Differential contribution of two organelles of endosymbiotic origin to iron-sulfur cluster synthesis and overall fitness in Toxoplasma.

Iron-sulfur (Fe-S) clusters are one of the most ancient and ubiquitous prosthetic groups, and they are required by a variety of proteins involved in important metabolic processes. Apicomplexan parasites have inherited different plastidic and mitochondrial Fe-S clusters biosynthesis pathways through endosymbiosis. We have investigated the relative contributions of these pathways to the fitness of Toxoplasma gondii, an apicomplexan parasite causing disease in humans, by generating specific mutants. Phenotypic analysis and quantitative proteomics allowed us to highlight notable differences in these mutants. Both Fe-S cluster synthesis pathways are necessary for optimal parasite growth in vitro, but their disruption leads to markedly different fates: impairment of the plastidic pathway leads to a loss of the organelle and to parasite death, while disruption of the mitochondrial pathway trigger differentiation into a stress resistance stage. This highlights that otherwise similar biochemical pathways hosted by different sub-cellular compartments can have very different contributions to the biology of the parasites, which is something to consider when exploring novel strategies for therapeutic intervention.

Leishmania amazonensis ferric iron reductase (LFR1) is a bifunctional enzyme: Unveiling a NADPH oxidase activity.

Leishmania amazonensis is one of leishmaniasis' causative agents, a disease that has no cure and leads to the appearance of cutaneous lesions. Recently, our group showed that heme activates a Na+/K+ ATPase in these parasites through a signaling cascade involving hydrogen peroxide (H2O2) generation. Heme has a pro-oxidant activity and signaling capacity, but the mechanism by which this molecule increases H2O2 levels in L. amazonensis has not been elucidated. Here we investigated the source of H2O2 stimulated by heme, ruling out the participation of mitochondria and raising the possibility of a role for a NADPH oxidase (Nox) activity. Despite the absence of a classical Nox sequence in trypanosomatid genomes, L. amazonensis expresses a surface ferric iron reductase (LFR1). Interestingly, Nox enzymes are thought to have evolved from ferric iron reductases because they share same core domain and are very similar in structure. The main difference is that Nox catalyses electron flow from NADPH to oxygen, generating reactive oxygen species (ROS), while ferric iron reductase promotes electron flow to ferric iron, generating ferrous iron. Using L. amazonensis overexpressing or knockout for LFR1 and heterologous expression of LFR1 in mammalian embryonic kidney (HEK 293) cells, we show that this enzyme is bifunctional, being able to generate both ferrous iron and H2O2. It was previously described that protozoans knockout for LFR1 have their differentiation to virulent forms (amastigote and metacyclic promastigote) impaired. In this work, we observed that LFR1 overexpression stimulates protozoan differentiation to amastigote forms, reinforcing the importance of this enzyme in L. amazonensis life cycle regulation. Thus, we not only identified a new source of ROS production in Leishmania, but also described, for the first time, an enzyme with both ferric iron reductase and Nox activities.

First Report of Echinococcus ortleppi in Human Cases of Cystic Echinococcosis in Poland.

Cystic echinococcosis is considered as an emerging zoonosis that can develop asymptomatically for years, clinically nonpathognomic. The disease is of public health importance due to often late, difficult diagnostics, uncertain results of treatment, the need to remove hydatid cysts surgically in advanced cases, and poor prognosis in untreated patients. Six Polish female patients with diagnosed cystic echinococcosis (CE) were examined. DNA extracted from the liver and lung samples served for amplification of mitochondrial nad1 gene fragment. Sequence alignments of 5 isolates showed identity with the pig strain, Echinococcus canadensis G7. One case was in 100% identical with Echinococcus ortleppi G5, the cattle strain. These data demonstrate first report of E. ortleppi, regarded as extinct species, causing human cystic echinococcosis in Poland, where the most frequent causative agent of human CE is E. canadensis.

