cross the human placenta. Furthermore, results from studies in pregnant mice and non-human primates are conflicting regarding the role of cross-reactive dengue virus (DENV) antibodies on transplacental ZIKV transmission. Elucidating how ZIKV can cross the placenta and which risk factors contribute to this is important for risk assessment and for potential intervention strategies for transplacental ZIKV transmission. In this study we use an ex vivo human placental perfusion model to study transplacental ZIKV transmission and the effect that cross-reactive DENV antibodies have on this transmission. By using this model, we demonstrate that DENV antibodies significantly increase ZIKV uptake in perfused human placentas and that this increased uptake is neonatal Fc-receptor-dependent. Furthermore, we show that cross-reactive DENV antibodies enhance ZIKV infection in term human placental explants and in primary fetal macrophages but not in primary trophoblasts. Our data supports the hypothesis that presence of cross-reactive DENV antibodies could be an important risk factor for transplacental ZIKV transmission. Furthermore, we demonstrate that the ex vivo placental perfusion model is a relevant and animal friendly model to study transplacental pathogen transmission.     

Toxoplasma gondii Migration within and Infection of Human Retina

Toxoplasmic retinochoroiditis is a common blinding retinal infection caused by the parasite, Toxoplasma gondii. Basic processes relating to establishment of infection in the human eye by T. gondii tachyzoites have not been investigated. To evaluate the ability of tachyzoites to navigate the human retina, we developed an ex vivo assay, in which a suspension containing 1.5×10⁷ parasites replaced vitreous in a posterior eyecup. After 8 hours, the retina was formalin-fixed and paraffin-embedded, and sections were immunostained to identify tachyzoites. To determine the preference of tachyzoites for human retinal neuronal versus glial populations, we infected dissociated retinal cultures, subsequently characterized by neuron-specific enolase or glial fibrillary acidic protein expression, and retinal cell lines, with YFP-expressing tachyzoites. In migration assays, retinas contained 110–250 live tachyzoites; 64.5–95.2% (mean  = 79.6%) were localized to the nerve fiber layer, but some were detected in the outer retina. Epifluorescence imaging of dissociated retinal cultures 24 hours after infection indicated preferential infection of glia. This observation was confirmed in growth assays, with significantly higher (p≤0.005) numbers of tachyzoites measured in glial verus neuronal cell lines. Our translational studies indicate that, after entering retina, tachyzoites may navigate multiple tissue layers. Tachyzoites preferentially infect glial cells, which exist throughout the retina. These properties may contribute to the success of T. gondii as a human pathogen.     

Silencing of GRA10 protein expression inhibits Toxoplasma gondii intracellular growth and development

Toxoplasma gondii dense granule proteins (GRAs) are secreted abundantly in both the tachyzoite and bradyzoite stages of the parasite and are known to localize to various compartments of the parasitophorous vacuole (PV) that interfaces with the host cell milieu. Thus, GRAs may play significant roles in the biogenesis of the PV that is important for survival of intracellular T. gondii. GRA10 is a dense granule protein whose role in T. gondii has not yet been characterized. Therefore, in this study, we endeavored to determine the role of GRA10 in the growth and survival of intracellular T. gondii by using phosphorodiamidate morpholino oligomers (PPMOs) antisense knockdown approach to disrupt the translation of GRA10 mRNA in the parasites. We expressed and purified a truncated recombinant GRA10 protein to generate anti-GRA10 polyclonal antibodies that we used to characterize GRA10 in T. gondii. We found that GRA10 is a soluble, dense granule-associated protein that is secreted into the parasite cytosol and the parasitophorous vacuole milieu. Using in vitro cultures, we found that knockdown of GRA10 results in severe inhibition of T. gondii growth in human fibroblasts and in ovine monocytic cells. Together, our findings define GRA10 as a dense granule protein that plays a significant role in the growth and propagation of intracellular T. gondii in human fibroblasts and in ovine monocytic cells.     

