Properties and predicted functions of large genes and proteins of apicomplexan parasites.

Evolutionary constraints greatly favor compact genomes that efficiently encode proteins. However, several eukaryotic organisms, including apicomplexan parasites such as Toxoplasma gondii, Plasmodium falciparum and Babesia duncani, the causative agents of toxoplasmosis, malaria and babesiosis, respectively, encode very large proteins, exceeding 20 times their average protein size. Although these large proteins represent <1% of the total protein pool and are generally expressed at low levels, their persistence throughout evolution raises important questions about their functions and possible evolutionary pressures to maintain them. In this study, we examined the trends in gene and protein size, function and expression patterns within seven apicomplexan pathogens. Our analysis revealed that certain large proteins in apicomplexan parasites harbor domains potentially important for functions such as antigenic variation, erythrocyte invasion and immune evasion. However, these domains are not limited to or strictly conserved within large proteins. While some of these proteins are predicted to engage in conventional metabolic pathways within these parasites, others fulfill specialized functions for pathogen-host interactions, nutrient acquisition and overall survival.

Babesia duncani, A Model Organism for Investigating Intraerythrocytic Parasitism and Novel Anti-Parasitic Therapeutic Strategies.

Pathogens such as Plasmodium, Babesia, and Theileria invade and multiply within host red blood cells, leading to the pathological consequences of malaria, babesiosis and theileriosis. Establishing continuous in vitro culture systems and suitable animal models is crucial for studying these pathogens. This review spotlights the B. duncani "in culture-in mouse (ICIM)" model as a promising resource for advancing research on the biology, pathogenicity, and virulence of intraerythrocytic parasites. The model offers practical benefits, encompassing well-defined culture conditions, ease of manipulation and a well-annotated genome. Moreover, B. duncani serves as a surrogate system for drug discovery, facilitating the evaluation of new antiparasitic drugs in vitro and in animals, elucidating their modes of action, and uncovering potential resistance mechanisms. The B. duncani ICIM model thus emerges as a multifaceted tool with profound implications, promising advancements in our understanding of parasitic biology and shaping the development of future therapies.

A Set of Diagnostic Tests for Detection of Active Babesia duncani Infection.

Human babesiosis is a rapidly emerging and potentially fatal tick-borne disease caused by intraerythrocytic apicomplexan parasites of the Babesia genus. Among the various species of Babesia that infect humans, B. duncani has been found to cause severe and life-threatening infections. Detection of active B. duncani infection is critical for accurate diagnosis and effective management of the disease. While molecular assays for the detection of B. duncani infection in blood are available, a reliable strategy to detect biomarkers of active infection has not yet been developed. Here, we report the development of the first B. duncani antigen capture assays that rely on the detection of two B. duncani -exported immunodominant antigens, BdV234 and BdV38. The assays were validated using blood samples from cultured parasites in human erythrocytes and B. duncani -infected laboratory mice at different parasitemia levels and following therapy. The assays display high specificity with no cross-reactivity with B. microti , B. divergens , Babesia MO1, or P. falciparum. The assay also demonstrates high sensitivity, detecting as low as 115 infected erythrocytes/µl of blood. Screening of 1,731 blood samples from diverse biorepositories, including previously identified Lyme and/or B. microti positive human samples and new specimens from field mice, showed no evidence of B. duncani infection in these samples. The assays could be useful in diverse diagnostic scenarios, including point-of-care testing for early B. duncani infection detection in patients, field tests for screening reservoir hosts, and high-throughput screening such as blood collected for transfusion. Short summary: We developed two ELISA-based assays, BdACA38 and BdACA234, for detecting B. duncani , a potentially fatal tick-borne parasite causing human babesiosis. The assays target two immunodominant antigens, BdV234 and BdV38, demonstrating high specificity (no cross-reactivity with other Babesia species or Plasmodium falciparum ) and sensitivity (detecting as low as 115 infected erythrocytes/µl). The assays were validated using in vitro-cultured parasites and infected mice. Screening diverse blood samples showed no evidence of B. duncani active infection among 1,731 human and field mice blood samples collected from the north-eastern, midwestern, and western US. These assays offer potential in diverse diagnostic scenarios, including early patient detection, reservoir animal screening, and transfusion-transmitted babesiosis prevention.