Leishmania donovani Parasites Are Inhibited by the Benzoxaborole AN2690 Targeting Leucyl-tRNA Synthetase.

Visceral leishmaniasis is an important public health threat in parts of India. It is caused by a protozoan parasite, Leishmania donovani Currently available drugs manifest severe side effects. Hence, there is a need to identify new drug targets and drugs. Aminoacyl-tRNA synthetases, required for protein synthesis, are known drug targets for bacterial and fungal pathogens. The aim of the present study was to obtain essentiality data for Leishmania donovani leucyl-tRNA synthetase (LdLRS) by gene replacement. Gene replacement studies indicate that this enzyme plays an essential role in the viability of this pathogenic organism and appears to be indispensable for its survival in vitro The heterozygous mutant parasites demonstrated a growth deficit and reduced infectivity in mouse macrophages compared to the wild-type cells. We also report that Leishmania donovani recombinant LRS displayed aminoacylation activity and that the protein localized to both the cytosol and the mitochondrion. A broad-spectrum antifungal, 5-fluoro-1,3-dihydro-1-hydroxy-2,1-benzoxaborole (AN2690), was found to inhibit parasite growth in both the promastigote and amastigote stages in vitro as well as in vivo in BALB/c mice. This compound exhibited low toxicity to mammalian cells. AN2690 was effective in inhibiting the aminoacylation activity of the recombinant LdLRS. We provide preliminary chemical validation of LdLRS as a drug target by showing that AN2690 is an inhibitor both of L. donovani LRS and of L. donovani cell growth.

Atovaquone enhances doxorubicin's efficacy via inhibiting mitochondrial respiration and STAT3 in aggressive thyroid cancer.

The clinical management of anaplastic thyroid carcinoma and follicular thyroid carcinoma is challenging and requires an alternative therapeutic strategy. Although atovaquone is an FDA-approved anti-malarial drug, studies has recently demonstrated its anti-cancer activities. In line with these efforts, our study shows that atovaquone is an attractive candidate for thyroid cancer treatment. We show that atovaquone significantly inhibits growth, migration and survival in a concentration-dependent manner in 8505C and FTC113 cells. Mechanistically, atovaquone inhibits mitochondrial complex III activity, leading to mitochondrial respiration inhibition and reduction of ATP production in thyroid cancer cells. The inhibitory effects of atovaquone is reversed in mitochondrial respiration-deficient 8505C ρ0 cells, confirming mitochondrial respiration as the mechanism of atovaquone's action in thyroid cancer. In addition, atovaquone suppresses phosphorylation of STAT3 in thyroid cancer wildype but not ρ0 cells, demonstrating that STAT3 phosphorylation inhibition by atovaquone is a consequence of mitochondrial respiration inhibition. Notably, we further demonstrate that atovaquone significantly augments doxorubicin's inhibitory effects via suppressing mitochondrial respiration and STAT3. Our findings suggest that atovaquone can be repurposed for thyroid cancer treatment. Our work also highlights that targeting mitochondrial respiration may represent potential therapeutic strategy in thyroid cancer.

A glutaredoxin in the mitochondrial intermembrane space has stage-specific functions in the thermo-tolerance and proliferation of African trypanosomes.

Trypanosoma brucei glutaredoxin 2 (Grx2) is a dithiol glutaredoxin that is specifically located in the mitochondrial intermembrane space. Bloodstream form parasites lacking Grx2 or both, Grx2 and the cytosolic Grx1, are viable in vitro and infectious to mice suggesting that neither oxidoreductase is needed for survival or infectivity to mammals. A 37 °C to 39 °C shift changes the cellular redox milieu of bloodstream cells to more oxidizing conditions and induces a significantly stronger growth arrest in wildtype parasites compared to the mutant cells. Grx2-deficient cells ectopically expressing the wildtype form of Grx2 with its C31QFC34 active site, but not the C34S mutant, regain the sensitivity of the parental strain, indicating that the physiological role of Grx2 requires both active site cysteines. In the procyclic insect stage of the parasite, Grx2 is essential. Both alleles can be replaced if procyclic cells ectopically express authentic or C34S, but not C31S/C34S Grx2, pointing to a redox role that relies on a monothiol mechanism. RNA-interference against Grx2 causes a virtually irreversible proliferation defect. The cells adopt an elongated morphology but do not show any significant alteration in the cell cycle. The growth retardation is attenuated by high glucose concentrations. Under these conditions, procyclic cells obtain ATP by substrate level phosphorylation suggesting that Grx2 might regulate a respiratory chain component.