Regulation of CD8 T cell responses to infection with parasitic protozoa

There are over 10,000 species of parasitic protozoa, a subset of which can cause considerable disease in humans. Here we examine in detail the complex immune response generated during infection with a subset of these parasites: Trypanosoma cruzi, Leishmania sp., Toxoplasma gondii, and Plasmodium sp. While these particular species perhaps represent the most studied parasites in terms of understanding how T cells function during infection, it is clear that the lessons learned from this body of work are also relevant to the other protozoa known to induce a CD8⁺ T cell response. This review will highlight some of the key studies that established that CD8⁺ T cells play a major role in protective immunity to protozoa, the factors that promote the generation as well as maintenance of the CD8⁺ T cell response during these infections, and draw attention to some of the gaps in our knowledge. Moreover, the development of new tools, including MHC-Class I tetramer reagents and the use of TCR transgenic mice or genetically modified parasites, has provided a better appreciation of how parasite specific CD8⁺ T cell responses are initiated and new insights into their phenotypic plasticity     

Impairment of T Cell Function in Parasitic Infections

In mammals subverted as hosts by protozoan parasites, the latter and/or the agonists they release are detected and processed by sensors displayed by many distinct immune cell lineages, in a tissue(s)-dependent context. Focusing on the T lymphocyte lineage, we review our present understanding on its transient or durable functional impairment over the course of the developmental program of the intracellular parasites Leishmania spp., Plasmodium spp., Toxoplasma gondii, and Trypanosoma cruzi in their mammalian hosts. Strategies employed by protozoa to down-regulate T lymphocyte function may act at the initial moment of naïve T cell priming, rendering T cells anergic or unresponsive throughout infection, or later, exhausting T cells due to antigen persistence. Furthermore, by exploiting host feedback mechanisms aimed at maintaining immune homeostasis, parasites can enhance T cell apoptosis. We will discuss how infections with prominent intracellular protozoan parasites lead to a general down-regulation of T cell function through T cell anergy and exhaustion, accompanied by apoptosis, and ultimately allowing pathogen persistence.     

The relationship between the presence of antibodies and direct detection of Toxoplasma gondii in slaughtered calves and cattle in four European countries

In cattle, antibodies to Toxoplasma gondii infection are frequently detected, but evidence for the presence of T. gondii tissue cysts in cattle is limited. To study the concordance between the presence of anti-T. gondii IgG and viable tissue cysts of T. gondii in cattle, serum, liver and diaphragm samples of 167 veal calves and 235 adult cattle were collected in Italy, the Netherlands, Romania and the United Kingdom. Serum samples were tested for anti-T. gondii IgG by the modified agglutination test and p30 immunoblot. Samples from liver were analyzed by mouse bioassay and PCR after trypsin digestion. In addition, all diaphragms of cattle that had tested T. gondii-positive (either in bioassay, by PCR on trypsin-digested liver or serologically by MAT) and a selection of diaphragms from cattle that had tested negative were analyzed by magnetic capture quantitative PCR (MC-PCR). Overall, 13 animals were considered positive by a direct detection method: seven out of 151 (4.6%) by MC-PCR and six out of 385 (1.6%) by bioassay, indicating the presence of viable parasites. As cattle that tested positive in the bioassay tested negative by MC-PCR and vice-versa, these results demonstrate a lack of concordance between the presence of viable parasites in liver and the detection of T. gondii DNA in diaphragm. In addition, the probability to detect T. gondii parasites or DNA in seropositive and seronegative cattle was comparable, demonstrating that serological testing by MAT or p30 immunoblot does not provide information about the presence of T. gondii parasites or DNA in cattle and therefore is not a reliable indicator of the risk for consumers.     

Trypanosoma cruzi: effects of adrenalectomy during the acute phase of experimental infection

Glucocorticoid hormones have been implicated as an important modulator of Trypanosoma cruzi pathogenesis. Since adrenal steroid hormones play a fundamental role in modulating the immune response, we hypothesized that adrenalectomy affect the course of the experimental T. cruzi infection. This study was undertaken to determine the effects of adrenalectomy during the acute phase of T. cruzi infection. Blood and tissue parasitism, macrophages, nitric oxide (NO) production and IFN-γ were evaluated in male Wistar rats infected with the Y strain of T. cruzi. Our results show that adrenalectomized rats displayed increased number of blood and heart parasites accompanied by decreases in the total number of peritoneal macrophages and IFN-γ when compared to controls. Adrenalectomy also reduced the levels of NO released from peritoneal macrophages of infected animals. These results suggest that adrenal corticosteroid insufficiency due to adrenalectomy could be considered an important factor during development of acute phases of experimental Chagas’ disease, enhancing pathogenesis through disturbance of the host’s immune system.     