Effectiveness of Two New Endochin-like Quinolones, ELQ-596 and ELQ-650, in Experimental Mouse Models of Human Babesiosis.

Endochin-like quinolones (ELQs) define a class of small molecule antimicrobials that target the mitochondrial electron transport chain of various human parasites by inhibiting their cytochrome bc1 complexes. The compounds have shown potent activity against a wide range of protozoan parasites, including the intraerythrocytic parasites Plasmodium and Babesia, the agents of human malaria and babesiosis, respectively. First-generation ELQ compounds were previously found to reduce infection by Babesia microti and Babesia duncani in animal models of human babesiosis but achieved a radical cure only in combination with atovaquone and required further optimization to address pharmacological limitations. Here, we report the identification of two second-generation 3-biaryl ELQ compounds, ELQ-596 and ELQ-650, with potent antibabesial activity in vitro and favorable pharmacological properties. In particular, ELQ-598, a prodrug of ELQ-596, demonstrated high efficacy as an orally administered monotherapy at 10 mg/kg. The compound achieved radical cure in both the chronic model of B. microti-induced babesiosis in immunocompromised mice and the lethal infection model induced by B. duncani in immunocompetent mice. Given its high potency, favorable physicochemical properties, and low toxicity profile, ELQ-596 represents a promising drug for the treatment of human babesiosis.

Multiomics analysis reveals B. MO1 as a distinct Babesia species and provides insights into its evolution and virulence.

Babesiosis, caused by protozoan parasites of the genus Babesia , is an emerging tick-borne disease of significance for both human and animal health. Babesia parasites infect erythrocytes of vertebrate hosts where they develop and multiply rapidly to cause the pathological symptoms associated with the disease. The identification of various Babesia species underscores the ongoing risk of new zoonotic pathogens capable of infecting humans, a concern amplified by anthropogenic activities and environmental shifts impacting the distribution and transmission dynamics of parasites, their vectors, and reservoir hosts. One such species, Babesia MO1, previously implicated in severe cases of human babesiosis in the midwestern United States, was initially considered closely related to B. divergens , the predominant agent of human babesiosis in Europe. Yet, uncertainties persist regarding whether these pathogens represent distinct variants of the same species or are entirely separate species. We show that although both B. MO1 and B. divergens share similar genome sizes, comprising three nuclear chromosomes, one linear mitochondrial chromosome, and one circular apicoplast chromosome, major differences exist in terms of genomic sequence divergence, gene functions, transcription profiles, replication rates and susceptibility to antiparasitic drugs. Furthermore, both pathogens have evolved distinct classes of multigene families, crucial for their pathogenicity and adaptation to specific mammalian hosts. Leveraging genomic information for B. MO1, B. divergens , and other members of the Babesiidae family within Apicomplexa provides valuable insights into the evolution, diversity, and virulence of these parasites. This knowledge serves as a critical tool in preemptively addressing the emergence and rapid transmission of more virulent strains.

Babesia BdFE1 esterase is required for the anti-parasitic activity of the ACE inhibitor fosinopril.

Effective and safe therapies for the treatment of diseases caused by intraerythrocytic parasites are impeded by the rapid emergence of drug resistance and the lack of novel drug targets. One such disease is human babesiosis, which is a rapidly emerging tick-borne illness caused by Babesia parasites. In this study, we identified fosinopril, a phosphonate-containing, FDA-approved angiotensin converting enzyme (ACE) inhibitor commonly used as a prodrug for hypertension and heart failure, as a potent inhibitor of Babesia duncani parasite development within human erythrocytes. Cell biological and mass spectrometry analyses revealed that the conversion of fosinopril to its active diacid molecule, fosinoprilat, is essential for its antiparasitic activity. We show that this conversion is mediated by a parasite-encoded esterase, BdFE1, which is highly conserved among apicomplexan parasites. Parasites carrying the L238H mutation in the active site of BdFE1 failed to convert the prodrug to its active moiety and became resistant to the drug. Our data set the stage for the development of this class of drugs for the therapy of vector-borne parasitic diseases.