Cardiomyocyte diffusible redox mediators control Trypanosoma cruzi infection: role of parasite mitochondrial iron superoxide dismutase.

Chagas disease (CD), caused by the protozoa Trypanosoma cruzi, is a chronic illness in which parasites persist in the host-infected tissues for years. T. cruzi invasion in cardiomyocytes elicits the production of pro-inflammatory mediators [TNF-α, IL-1ß, IFN-γ; nitric oxide (·NO)], leading to mitochondrial dysfunction with increased superoxide radical (O2·-), hydrogen peroxide (H2O2) and peroxynitrite generation. We hypothesize that these redox mediators may control parasite proliferation through the induction of intracellular amastigote programmed cell death (PCD). In this work, we show that T. cruzi (CL-Brener strain) infection in primary cardiomyocytes produced an early (24 h post infection) mitochondrial dysfunction with H2O2 generation and the establishment of an oxidative stress evidenced by FoxO3 activation and target host mitochondrial protein expression (MnSOD and peroxiredoxin 3). TNF-α/IL-1ß-stimulated cardiomyocytes were able to control intracellular amastigote proliferation compared with unstimulated cardiomyocytes. In this condition leading to oxidant formation, an enhanced number of intracellular apoptotic amastigotes were detected. The ability of H2O2 to induce T. cruzi PCD was further confirmed in the epimastigote stage of the parasite. H2O2 treatment induced parasite mitochondrial dysfunction together with intra-mitochondrial O2·- generation. Importantly, parasites genetically engineered to overexpress mitochondrial Fe-superoxide dismutase (Fe-SODA) were more infective to TNF-α/IL-1ß-stimulated cardiomyocytes with less apoptotic amastigotes; this result underscores the role of this enzyme in parasite survival. Our results indicate that cardiomyocyte-derived diffusible mediators are able to control intracellular amastigote proliferation by triggering T. cruzi PCD and that parasite Fe-SODA tilts the process toward survival as part of an antioxidant-based immune evasion mechanism.

Antileishmanial Activity, Cytotoxicity and Mechanism of Action of Clioquinol Against Leishmania infantum and Leishmania amazonensis Species.

