Global Metabolomic Profiling of Host Red Blood Cells Infected with Babesia divergens Reveals Novel Antiparasitic Target Pathways.

Babesia divergens is an apicomplexan parasite that infects human red blood cells (RBCs), initiating cycles of invasion, replication, and egress, resulting in extensive metabolic modification of the host cells. Babesia is an auxotroph for most of the nutrients required to sustain these cycles. There are currently limited studies on the biochemical pathways that support these critical processes, necessitating the high-resolution global metabolomics approach described here to uncover the metabolic interactions between parasite and host RBC. Our results reveal an extensive parasite-mediated modulation of RBC metabolite levels of all classes, including lipids, amino acids, carbohydrates, and nucleotides, with numerous metabolic species varying in proportion to the level of infection. Many of these molecules are scavenged from the host RBCs. This is in accord with the needs of a rapidly proliferating parasite with limited biosynthetic capabilities. Probing these pathways in depth, we used growth inhibition assays to quantitate parasite susceptibility to drugs targeting these pathways and stimulated emission depletion (STED) microscopy to obtain high-resolution images of drug-treated parasites to correlate changes in morphology with specific metabolic blocks in order to validate the data generated by the untargeted metabolomics platform. Thus, interruption of cholesterol scavenging from the host cell led to premature parasite egress, while chemical targeting of the hydrolysis of acyl glycerides led to the buildup of malformed parasites that could not successfully egress. This is the first report detailing the global metabolomic profile of the B. divergens-infected RBC. Besides deciphering diverse aspects of the host-parasite relationship, our results can be exploited by others to uncover further drug targets in the host-parasite biochemical network. IMPORTANCE Human babesiosis is caused by apicomplexan parasites of the Babesia genus and is associated with transfusion-transmitted illness and relapsing disease in immunosuppressed populations. Through its continuous cycles of invasion, proliferation, and egress, B. divergens radically changes the metabolic environment of the host red blood cell, allowing us opportunities to study potential chemical vulnerabilities that can be targeted by drugs. This is the first global metabolomic profiling of Babesia-infected human red blood cells, and our analysis revealed perturbation in all biomolecular classes at levels proportional to the level of infection. In particular, lipids and energy flux pathways in the host cell were altered by infection. We validated the changes in key metabolic pathways by performing inhibition assays accompanied by high-resolution microscopy. Overall, this global metabolomics analysis of Babesia-infected red blood cells has helped to uncover novel aspects of parasite biology and identified potential biochemical pathways that can be targeted for chemotherapeutic intervention.

Elucidating parasite and host-cell factors enabling Babesia infection in sickle red cells under hypoxic/hyperoxic conditions.

Sickle red blood cells (RBCs) represent a naturally existing host-cell resistance mechanism to hemoparasite infections. We investigate the basis of this resistance using Babesia divergens grown in sickle (SS) and sickle trait (AS) cells. We found that oxygenation and its corresponding effect on RBC sickling, frequency of fetal hemoglobin positive (HbF+) cells, cellular redox environment, and parasite proliferation dynamics, all played a role in supporting or inhibiting Babesia proliferation. To identify cellular determinants that supported infection, an image flow cytometric tool was developed that could identify sickled cells and constituent Hb. We showed that hypoxic conditions impaired parasite growth in both SS and AS cells. Furthermore, cell sickling was alleviated by oxygenation (hyperoxic conditions), which decreased inhibition of parasite proliferation in SS cells. Interestingly, our tool identified HbF+-SS as host-cells of choice under both hypoxic and hyperoxic conditions, which was confirmed using cord RBCs containing high amounts of HbF+ cells. Uninfected SS cells showed a higher reactive oxygen species-containing environment, than AA or AS cells, which was further perturbed on infection. In hostile SS cells we found that Babesia alters its subpopulation structure, with 1N dominance under hypoxic conditions yielding to equivalent ratios of all parasite forms at hyperoxic conditions, favorable for growth. Multiple factors, including oxygenation and its impact on cell shape, HbF positivity, redox status, and parasite pleiotropy allow Babesia propagation in sickle RBCs. Our studies provide a cellular and molecular basis of natural resistance to Babesia, which will aid in defining novel therapies against human babesiosis.

Identification and characterization of extracellular vesicles from red cells infected with Babesia divergens and Babesia microti.