The human malaria parasite Plasmodium falciparum can sense environmental changes and respond by antigenic switching.

The primary antigenic and virulence determinant of the human malaria parasite Plasmodium falciparum is a variant surface protein called PfEMP1. Different forms of PfEMP1 are encoded by a multicopy gene family called var, and switching between active genes enables the parasites to evade the antibody response of their human hosts. var gene switching is key for the maintenance of chronic infections; however, what controls switching is unknown, although it has been suggested to occur at a constant frequency with little or no environmental influence. var gene transcription is controlled epigenetically through the activity of histone methyltransferases (HMTs). Studies in model systems have shown that metabolism and epigenetic control of gene expression are linked through the availability of intracellular S-adenosylmethionine (SAM), the principal methyl donor in biological methylation modifications, which can fluctuate based on nutrient availability. To determine whether environmental conditions and changes in metabolism can influence var gene expression, P. falciparum was cultured in media with altered concentrations of nutrients involved in SAM metabolism. We found that conditions that influence lipid metabolism induce var gene switching, indicating that parasites can respond to changes in their environment by altering var gene expression patterns. Genetic modifications that directly modified expression of the enzymes that control SAM levels similarly led to profound changes in var gene expression, confirming that changes in SAM availability modulate var gene switching. These observations directly challenge the paradigm that antigenic variation in P. falciparum follows an intrinsic, programed switching rate, which operates independently of any external stimuli.

Maturation of the malarial phosphatidylserine decarboxylase is mediated by high affinity binding to anionic phospholipids.

Decarboxylation of phosphatidylserine (PS) to form phosphatidylethanolamine by PS decarboxylases (PSDs) is an essential process in most eukaryotes. Processing of a malarial PSD proenzyme into its active alpha and beta subunits is by an autoendoproteolytic mechanism regulated by anionic phospholipids, with PS serving as an activator and phosphatidylglycerol (PG), phosphatidylinositol, and phosphatidic acid acting as inhibitors. The biophysical mechanism underlying this regulation remains unknown. We used solid phase lipid binding, liposome-binding assays, and surface plasmon resonance to examine the binding specificity of a processing-deficient Plasmodium PSD (PkPSDS308A) mutant enzyme and demonstrated that the PSD proenzyme binds strongly to PS and PG but not to phosphatidylethanolamine and phosphatidylcholine. The equilibrium dissociation constants (Kd) of PkPSD with PS and PG were 80.4 nM and 66.4 nM, respectively. The interaction of PSD with PS is inhibited by calcium, suggesting that the binding mechanism involves ionic interactions. In vitro processing of WT PkPSD proenzyme was also inhibited by calcium, consistent with the conclusion that PS binding to PkPSD through ionic interactions is required for the proenzyme processing. Peptide mapping identified polybasic amino acid motifs in the proenzyme responsible for binding to PS. Altogether, the data demonstrate that malarial PSD maturation is regulated through a strong physical association between PkPSD proenzyme and anionic lipids. Inhibition of the specific interaction between the proenzyme and the lipids can provide a novel mechanism to disrupt PSD enzyme activity, which has been suggested as a target for antimicrobials, and anticancer therapies.

FT-GPI, a highly sensitive and accurate predictor of GPI-anchored proteins, reveals the composition and evolution of the GPI proteome in Plasmodium species.

BACKGROUND: Protozoan parasites are known to attach specific and diverse group of proteins to their plasma membrane via a GPI anchor. In malaria parasites, GPI-anchored proteins (GPI-APs) have been shown to play an important role in host-pathogen interactions and a key function in host cell invasion and immune evasion. Because of their immunogenic properties, some of these proteins have been considered as malaria vaccine candidates. However, identification of all possible GPI-APs encoded by these parasites remains challenging due to their sequence diversity and limitations of the tools used for their characterization. METHODS: The FT-GPI software was developed to detect GPI-APs based on the presence of a hydrophobic helix at both ends of the premature peptide. FT-GPI was implemented in C ++and applied to study the GPI-proteome of 46 isolates of the order Haemosporida. Using the GPI proteome of Plasmodium falciparum strain 3D7 and Plasmodium vivax strain Sal-1, a heuristic method was defined to select the most sensitive and specific FT-GPI software parameters. RESULTS: FT-GPI enabled revision of the GPI-proteome of P. falciparum and P. vivax, including the identification of novel GPI-APs. Orthology- and synteny-based analyses showed that 19 of the 37 GPI-APs found in the order Haemosporida are conserved among Plasmodium species. Our analyses suggest that gene duplication and deletion events may have contributed significantly to the evolution of the GPI proteome, and its composition correlates with speciation. CONCLUSION: FT-GPI-based prediction is a useful tool for mining GPI-APs and gaining further insights into their evolution and sequence diversity. This resource may also help identify new protein candidates for the development of vaccines for malaria and other parasitic diseases.