In this study, a quinoline derivate, clioquinol (5-chloro-7-iodoquinolin-8-ol), was evaluated against Leishmania amazonensis and Leishmania infantum promastigotes and amastigotes. The cytotoxicity in murine macrophages and human red blood cells, as well as the efficacy in treating infected macrophages and the inhibition of infection using pre-treated parasites were also evaluated. Results showed that clioquinol inhibited L. amazonensis and L. infantum promastigotes with effective concentration 50% (EC50 ) values of 2.55 ± 0.25 and 1.44 ± 0.35 µg/mL, respectively, and of 1.88 ± 0.13 and 0.98 ± 0.17 µg/mL against axenic amastigotes, respectively. The cytotoxic EC50 concentrations of clioquinol in murine macrophages and human red blood cells were, respectively, 255 ± 23 and 489 ± 20 µg/mL. With these results, the selectivity index was calculated, showing values of 99.9 and 177.1 against promastigotes, respectively, and of 135.6 and 260.1 against axenic amastigotes, respectively. Significant reductions in the percentage of infected macrophages after treatment using clioquinol were also observed, as well as when parasites were pre-treated with clioquinol and used to infect murine macrophages. The mechanism of action of clioquinol was investigated in L. amazonensis, and results revealed morphological and biochemical alterations in the clioquinol-treated parasites, including reduction in cell volume, loss of mitochondrial membrane potential, increase in the ROS production and rupture of the plasma membrane. The externalization of phosphatidylserine (PS) at the cell surface was evaluated in treated parasites that had been doubly labelled with annexin and propidium iodide (PI). The results showed no significant difference for PS exposure when compared to the untreated control, although a significant increase in the PI/annexin V-labelled cell population was found in the treated parasites. Results suggest that clioquinol induces a discontinuity of the parasite membrane, possibly related to a characteristic event of cell death caused by necrosis. This study demonstrates, for the first time, the antileishmanial activity of clioquinol against two relevant Leishmania species and suggests that the mitochondria of the parasites may be a possible biological target leading to parasite necrosis. Our findings suggest that clioquinol may have a potential application in treatment of leishmaniasis and further studies should be performed in infected mammalian hosts.

Leishmania donovani activates SREBP2 to modulate macrophage membrane cholesterol and mitochondrial oxidants for establishment of infection.

Establishment of infection by an intracellular pathogen depends on successful internalization with a concomitant neutralization of host defense machinery. Leishmania donovani, an intramacrophage pathogen, targets host SREBP2, a critical transcription factor, to regulate macrophage plasma membrane cholesterol and mitochondrial reactive oxygen species generation, favoring parasite invasion and persistence. Leishmania infection triggered membrane-raft reorientation-dependent Lyn-PI3K/Akt pathway activation which in turn deactivated GSK3ß to stabilize nuclear SREBP2. Moreover, cells perceiving less available intracellular cholesterol due to its sequestration at the plasma membrane resulted in the deregulation of the ER-residing SCAP-SREBP2-Insig circuit thereby assisting increased nuclear translocation of SREBP2. Both increased nuclear transport and stabilization of SREBP2 caused HMGCR-catalyzed cholesterol biosynthesis-mediated plasma membrane cholesterol enrichment leading to decreased membrane-fluidity and plausibly assisting delay in phagosomal acidification. Parasite survival ensuing entry was further ensured by SREBP2-dependent transcriptional up-regulation of UCP2, which suppressed mitochondrial ROS generation, one of the primary microbicidal molecules in macrophages recognized for its efficacy against Leishmania. Functional knock-down of SREBP2 both in vitro and in vivo was associated with reduction in macrophage plasma membrane cholesterol, increased ROS production and lower parasite survival. To our knowledge, this study, for the first time, reveals that Leishmania exploits macrophage cholesterol-dependent SREBP2 circuit to facilitate its entry and survival within the host.

Sustained activation of Akt elicits mitochondrial dysfunction to block Plasmodium falciparum infection in the mosquito host.

The overexpression of activated, myristoylated Akt in the midgut of female transgenic Anopheles stephensi results in resistance to infection with the human malaria parasite Plasmodium falciparum but also decreased lifespan. In the present study, the understanding of mitochondria-dependent midgut homeostasis has been expanded to explain this apparent paradox in an insect of major medical importance. Given that Akt signaling is essential for cell growth and survival, we hypothesized that sustained Akt activation in the mosquito midgut would alter the balance of critical pathways that control mitochondrial dynamics to enhance parasite killing at some cost to survivorship. Toxic reactive oxygen and nitrogen species (RNOS) rise to high levels in the midgut after blood feeding, due to a combination of high NO production and a decline in FOXO-dependent antioxidants. Despite an apparent increase in mitochondrial biogenesis in young females (3 d), energy deficiencies were apparent as decreased oxidative phosphorylation and increased [AMP]/[ATP] ratios. In addition, mitochondrial mass was lower and accompanied by the presence of stalled autophagosomes in the posterior midgut, a critical site for blood digestion and stem cell-mediated epithelial maintenance and repair, and by functional degradation of the epithelial barrier. By 18 d, the age at which An. stephensi would transmit P. falciparum to human hosts, mitochondrial dysfunction coupled to Akt-mediated repression of autophagy/mitophagy was more evident and midgut epithelial structure was markedly compromised. Inhibition of RNOS by co-feeding of the nitric-oxide synthase inhibitor L-NAME at infection abrogated Akt-dependent killing of P. falciparum that begins within 18 h of infection in 3-5 d old mosquitoes. Hence, Akt-induced changes in mitochondrial dynamics perturb midgut homeostasis to enhance parasite resistance and decrease mosquito infective lifespan. Further, quality control of mitochondrial function in the midgut is necessary for the maintenance of midgut health as reflected in energy homeostasis and tissue repair and renewal.