Babesiosis is a zoonosis and an important blood-borne human parasitic infection that has gained attention because of its growing infection rate in humans by transfer from animal reservoirs. Babesia represents a potential threat to the blood supply because asymptomatic infections in man are common, and blood from such donors can cause severe disease in certain recipients. Extracellular vesicles (EVs) are vesicles released by cells that contain a complex mixture of proteins, lipids, glycans, and genetic information that have been shown to play important roles in disease pathogenesis and susceptibility, as well as cell-cell communication and immune responses. In this article, we report on the identification and characterization of EVs released from red blood cells (RBCs) infected by two major human Babesia species-Babesia divergens from in vitro culture and those from an in vivo B. microti mouse infection. Using nanoparticle tracking analysis, we show that there is a range of vesicle sizes from 30 to 1,000 nm, emanating from the Babesia-infected RBC. The study of these EVs in the context of hemoparasite infection is complicated by the fact that both the parasite and the host RBC make and release vesicles into the extracellular environment. However, the EV frequency is 2- to 10-fold higher in Babesia-infected RBCs than uninfected RBCs, depending on levels of parasitemia. Using parasite-specific markers, we were able to show that ~50%-60% of all EVs contained parasite-specific markers on their surface and thus may represent the specific proportion of EVs released by infected RBCs within the EV population. Western blot analysis on purified EVs from both in vivo and in vitro infections revealed several parasite proteins that were targets of the host immune response. In addition, microRNA analysis showed that infected RBC EVs have different microRNA signature from uninfected RBC EVs, indicating a potential role as disease biomarkers. Finally, EVs were internalized by other RBCs in culture, implicating a potential role for these vesicles in cellular communication. Overall, our study points to the multiple functional implications of EVs in Babesia-host interactions and support the potential that EVs have as agents in disease pathogenesis.

Sickle Cell Anemia and Babesia Infection.

Babesia is an intraerythrocytic, obligate Apicomplexan parasite that has, in the last century, been implicated in human infections via zoonosis and is now widespread, especially in parts of the USA and Europe. It is naturally transmitted by the bite of a tick, but transfused blood from infected donors has also proven to be a major source of transmission. When infected, most humans are clinically asymptomatic, but the parasite can prove to be lethal when it infects immunocompromised individuals. Hemolysis and anemia are two common symptoms that accompany many infectious diseases, and this is particularly true of parasitic diseases that target red cells. Clinically, this becomes an acute problem for subjects who are prone to hemolysis and depend on frequent transfusions, like patients with sickle cell anemia or thalassemia. Little is known about Babesia's pathogenesis in these hemoglobinopathies, and most parallels are drawn from its evolutionarily related Plasmodium parasite which shares the same environmental niche, the RBCs, in the human host. In vitro as well as in vivo Babesia-infected mouse sickle cell disease (SCD) models support the inhibition of intra-erythrocytic parasite proliferation, but mechanisms driving the protection of such hemoglobinopathies against infection are not fully studied. This review provides an overview of our current knowledge of Babesia infection and hemoglobinopathies, focusing on possible mechanisms behind this parasite resistance and the clinical repercussions faced by Babesia-infected human hosts harboring mutations in their globin gene.

Metabolic regulation of sexual commitment in Plasmodium falciparum.

For malaria parasites regulating sexual commitment, the frequency with which asexual bloodstream forms differentiate into non-replicative male and female gametocytes, is critical because asexual replication is required to maintain a persistent infection of the human host while gametocytes are essential for infection of the mosquito vector and transmission. Here, we describe recent advances in understanding of the regulatory mechanisms controlling this key developmental decision. These include new insights into the mechanistic roles of the transcriptional master switch AP2-G and the epigenetic modulator GDV1, as well as the identification of defined metabolic signals that modulate their activity. Many of these metabolites are linked to parasite phospholipid biogenesis and we propose a model linking this pathway to the epigenetic regulation underlying sexual commitment in P. falciparum.

Insights into physiological roles of unique metabolites released from Plasmodium-infected RBCs and their potential as clinical biomarkers for malaria.

Plasmodium sp. are obligate intracellular parasites that derive most of their nutrients from their host meaning the metabolic circuitry of both are intricately linked. We employed untargeted, global mass spectrometry to identify metabolites present in the culture supernatants of P. falciparum-infected red blood cells synchronized at ring, trophozoite and schizont developmental stages. This revealed a temporal regulation in release of a distinct set of metabolites compared with supernatants of non-infected red blood cells. Of the distinct metabolites we identified pipecolic acid to be abundantly present in parasite lysate, infected red blood cells and infected culture supernatant. Further, we performed targeted metabolomics to quantify pipecolic acid concentrations in both the supernatants of red blood cells infected with P. falciparum, as well as in the plasma and infected RBCs of P. berghei-infected mice. Measurable and significant hyperpipecolatemia suggest that pipecolic acid has the potential to be a diagnostic marker for malaria.

Demonstration and Characterization of Cyst-Like Structures in the Life Cycle of Trichomonas vaginalis.