Plasmodium infection is associated with cross-reactive antibodies to carbohydrate epitopes on the SARS-CoV-2 Spike protein.

Sero-surveillance can monitor and project disease burden and risk. However, SARS-CoV-2 antibody test results can produce false positive results, limiting their efficacy as a sero-surveillance tool. False positive SARS-CoV-2 antibody results are associated with malaria exposure, and understanding this association is essential to interpret sero-surveillance results from malaria-endemic countries. Here, pre-pandemic samples from eight malaria endemic and non-endemic countries and four continents were tested by ELISA to measure SARS-CoV-2 Spike S1 subunit reactivity. Individuals with acute malaria infection generated substantial SARS-CoV-2 reactivity. Cross-reactivity was not associated with reactivity to other human coronaviruses or other SARS-CoV-2 proteins, as measured by peptide and protein arrays. ELISAs with deglycosylated and desialated Spike S1 subunits revealed that cross-reactive antibodies target sialic acid on N-linked glycans of the Spike protein. The functional activity of cross-reactive antibodies measured by neutralization assays showed that cross-reactive antibodies did not neutralize SARS-CoV-2 in vitro. Since routine use of glycosylated or sialated assays could result in false positive SARS-CoV-2 antibody results in malaria endemic regions, which could overestimate exposure and population-level immunity, we explored methods to increase specificity by reducing cross-reactivity. Overestimating population-level exposure to SARS-CoV-2 could lead to underestimates of risk of continued COVID-19 transmission in sub-Saharan Africa.

High-resolution crystal structure and chemical screening reveal pantothenate kinase as a new target for antifungal development.

Fungal infections are the leading cause of mortality by eukaryotic pathogens, with an estimated 150 million severe life-threatening cases and 1.7 million deaths reported annually. The rapid emergence of multidrug-resistant fungal isolates highlights the urgent need for new drugs with new mechanisms of action. In fungi, pantothenate phosphorylation, catalyzed by PanK enzyme, is the first step in the utilization of pantothenic acid and coenzyme A biosynthesis. In all fungi sequenced so far, this enzyme is encoded by a single PanK gene. Here, we report the crystal structure of a fungal PanK alone as well as with high-affinity inhibitors from a single chemotype identified through a high-throughput chemical screen. Structural, biochemical, and functional analyses revealed mechanisms governing substrate and ligand binding, dimerization, and catalysis and helped identify new compounds that inhibit the growth of several Candida species. The data validate PanK as a promising target for antifungal drug development.

Specific and Sensitive Diagnosis of Babesia microti Active Infection Using Monoclonal Antibodies to the Immunodominant Antigen BmGPI12.

The apicomplexan pathogen Babesia microti is responsible for most cases of human babesiosis worldwide. The disease, which presents as a malaria-like illness, is potentially fatal in immunocompromised or elderly patients, making the need for its accurate and early diagnosis an urgent public health concern. B. microti is transmitted primarily by Ixodes ticks but can also be transmitted via blood transfusion. The parasite completes its asexual reproduction in the host red blood cell, where each invading merozoite develops and multiplies to produce four daughter parasites. While various techniques, such as microscopy, PCR, and indirect fluorescence, have been used over the years for babesiosis diagnosis, detection of the secreted B. microti immunodominant antigen BmGPI12 using specific polyclonal antibodies was found to be the most effective method for the diagnosis of active infection and for evaluation of clearance following drug treatment. Here, we report the development of a panel of 16 monoclonal antibodies against BmGPI12. These antibodies detected secreted BmGPI12 in the plasma of infected humans. Antigen capture assays identified a combination of two monoclonal antibodies, 4C8 and 1E11, as a basis for a monoclonal antibody-based BmGPI12 capture assay (mGPAC) to detect active B. microti infection. Using a collection of 105 previously characterized human plasma samples, the mGPAC assay showed 97.1% correlation with RNA-based PCR (transcription-mediated amplification [TMA]) for positive and negative samples. The mGPAC assay also detected BmGPI12 in the plasma of six babesiosis patients at the time of diagnosis but not in three matched posttreatment samples. The mGPAC assay could thus be used alone or in combination with other assays for accurate detection of active B. microti infection.