ATG5 is essential for ATG8-dependent autophagy and mitochondrial homeostasis in Leishmania major.

Macroautophagy has been shown to be important for the cellular remodelling required for Leishmania differentiation. We now demonstrate that L. major contains a functional ATG12-ATG5 conjugation system, which is required for ATG8-dependent autophagosome formation. Nascent autophagosomes were found commonly associated with the mitochondrion. L. major mutants lacking ATG5 (Δatg5) were viable as promastigotes but were unable to form autophagosomes, had morphological abnormalities including a much reduced flagellum, were less able to differentiate and had greatly reduced virulence to macrophages and mice. Analyses of the lipid metabolome of Δatg5 revealed marked elevation of phosphatidylethanolamines (PE) in comparison to wild type parasites. The Δatg5 mutants also had increased mitochondrial mass but reduced mitochondrial membrane potential and higher levels of reactive oxygen species. These findings indicate that the lack of ATG5 and autophagy leads to perturbation of the phospholipid balance in the mitochondrion, possibly through ablation of membrane use and conjugation of mitochondrial PE to ATG8 for autophagosome biogenesis, resulting in a dysfunctional mitochondrion with impaired oxidative ability and energy generation. The overall result of this is reduced virulence.

Leishmania mitochondrial peroxiredoxin plays a crucial peroxidase-unrelated role during infection: insight into its novel chaperone activity.

Two-cysteine peroxiredoxins are ubiquitous peroxidases that play various functions in cells. In Leishmania and related trypanosomatids, which lack catalase and selenium-glutathione peroxidases, the discovery of this family of enzymes provided the molecular basis for peroxide removal in these organisms. In this report the functional relevance of one of such enzymes, the mitochondrial 2-Cys peroxiredoxin (mTXNPx), was investigated along the Leishmania infantum life cycle. mTXNPx null mutants (mtxnpx(-)) produced by a gene replacement strategy, while indistinguishable from wild type promastigotes, were found unable to thrive in a murine model of infection. Unexpectedly, however, the avirulent phenotype of mtxnpx(-) was not due to lack of the peroxidase activity of mTXNPx as these behaved like controls when exposed to oxidants added exogenously or generated by macrophages during phagocytosis ex vivo. In line with this, mtxnpx(-) were also avirulent when inoculated into murine hosts unable to mount an effective oxidative phagocyte response (B6.p47(phox-/-) and B6.RAG2(-/-) IFN-γ(-/-) mice). Definitive conclusion that the peroxidase activity of mTXNPx is not required for parasite survival in mice was obtained by showing that a peroxidase-inactive version of this protein was competent in rescuing the non-infective phenotype of mtxnpx(-). A novel function is thus proposed for mTXNPx, that of a molecular chaperone, which may explain the impaired infectivity of the null mutants. This premise is based on the observation that the enzyme is able to suppress the thermal aggregation of citrate synthase in vitro. Also, mtxnpx(-) were more sensitive than controls to a temperature shift from 25°C to 37°C, a phenotype reminiscent of organisms lacking specific chaperone genes. Collectively, the findings reported here change the paradigm which regards all trypanosomatid 2-Cys peroxiredoxins as peroxide-eliminating devices. Moreover, they demonstrate, for the first time, that these 2-Cys peroxiredoxins can be determinant for pathogenicity independently of their peroxidase activity.