Trichomonas vaginalis is the parasitic protozoan residing in human urogenital tract causing trichomoniasis, which is the leading non-viral sexually transmitted disease. It has cosmopolitan distribution throughout the globe and affects both men and women. Lifecycle of the parasite has been traditionally described as consisting of motile and symptom-causing trophozoites. Chemical and temperature perturbations in trophozoites have been shown to aid conversion to pseudocysts, which is poorly investigated. In the current study, we show the formation of viable cyst-like structures (CLS) in stationary phase of T. vaginalis axenic culture. We used a fluorescent stain called calcofluor white, which specifically binds to chitin and cellulose-containing structures, to score for T. vaginalis CLS. Using flow cytometry, we demonstrated and quantitated the processes of encystation as well as excystation; thus, completing the parasite's lifecycle in vitro without any chemical/temperature alterations. Like cysts from other protozoan parasites such as Entamoeba histolytica and Giardia lamblia, T. vaginalis CLS appeared spherical, immotile, and resistant to osmotic lysis and detergent treatments. Ultrastructure of CLS demonstrated by Transmission Electron Microscopy showed a thick electron-dense deposition along its outer membrane. To probe the physiological role of CLS, we exposed parasites to vaginal pH and observed that trophozoites took this as a cue to convert to CLS. Further, upon co- culturing with cells of cervical origin, CLS rapidly excysted to form trophozoites which abrogated the cervical cell monolayer in a dose-dependent manner. To further corroborate the presence of two distinct forms in T. vaginalis, we performed two-dimensional gel electrophoresis and global, untargeted mass spectrometry to highlight differences in the proteome with trophozoites. Interestingly, CLS remained viable in chlorinated swimming pool water implicating the possibility of its role as environmentally resistant structures involved in non-sexual mode of parasite transmission. Finally, we showed that symptomatic human patient vaginal swabs had both T. vaginalis trophozoites and CLS; thus, highlighting its importance in clinical infections. Overall, our study highlights the plasticity of the pathogen and its rapid adaption when subjected to stressful environmental cues and suggests an important role of CLS in the parasite's life cycle, pathogenesis and transmission.

Commit, hide and escape: the story of Plasmodium gametocytes.

Malaria is the major cause of mortality and morbidity in tropical countries. The causative agent, Plasmodium sp., has a complex life cycle and is armed with various mechanisms which ensure its continuous transmission. Gametocytes represent the sexual stage of the parasite and are indispensable for the transmission of the parasite from the human host to the mosquito. Despite its vital role in the parasite's success, it is the least understood stage in the parasite's life cycle. The presence of gametocytes in asymptomatic populations and induction of gametocytogenesis by most antimalarial drugs warrants further investigation into its biology. With a renewed focus on malaria elimination and advent of modern technology available to biologists today, the field of gametocyte biology has developed swiftly, providing crucial insights into the molecular mechanisms driving sexual commitment. This review will summarise key current findings in the field of gametocyte biology and address the associated challenges faced in malaria detection, control and elimination.

A secreted Heat shock protein 90 of Trichomonas vaginalis.

Trichomonas vaginalis is a causative agent of Trichomoniasis, a leading non-viral sexually transmitted disease worldwide. In the current study, we show Heat shock protein 90 is essential for its growth. Upon genomic analysis of the parasite, it was found to possess seven ORFs which could potentially encode Hsp90 isoforms. We identified a cytosolic Hsp90 homolog, four homologs which can align to truncated cytosolic Hsp90 gene products along with two Grp94 homologs (ER isoform of Hsp90). However, both Grp94 orthologs lacked an ER retention motif. In cancer cells, it is very well established that Hsp90 is secreted and regulates key clients involved in metastases, migration, and invasion. Since Trichomonas Grp94 lacks ER retention motif, we examined the possibility of its secretion. By using cell biology and biochemical approaches we show that the Grp94 isoform of Hsp90 is secreted by the parasite by the classical ER-Golgi pathway. This is the first report of a genome encoded secreted Hsp90 in a clinically important parasitic protozoan.

A disrupted transsulphuration pathway results in accumulation of redox metabolites and induction of gametocytogenesis in malaria.

Intra-erythrocytic growth of malaria parasite is known to induce redox stress. In addition to haem degradation which generates reactive oxygen species (ROS), the parasite is also thought to efflux redox active homocysteine. To understand the basis underlying accumulation of homocysteine, we have examined the transsulphuration (TS) pathway in the parasite, which is known to convert homocysteine to cysteine in higher eukaryotes. Our bioinformatic analysis revealed absence of key enzymes in the biosynthesis of cysteine namely cystathionine-ß-synthase and cystathionine-γ-lyase in the parasite. Using mass spectrometry, we confirmed the absence of cystathionine, which is formed by enzymatic conversion of homocysteine thereby confirming truncation of TS pathway. We also quantitated levels of glutathione and homocysteine in infected erythrocytes and its spent medium. Our results showed increase in levels of these metabolites intracellularly and in culture supernatants. Our results provide a mechanistic basis for the long-known occurrence of hyperhomocysteinemia in malaria. Most importantly we find that homocysteine induces the transcription factor implicated in gametocytogenesis namely AP2-G and consequently triggers sexual stage conversion. We confirmed this observation both in vitro using Plasmodium falciparum cultures, and in vivo in the mouse model of malaria. Our study implicates homocysteine as a potential physiological trigger of gametocytogenesis.