Unveiling the Intracellular Survival Gene Kit of Trypanosomatid Parasites

<div><p>Trypanosomatids are unicellular protozoans of medical and economical relevance since they are the etiologic agents of infectious diseases in humans as well as livestock. Whereas <i>Trypanosoma cruzi</i> and different species of <i>Leishmania</i> are obligate intracellular parasites, <i>Trypanosoma brucei</i> and other trypanosomatids develop extracellularly throughout their entire life cycle. After their genomes have been sequenced, various comparative genomic studies aimed at identifying sequences involved with host cell invasion and intracellular survival have been described. However, for only a handful of genes, most of them present exclusively in the <i>T. cruzi</i> or <i>Leishmania</i> genomes, has there been any experimental evidence associating them with intracellular parasitism. With the increasing number of published complete genome sequences of members of the trypanosomatid family, including not only different <i>Trypanosoma</i> and <i>Leishmania</i> strains and subspecies but also trypanosomatids that do not infect humans or other mammals, we may now be able to contemplate a slightly better picture regarding the specific set of parasite factors that defines each organism's mode of living and the associated disease phenotypes. Here, we review the studies concerning <i>T. cruzi</i> and <i>Leishmania</i> genes that have been implicated with cell invasion and intracellular parasitism and also summarize the wealth of new information regarding the mode of living of intracellular parasites that is resulting from comparative genome studies that are based on increasingly larger trypanosomatid genome datasets.</p></div

Common genes present exclusively in intracellular parasites.

<p>After identifying orthologous proteins, by performing an all-versus-all alignment between the amino acid sequences, the results of the pairwise alignments were used as input to the OrthoMCL software V1.4 with its default parameters. Specific OrthoMCL clusters of intracellular and extracellular/apathogenic trypanosomatids and functional enrichment analysis based in genome annotation were performed using in-house PERL scripts.</p

The distinct life cycles of tritryp parasites.

<p>Panels A–C show the life cycles of <i>T. cruzi</i>, <i>Leishmania</i> spp, and <i>T. brucei</i>, respectively. In each panel, some of the parasite stages present in their insect vectors, <i>T. cruzi</i> epimastigotes, <i>Leishmania</i> promastigotes, and <i>T. brucei</i> procyclic forms, are shown on the left. Different sand fly species of the genera <i>Lutzomyia</i> and <i>Phlebotomus</i> are vectors for Leishmania. <i>Triatoma infestans</i> and <i>Rhodnius prolixus</i> are the most important vector species in the transmission of <i>T. cruzi</i> to man, whereas different species of <i>Glossina</i>, also known as tse-tse fly, are vectors of African trypanosomes. Leishmania and <i>T. brucei</i> parasites move from the fly midgut up to the mouthparts before being inoculated into the human host as metacyclic, infective forms. Although Leishmania promastigotes achieve their journey in sand flies by being regurgitated from the stomodeal valve to the mouthparts, <i>T. brucei</i> epimastigotes do not stay in the mouthparts, as they have to first migrate from the proventriculus to the salivary glands where they develop into metacyclic forms and are expelled with the insect saliva. In contrast, <i>T. cruzi</i> infective metacyclic trypomastigotes develop in the hindgut of the triatomine bug and, after being excreted with the insect feces, gain access to the mammalian host bloodstream through skin wounds or the mucous membranes. On the right side of each panel, parasite forms present in the mammalian host, <i>T. cruzi</i> trypomastigotes, and intracellular amastigotes, <i>Leishmania</i> intracellular amastigotes, and <i>T. brucei</i> bloodstream forms are shown. Whereas <i>Leishmania</i> promastigotes are internalized by host phagocytes and reside into the phagolysosome, <i>T. cruzi</i> trypomastigotes actively invade a variety of nonphagocytic cells and are able to escape from the phagocytic vacuole and multiply in the host cell cytoplasm. Although distinct developmental forms of <i>T. brucei</i> are found in the mammalian host, namely stumpy and slender trypomastigotes, they remain extracellular during the entire parasite life cycle and were represented here as bloodstream trypomastigotes. Panel D shows a phylogenetic analysis inferred from glycosomal glyceraldehyde 3-phosphate dehydrogenase (gapdh) nucleotide sequences from 16 trypanosomatid species, with the species that have an intracellular stage shown with a light blue color. The maximum likelihood tree was constructed with 849 nt (80% of gapdh coding sequences), using SeaView v.04 and rooted at the <i>Crithidia fasciculata</i>/<i>A. deanei</i> clade, with the bootstrap values for 1,000 replicates shown in the major basal nodes.</p

<i>T. cruzi</i> and <i>Leishmania</i> genes involved with host cell invasion and intracellular survival.