HIV-1 Expressing the Envelopes of Transmitted/Founder or Control/Reference Viruses Have Similar Infection Patterns of CD4 T-Cells in Human Cervical Tissue Ex Vivo

Recently, it was found that 80% of sexual HIV-1 transmissions are established by a single virion/viral genome. To investigate whether the transmitted/founder (T/F) viruses have specific biological properties favoring sexual transmission, we inoculated human cervical tissue explants with isogenic HIV-1 viruses encoding Env sequences from T/F and control reference (C/R) HIV-1 variants as well as with full length T/F HIV-1 and compared their replication efficiencies, T cell depletion, and the activation status of infected cells. We found that all the HIV-1 variants were capable of transmitting infection to cervical tissue ex vivo and in this system preferentially replicate in activated CD4 T cells and deplete these cells. There was no difference in the biological properties of T/F and C/R HIV-1 variants as evaluated in ex vivo cervical tissue.     

Toxoplasma gondii: Effects of Artemisia annua L. on susceptibility to infection in experimental models in vitro and in vivo

Considering that the treatment for toxoplasmosis is based on drugs that show limited efficacy due to their substantial side effects, the purpose of the present study was to evaluate the effects of Artemisia annua on in vitro and in vivo Toxoplasma gondii infection. A. annua infusion was prepared from dried herb and tested in human foreskin fibroblasts (HFF) or mice that were infected with the parasite and compared with sulfadiazine treatment. For in vitro experiments, treatment was done on parasite before HFF infection or on cells previously infected with T. gondii and the inhibitory concentration (IC₅₀) values for each treatment condition were determined. Viability of HFF cells in the presence of different concentrations of A. annua infusion and sulfadiazine was above 72%, even when the highest concentrations from both treatments were tested. Also, the treatment of T. gondii tachyzoites with A. annua infusion before infection in HFF cells showed a dose-response inhibitory curve that reached up to 75% of inhibition, similarly to the results observed when parasites were treated with sulfadiazine. In vivo experiments with a cystogenic T. gondii strain demonstrated an effective control of infection using A. annua infusion. In conclusion, our results indicate that A. annua infusion is useful to control T. gondii infection, due to its low toxicity and its inhibitory action directly against the parasite, resulting in a well tolerated therapeutic tool.     

Trypanosoma cruzi TcSMUG L-surface Mucins Promote Development and Infectivity in the Triatomine Vector Rhodnius prolixus

Background

TcSMUG L products were recently identified as novel mucin-type glycoconjugates restricted to the surface of insect-dwelling epimastigote forms of Trypanosoma cruzi, the etiological agent of Chagas disease. The remarkable conservation of their predicted mature N-terminal region, which is exposed to the extracellular milieu, suggests that TcSMUG L products may be involved in structural and/or functional aspects of the interaction with the insect vector.

Methodology and Principal Findings

Here, we investigated the putative roles of TcSMUG L mucins in both in vivo development and ex vivo attachment of epimastigotes to the luminal surface of the digestive tract of Rhodnius prolixus. Our results indicate that the exogenous addition of TcSMUG L N-terminal peptide, but not control T. cruzi mucin peptides, to the infected bloodmeal inhibited the development of parasites in R. prolixus in a dose-dependent manner. Pre-incubation of insect midguts with the TcSMUG L peptide impaired the ex vivo attachment of epimastigotes to the luminal surface epithelium, likely by competing out TcSMUG L binding sites on the luminal surface of the posterior midgut, as revealed by fluorescence microscopy.

Conclusion and Significance

Together, these observations indicate that TcSMUG L mucins are a determinant of both adhesion of T. cruzi epimastigotes to the posterior midgut epithelial cells of the triatomine, and the infection of the insect vector, R. prolixus.     

