Plasmodium vivax Infection Alters Mitochondrial Metabolism in Human Monocytes.

Monocytes play an important role in the host defense against Plasmodium vivax as the main source of inflammatory cytokines and mitochondrial reactive oxygen species (mROS). Here, we show that monocyte metabolism is altered during human P. vivax malaria, with mitochondria playing a major function in this switch. The process involves a reprograming in which the cells increase glucose uptake and produce ATP via glycolysis instead of oxidative phosphorylation. P. vivax infection results in dysregulated mitochondrial gene expression and in altered membrane potential leading to mROS increase rather than ATP production. When monocytes were incubated with P. vivax-infected reticulocytes, mitochondria colocalized with phagolysosomes containing parasites representing an important source mROS. Importantly, the mitochondrial enzyme superoxide dismutase 2 (SOD2) is simultaneously induced in monocytes from malaria patients. Taken together, the monocyte metabolic reprograming with an increased mROS production may contribute to protective responses against P. vivax while triggering immunomodulatory mechanisms to circumvent tissue damage. IMPORTANCE Plasmodium vivax is the most widely distributed causative agent of human malaria. To achieve parasite control, the human immune system develops a substantial inflammatory response that is also responsible for the symptoms of the disease. Among the cells involved in this response, monocytes play an important role. Here, we show that monocyte metabolism is altered during malaria, with its mitochondria playing a major function in this switch. This change involves a reprograming process in which the cells increase glucose uptake and produce ATP via glycolysis instead of oxidative phosphorylation. The resulting altered mitochondrial membrane potential leads to an increase in mitochondrial reactive oxygen species rather than ATP. These data suggest that agents that change metabolism should be investigated and used with caution during malaria.

Autophagy: A necessary process during the Trypanosoma cruzi life-cycle.

Autophagy is a well-conserved process of self-digestion of intracellular components. T. cruzi is a protozoan parasite with a complex life-cycle that involves insect vectors and mammalian hosts. Like other eukaryotic organisms, T. cruzi possesses an autophagic pathway that is activated during metacyclogenesis, the process that generates the infective forms of parasites. In addition, it has been demonstrated that mammalian autophagy has a role during host cell invasion by T. cruzi, and that T. cruzi can modulate this process to its own benefit. This review describes the latest findings concerning the participation of autophagy in both the T. cruzi differentiation processes and during the interaction of parasites within the host cells. Data to date suggest parasite autophagy is important for parasite survival and differentiation, which offers interesting prospects for therapeutic strategies. Additionally, the interruption of mammalian autophagy reduces the parasite infectivity, interfering with the intracellular cycle of T. cruzi inside the host. However, the impact on other stages of development, such as the intracellular replication of parasites is still not clearly understood. Further studies in this matter are necessaries to define the integral effect of autophagy on T. cruzi infection with both in vitro and in vivo approaches.

The autophagic pathway is a key component in the lysosomal dependent entry of Trypanosoma cruzi into the host cell.

The etiologic agent of Chagas disease, Trypanosoma cruzi, infects mammalian cells activating a signal transduction cascade that leads to the formation of its parasitophorous vacuole. Previous works have demonstrated the crucial role of lysosomes in the establishment of T. cruzi infection. In this work we have studied the possible relationship between this parasite and the host cell autophagy. We show, for the first time, that the vacuole containing T. cruzi (TcPV) is decorated by the host cell autophagic protein LC3. Furthermore, live cell imaging experiments indicate that autolysosomes are recruited to parasite entry sites. Interestingly, starvation or pharmacological induction of autophagy before infection significantly increased the number of infected cells whereas inhibitors of this pathway reduced the invasion. In addition, the absence of Atg5 or the reduced expression of Beclin 1 -- two proteins required at the initial steps of autophagosome formation -- limited parasite entry and reduced the association between TcPV and the classical lysosomal marker Lamp-1. These results indicate that mammalian autophagy is a key process that favors the colonization of T. cruzi in the host cell.

Do microtubules around the Toxoplasma gondii-containing parasitophorous vacuole in skeletal muscle cells form a barrier for the phagolysosomal fusion?

The intracellular fate of Toxoplasma gondii was studied in primary cultures of skeletal muscle cells (SMC). The labelling of secondary lysosomes with BSA-Au particles showed no phagolysosomal fusion with the vacuole containing the parasite. After internalization of the parasites, the parasitophorous vacuole became involved by closely apposed endoplasmic reticulum (ER) and mitochondria; within 18 h of interaction, microtubules were visualized in association with the parasitophorous vacuole, suggesting that they could form a barrier for the phagolysosomal fusion.