<p><i>T. cruzi</i> and <i>Leishmania</i> genes involved with host cell invasion and intracellular survival.</p

Surface proteins present in <i>Leishmania</i> and <i>T. cruzi</i>.

<p>The figure shows six different surface molecules known to be present in promastigote and amastigote forms of <i>Leishmania</i> (left) and trypomastigote and amastigote forms of <i>T. cruzi</i> (right). Each protein is represented by the symbols indicated below the figure.</p

Analysis of the cellular and humoral response and of the involvement of IL-12, CD4 and CD8 T cells in the IFN-γ production after <i>L. infantum</i> challenge.

<p>Single cells suspensions were obtained from the spleens of mice, 10 weeks after infection. Cells were non-stimulated (medium; background control) or stimulated with <i>L. infantum</i> SLA (25 µg mL⁻¹) for 48 h at 37°C, 5% CO₂. Levels of IFN-γ, IL-12, GM-CSF, IL-4 and IL-10 were measured in culture supernatants by capture ELISA. Mean ± standard deviation (SD) of the cytokines levels determined in four individual mice per group is shown (<b>A</b>). Statistically significant differences between the rLiHyp1 plus saponin group and the control mice (saline and saponin groups) were observed (*** <i>P<0.0001</i>). The analysis of the involvement of IL-12 and CD4 and CD8 T cells in the IFN-γ production is showed (<b>B</b>). Levels of IFN-γ in the supernatants of spleen cells cultures stimulated with SLA, as explained above, in the absence (positive control) or in the presence of anti-IL-12, anti-CD4, or anti-CD8 monoclonal antibodies were measured. Statistically significant differences between non-treated control cells and cultures incubated with anti-CD4 and anti-IL-12 monoclonal antibodies were observed (*** <i>P</i><0.0001). The ratio between IFN-γ/IL-10 and IFN-γ/IL-4 levels (<b>C</b>), and between IL-12/IL-10 and IL-12/IL-4 levels (<b>D</b>), are also showed. Statistically significant differences between the rLiHyp1 plus saponin group and the control groups were observed (***<i>P</i><0.0001). The ratio between SLA-specific IgG1 and IgG2a antibodies levels were calculated for sera of each individual mouse within their respective vaccination group and statistically significant difference between the rLiHyp1 plus saponin group and the control groups was also observed (* <i>P</i><0.005) (<b>E</b>).</p

Cellular and humoral response induced in BALB/c mice by immunization with rLiHyp1 plus saponin.

<p>Single cells suspensions were obtained from the spleens of mice, four weeks after vaccination. Cells were non-stimulated (medium; background control) or stimulated with rLiHyp1 (20 µg mL⁻¹) for 48 h at 37°C, 5% CO₂. IFN-γ, IL-12, GM-CSF, IL-4, and IL-10 levels were measured in culture supernatants by capture ELISA (<b>A</b>). Each bar represents the mean ± standard deviation (SD) of data from four individual mice per group. Statistically significant differences in the IFN-γ, IL-12 and GM-CSF levels between the rLiHyp1 plus saponin group and control mice (saline and saponin groups) were observed (*** <i>P</i><0.0001). The ratio between IFN-γ/IL-10 and IFN-γ/IL-4 levels (<b>B</b>); and between IL-12/IL-10 and IL-12/IL-4 levels (<b>C</b>) are also showed. Statistically significant differences in the ratios between the rLiHyp1 plus saponin group and control groups were observed (*** <i>P</i><0.0001). The ratio between rLiHyp1-specific IgG1 and IgG2a antibodies was obtained for sera of each individual mouse within their respective vaccination group and statistically significant difference between the rLiHyp1 plus saponin group and control groups was also observed (* <i>P</i><0.005) (<b>D</b>).</p

Protection of BALB/c mice vaccinated with rLiHyp1 plus saponin against <i>L. infantum</i>.

<p>Mice inoculated with saline, saponin, or rLiHyp1 plus saponin were subcutaneously infected with virulent 1×10⁷ stationary-phase promastigotes of <i>L. infantum</i>. The number of parasites in the liver (<b>A</b>), spleen (<b>B</b>), bone marrow (<b>C</b>), and paws' draining lymph nodes (<b>D</b>) was measured, 10 weeks after challenge by a limiting-dilution technique. Mean ± standard deviation (SD) of four mice in each group is shown. Statistically significant differences in the parasite load in all evaluated organs between the rLiHyp1 plus saponin group and control mice (saline and saponin groups) are showed (in numbers). Data shown in this study are representative of two independent experiments, which presented similar results.</p