The Receptor Slamf1 on the Surface of Myeloid Lineage Cells Controls Susceptibility to Infection by Trypanosoma cruzi

Trypanosoma cruzi, the protozoan parasite responsible for Chagas'' disease, causes severe myocarditis often resulting in death. Here, we report that Slamf1−/− mice, which lack the hematopoietic cell surface receptor Slamf1, are completely protected from an acute lethal parasite challenge. Cardiac damage was reduced in Slamf1−/− mice compared to wild type mice, infected with the same doses of parasites, as a result of a decrease of the number of parasites in the heart even the parasitemia was only marginally less. Both in vivo and in vitro experiments reveal that Slamf1-defIcient myeloid cells are impaired in their ability to replicate the parasite and show altered production of cytokines. Importantly, IFN-γ production in the heart of Slamf1 deficient mice was much lower than in the heart of wt mice even though the number of infiltrating dendritic cells, macrophages, CD4 and CD8 T lymphocytes were comparable. Administration of an anti-Slamf1 monoclonal antibody also reduced the number of parasites and IFN-γ in the heart. These observations not only explain the reduced susceptibility to in vivo infection by the parasite, but they also suggest human Slamf1 as a potential target for therapeutic target against T. cruzi infection.     

Analysis of Toxoplasma gondii surface antigen 2 gene (SAG2). Relevance of genotype I in clinical toxoplasmosis

Toxoplasma gondii is one of the most successful protozoan parasites given its ability to manipulate the immune system and establish a chronic infection. It is a parasite with a significant impact on human health, mainly in immunocompromised patients. In Europe and North America, only a few clonal genotypes (I, II and III) seem to be responsible for the vast majority of Toxoplasma infections. Surface antigen 2 gene (SAG2) has been extensively used for genotyping T. gondii isolates. The analysis of this locus reveals that in Northern hemisphere, human disease causing isolates are mainly type II, whereas T. gondii isolated from different animals are both type II and III. Since the immune response depends on parasite genotype, it seems relevant to characterize parasites producing human toxoplasmosis in different geographical areas. The growing information about the prevalent T. gondii genotypes in South America mostly refers to domestic animals. This is the first report of genetic characterization of T. gondii isolates from clinical samples in Chile, South America. All the samples analyzed corresponded to SAG2 type I isolates, and they differ from classic SAG2 type I by genetic polymorphisms. This study contributes to the scarce available information on T. gondii at South America, and reinforces an emerging concept suggesting that SAG2 type I, rather than II, parasites are a frequent cause of clinical toxoplasmosis in this continent.     

Toxoplasma gondii infection in meat-producing small ruminants: Meat juice serology and genotyping

The consumption of ovine and caprine meat is considered one of the major transmission routes for Toxoplasma gondii infection in humans. The present study aimed at obtaining epidemiological and molecular data on T. gondii infection in small ruminants slaughtered or commercialized in Italy. Meat juices from 227 sheep and 51 goats were analyzed with a commercial ELISA and antibodies were detected in 28.6% sheep and 27.5% goats. A significant difference was highlighted between adult sheep and the other considered categories (young sheep, young and adult goats) concerning the detection of antibodies (94.1%; p-value = .008). Muscles of positives samples were submitted to molecular analysis, and T. gondii DNA was detected in 15 sheep and three goats; sequencing of B1 gene showed that all belonged to Type II. The present study confirmed small ruminants' meat as a possible source of T. gondii infection for consumers eating raw or undercooked meat, particularly in those countries where the consumption of sheep and goats' meat products is a traditional gastronomic habit.     

Trypanosoma cruzi amastigotes that persist in the colon during chronic stage murine infections have a reduced replication rate

Chronic Trypanosoma cruzi infections are typically lifelong, with small numbers of parasites surviving in restricted tissue sites, which include the gastrointestinal tract. There is considerable debate about the replicative status of these persistent parasites and whether there is a role for dormancy in long-term infection. Here, we investigated T. cruzi proliferation in the colon of chronically infected mice using 5-ethynyl-2′deoxyuridine incorporation into DNA to provide ‘snapshots’ of parasite replication status. Highly sensitive imaging of the extremely rare infection foci, at single-cell resolution, revealed that parasites are three times more likely to be in S-phase during the acute stage than during the chronic stage. By implication, chronic infections of the colon are associated with a reduced rate of parasite replication. Despite this, very few host cells survived infection for more than 14 days, suggesting that T. cruzi persistence continues to involve regular cycles of replication, host cell lysis and re-infection. We could find no evidence for wide-spread dormancy in parasites that persist in this tissue reservoir.