Apoptosis as the adaptation mechanism in survival of Trichinella spiralis in the host.

The study evaluates the role of apoptosis-inducing factor (AIF) in the process of striated muscle cell transformation after occupation by Trichinella spiralis. Its relationship with other apoptosis-related factors [apoptotic protease-activating factor 1, Bcl-2 associated protein X (BAX), Bcl-2, caspase 3, survivin, poly (ADP-ribose) polymerase-1 (PARP-1), and endothelial and inducible (iNOS) nitric oxide synthase] was evaluated by immunohistochemistry. In the context of low BAX and caspase 3 expression and strong distribution of AIF in the sarcoplasm and nucleus at the very early stage of infection, we suppose that AIF-mediated signaling is involved in the apoptosis activation in the area of Trichinella occupation. In the time course of nurse cell formation, survivin and caspase 3 migrated into the enlarged nuclei with strong PARP-1 expression. In the end of encapsulation of Trichinella, expression of all proapoptotic factors ceased and only survivin in the nuclei and Bcl-2 positivity in the cytoplasm persisted in the formed nurse cell. The expression of sarcoplasmic iNOS was absent during the process of muscle cell de-differentiation and reappeared within the nurse cell. It seems that upregulation and downregulation of factors of apoptosis in the skeletal muscle cell represents an adaptive mechanism providing a comfortable niche for the parasite.

Mitochondria in the midgut epithelial cells of sugarcane borer parasitized by Cotesia flavipes (Cameron, 1891).

The sugarcane borer Diatraea saccharalis (Lepidoptera: Crambidae) has been controlled by Cotesia flavipes (Hymenoptera: Braconidae); however, very little is known about the effect of the parasitism in the host organs, including the midgut. This work aims to verify mitochondrial alteration in the different midgut epithelial cells of D. saccharalis parasitized by C. flavipes. Midgut fragments (anterior and posterior region) of both non-parasitized and parasitized larvae were processed for transmission electron microscopy. The mitochondria of midgut epithelial cell in the parasitized larvae exhibit morphological alteration, represented by matrix rarefaction and vacuolisation. These mitochondrial alterations are more pronounced in the anterior midgut region during the parasitism process, mainly in the columnar cell.

Mitochondria in the midgut epithelial cells of sugarcane borer parasitized by Cotesia flavipes (Cameron, 1891) / Mitocôndria nas Células Epiteliais do Intestino Médio da Broca-da-cana Parasitada por Cotesia flavipes

The sugarcane borer Diatraea saccharalis (Lepidoptera: Crambidae) has been controlled by Cotesia flavipes (Hymenoptera: Braconidae); however, very little is known about the effect of the parasitism in the host organs, including the midgut. This work aims to verify mitochondrial alteration in the different midgut epithelial cells of D. saccharalis parasitized by C. flavipes. Midgut fragments (anterior and posterior region) of both non-parasitized and parasitized larvae were processed for transmission electron microscopy. The mitochondria of midgut epithelial cell in the parasitized larvae exhibit morphological alteration, represented by matrix rarefaction and vacuolisation. These mitochondrial alterations are more pronounced in the anterior midgut region during the parasitism process, mainly in the columnar cell.