Surface Zn-proteinase as a molecule for defense of Leishmania mexicana amazonensis promastigotes against cytolysis inside macrophage phagolysosomes.

The role of the surface membrane Zn-proteinase in protecting the cellular integrity of the macrophage parasite Leishmania mexicana amazonensis from intraphagolysosomal cytolysis was studied. These cells lose their infectivity to host macrophages after prolonged cultivation in axenic growth medium. The virulent and attenuated variants of the parasite cells were cloned. Failure of these attenuated parasite cells to survive inside macrophage phagolysosomes is associated with 20- to 50-fold reduction in the expression of surface gp63 protein. In situ inhibition of gp63 proteinase activity inside Leishmania-infected macrophage phagolysosomes with targeted delivery of an inhibitor of gp63 proteinase activity, 1,10-phenanthroline, selectively eliminated intracellular Leishmania amastigotes, further suggesting the importance of this proteinase in phagolysosomal survival of the parasite. An upstream sequence (US) of the gp63 gene was cloned in front of the bacterial chloramphenicol acetyltransferase (CAT) gene in plasmid pCATbasic. Transfection of L. mexicana amazonensis cells with this recombinant plasmid showed that expression of the CAT gene from this US is 15- to 20-fold higher in virulent clones than in avirulent clones of the parasite. Band shift analysis with the cloned US also showed that binding of protein(s) was 15- to 20-fold higher in virulent cell extract than in avirulent cell extract. Coating of attenuated cells or liposomes with proteolytically active gp63 protects them from degradation inside macrophage phagolysosomes. These results suggest a novel mechanism of survival of this phagolysosomal parasite with the help of its surface Zn-proteinase.

Differential expression of the plasma membrane Mg2+ ATPase and Ca2+ ATPase activity during adhesion and interiorization of Leishmania amazonensis in fibroblasts in vitro.

With ultrastructural cytochemistry we localized the activity of the plasma membrane enzyme markers Mg2+ ATPase and Ca2+ ATPase during the interaction between Leishmania amazonensis and in vitro primary culture fibroblasts. The expression of the enzymes was followed during the parasite adhesion and its interiorization. After the interiorization step, a striking difference was seen between the two enzymes studied when the parasite was found within the parasitophorous vacuole in the fibroblast cytoplasm. The activity of the Ca2+ ATPase found at the Leishmania amazonensis plasma membrane during the attachment step of the infection remained also present inside the phagosome, whereas the Mg2+ ATPase activity disappeared. So far, all the reports in the literature referred the presence of Ca2+ ATPase in Leishmania parasite only in the crude ghost plasma membrane. The Ca2+ ATPase present at the parasite plasma membrane may be involved in the regulation of calcium levels inside the phagosome. Further characterization of this Ca2+ ATPase at the plasma membrane of the parasite, when still inside the phagosome, should permit a better understanding of its functional role in maintaining the parasite surface membrane structure necessary for its existence.

Surface acid proteinase (gp63) of Leishmania mexicana. A metalloenzyme capable of protecting liposome-encapsulated proteins from phagolysosomal degradation by macrophages.

Acid proteinase activity is associated with the major surface glycoprotein (gp63) of both extracellular promastigotes and intracellular amastigotes of the parasitic protozoan, Leishmania mexicana. The enzyme purified by monoclonal affinity chromatography from promastigotes is strongly inhibited by metal ion chelators, which is reversible by the addition of Zn(II). This proteinase loses its activity after dialysis against 1,10-phenanthroline. The apoenzyme thus prepared is reactivated substantially by Zn(II) and partially by Cu(II), Cd(II), Co(II), or Ni(II). From the recently published structure of the gene encoding gp63, we identify hitherto unrecognized sequences, which can be aligned to the consensus zinc-binding sites of other known metalloproteinases. Anti-gp63 polyclonal antibodies, but not the monoclonals, precipitate similar molecules from amastigotes. These molecules differ slightly from gp63 in electrophoretic mobility but have similar endopeptidase activity. Phagolysosomal degradation by macrophages of proteins entrapped in liposomes is prevented by coating them with native gp63. This protection is lost with heat denaturation of gp63 to kill its enzymatic activity. The proteolytic activity of the metalloenzyme on the surface of these parasites may thus protect their membrane from cytolytic damages during their survival, differentiation, and multiplication in the phagolysosomes of macrophages.