Diatraea saccharalis (Lepidoptera: Crambidae), broca da cana-de-açúcar, tem sido controlada por Cotesia flavipes (Hymenoptera: Braconidae); pouco se sabe sobre o efeito do parasitismo nos diferentes órgãos do inseto hospedeiro, principalmente no intestino médio. O objetivo desse trabalho foi verificar as alterações mitocondriais das diferentes células epiteliais do intestino médio de larvas de D. saccharalis parasitadas por C. flavipes. Fragmentos do intestino médio (regiões anterior e posterior) de larvas de D. saccharalis não-parasitadas e parasitadas foram processados para microscopia eletrônica de transmissão. As mitocôndrias das células epiteliais do intestino médio de larvas parasitadas exibem alterações, especialmente rarefação e vacuolização da matriz, que foram mais pronunciadas nas células epiteliais da região anterior do intestino médio na vigência do parasitismo, em especial nas células colunares.

Toxoplasma gondii infection confers resistance against BimS-induced apoptosis by preventing the activation and mitochondrial targeting of pro-apoptotic Bax.

In order to accomplish their life style, intracellular pathogens, including the apicomplexan Toxoplasma gondii, subvert the innate apoptotic response of infected host cells. However, the precise mechanisms of parasite interference with the mitochondrial apoptotic pathway remain unknown. Here, we used the conditional expression of the BH3-only protein Bim(S) to pinpoint the interaction of T. gondii with the intrinsic pathway of apoptosis. Infection of epithelial cells with T. gondii dose-dependently abrogated Bim(S)-triggered release of cytochrome c from host-cell mitochondria into the cytosol, induction of activity of caspases 3, 7 and 9, and chromatin condensation. Furthermore, inhibition of apoptosis in parasite-infected lymphocytes counteracted death of Toxoplasma-infected host cells. Although total cellular levels and mitochondrial targeting of Bim(S) was not altered by the infection, the activation of pro-apoptotic effector proteins Bax and Bak was strongly impaired. Inhibition of Bax and Bak activation by T. gondii was seen with regard to their conformational changes, the cytosol-to-mitochondria targeting and the oligomerization of Bax but not their cellular protein levels. Blockade of Bax and Bak activation was not mediated by the upregulation of anti-apoptotic Bcl-2-like proteins following infection. Further, the BH3-mimetic ABT-737 failed to overcome the Toxoplasma-imposed inhibition of Bim(S)-triggered apoptosis. These results indicate that T. gondii targets activation of pro-apoptotic Bax and Bak to inhibit the apoptogenic function of mitochondria and to increase host-cell viability.

Blood stage malaria antigens induce different activation-induced cell death programs in splenic CD4+T cells.

CD4(+) T cells respond to antigen immunization through a process of activation, clonal expansion to generate activated effector T cells followed by activation-induced clonal deletion of the responding T cells. While loss of responding T cells in post-activation death by apoptosis is a major factor regulating immune homeostasis, the precise pathways involved in downsizing of Plasmodium falciparum antigen-induced T cell expansions are not well characterized. We report in this study that splenic CD4(+) T cells from mice immunized with nonreplicating immunogens like OVA or recombinant blood stage P. falciparum antigens, PfMSP-3 and PfMSP-1(19) or crude parasite antigen (PfAg) undergo sequential T cell activation, proliferation followed by activation-induced cell death (AICD) in a dose- and time-dependent manner after Ag restimulation. While PfMSP-3 and OVA-induced AICD was mediated through a death receptor-dependent apoptotic program, PfMSP-1(19) and PfAg-induced AICD was via a mechanism dependent on the activation of mitochondria apoptosis signalling pathway through Bax activation. These results provide insights into the mechanism through which two blood stage merozoite antigens trigger different apoptotic programs of AICD in splenic CD4(+) T cells.

New host nuclear functions are not required for the modifications of the parasitophorous vacuole of Toxoplasma.

The obligate intracellular parasite Toxoplasma develops within a parasitophorous vacuole (PV) uniquely adapted for its survival in mammalian cells. Post-invasion events extensively modify the PV, resulting in interactions with host cell structures. Recent studies emphasized that Toxoplasma is able to co-opt host gene expression, suggesting that host transcriptional activities are required for parasite infection. By using an experimental enucleation model, we investigated the potential need for Toxoplasma to modify its PV by modulating gene expression in the cell wherein it resides. Unexpectedly, cytoplasts can be actively invaded by Toxoplasma and sustain its replication inside a vacuole until egress and transmission to neighbouring cells. Although randomly distributed in the cytoplast, the PV associates with host centrosomes and the Golgi, is surrounded by host microtubules, and recruits host endoplasmic reticulum and mitochondria. Parasites are proficient in diverting exogenous nutrients from the endocytic network of cytoplasts. In enucleated cells invaded by an avirulent strain of T. gondii, the PV can normally transform into cysts. These observations suggest that new host nuclear functions are not proximately required for the post-invasion events underlying the remodelling of the host cell in which the parasites are confined, and therefore for the generation of infectious parasites in vitro.

Apoptosis of macrophages induced by Trichomonas vaginalis through the phosphorylation of p38 mitogen-activated protein kinase that locates at downstream of mitochondria-dependent caspase activation.

Trichomonas vaginalis, a flagellated protozoan parasite, is the causative organism of trichomoniasis. We have recently demonstrated that T. vaginalis induces apoptotic cell death via a Bcl-x(L)-dependent pathway in RAW264.7 macrophages. In this study, we attempted to characterize in detail the signaling cascades resulting in T. vaginalis-induced macrophage apoptosis, focusing particularly on mitochondrial changes and the role of p38 mitogen-activated protein kinase (p38 MAPK) activation. We found that T. vaginalis induced mitochondrial changes including the release of cytochrome c and the serial activation of caspases, leading to the activation of p38 MAPK in macrophages. These biochemical changes culminated in the apoptosis of the host cells. Caspase inhibitors induced a significant inhibition of T. vaginalis-induced nuclear damage, as well as the activation of p38 MAPK. Treatment with the p38 MAPK inhibitor, SB203580, or the overexpression of kinase-inactive p38 MAPK, induced an attenuation of T. vaginalis-induced apoptosis but not cytochrome c release, the activation of caspase-9 and caspase-3, or PARP cleavage. Furthermore, SB203580 treatment to human macrophages consistently blocked T. vaginalis-induced apoptosis. Collectively, our findings indicate that p38 MAPK signaling cascade is requisite to apoptosis of T. vaginalis-infected macrophage, and this apoptotic process occurs via the phosphorylation of p38 MAPK, which is located downstream of mitochondria-dependent caspase activation, conferring insight into the plausible molecular mechanism of T. vaginalis-immune evasion from macrophage attack.

Relationship between the host cell mitochondria and the parasitophorous vacuole in cells infected with Encephalitozoon microsporidia.

Encephalitozoon microsporidia proliferate and differentiate within a parasitophorous vacuole. Using the fluorescent probe, calcein, and the mitochondrial probe, MitoTracker-CMXRos, a vital method was developed that confirmed ultrastructural reports that the host cell mitochondria frequently lie in immediate proximity to the parasitophorous vacuole. Morphometry failed to demonstrate any infection-induced increase in host cell mitochondria as there was no correlation between the mitochondrial volume and the extent of infection as judged by the parasitophorous vacuole volume. The total ATP concentration of infected cells did not differ from that of uninfected cells in spite of the increased metabolic demands of the infection. Treatment with 10(-6) M albendazole, more than ten times the antiparasitic IC50 dose, and demecolcine had no subjective effect on the proximity of mitochondria to the parasitophorous vacuole membrane when studied by either transmission electron microscopy or by confocal microscopy even though these drug concentrations affected microtubule structure. Thus, once the association between mitochondria and the parasitophorous vacuole has been established, host cell microtubule integrity is probably not required for its maintenance. It is unlikely that the antimicrosporidial action of albendazole involves physically uncoupling developing parasite stages from host cell organelle metabolic support.