Babesia duncani as a Model Organism to Study the Development, Virulence, and Drug Susceptibility of Intraerythrocytic Parasites In Vitro and In Vivo.

Human babesiosis is a malaria-like illness caused by tick-borne intraerythrocytic Babesia parasites of the Apicomplexa phylum. Whereas several species of Babesia can cause severe disease in humans, the ability to propagate Babesia duncani both in vitro in human erythrocytes and in mice makes it a unique pathogen to study Babesia biology and pathogenesis. Here we report an optimized B. duncani in culture-in mouse (ICIM) model that combines continuous in vitro culture of the parasite with a precise model of lethal infection in mice. We demonstrate that B. duncani-infected erythrocytes as well as free merozoites can cause lethal infection in C3H/HeJ mice. Highly reproducible parasitemia and survival outcomes could be established using specific parasite loads in different mouse genetic backgrounds. Using the ICIM model, we discovered 2 new endochin-like quinolone prodrugs (ELQ-331 and ELQ-468) that alone or in combination with atovaquone are highly efficacious against B. duncani and Babesia microti.

Redesigning therapies for pantothenate kinase-associated neurodegeneration.

Pantothenate kinase-associated neurodegeneration (PKAN) is an incurable rare genetic disorder of children and young adults caused by mutations in the PANK2 gene, which encodes an enzyme critical for the biosynthesis of coenzyme A. Although PKAN affects only a small number of patients, it shares several hallmarks of more common neurodegenerative diseases of older adults such as Alzheimer's disease and Parkinson's disease. Advances in etiological understanding and treatment of PKAN could therefore have implications for our understanding of more common diseases and may shed new lights on the physiological importance of coenzyme A, a cofactor critical for the operation of various cellular metabolic processes. The large body of knowledge that accumulated over the years around PKAN pathology, including but not limited to studies of various PKAN models and therapies, has contributed not only to progress in our understanding of the disease but also, importantly, to the crystallization of key questions that guide future investigations of the disease. In this review, we will summarize this knowledge and demonstrate how it forms the backdrop to new avenues of research.

Evidence for a Conserved Function of Eukaryotic Pantothenate Kinases in the Regulation of Mitochondrial Homeostasis and Oxidative Stress.

Human PANK1, PANK2, and PANK3 genes encode several pantothenate kinase isoforms that catalyze the phosphorylation of vitamin B5 (pantothenic acid) to phosphopantothenate, a critical step in the biosynthesis of the major cellular cofactor, Coenzyme A (CoA). Mutations in the PANK2 gene, which encodes the mitochondrial pantothenate kinase (PanK) isoform, have been linked to pantothenate-kinase associated neurodegeneration (PKAN), a debilitating and often fatal progressive neurodegeneration of children and young adults. While the biochemical properties of these enzymes have been well-characterized in vitro, their expression in a model organism such as yeast in order to probe their function under cellular conditions have never been achieved. Here we used three yeast mutants carrying missense mutations in the yeast PanK gene, CAB1, which are associated with defective growth at high temperature and iron, mitochondrial dysfunction, increased iron content, and oxidative stress, to assess the cellular function of human PANK genes and functional conservation of the CoA-controlled processes between humans and yeast. Overexpression of human PANK1 and PANK3 in these mutants restored normal cellular activity whereas complementation with PANK2 was partial and could only be achieved with an isoform, PanK2mtmΔ, lacking the mitochondrial transit peptide. These data, which demonstrate functional conservation of PanK activity between humans and yeast, set the stage for the use of yeast as a model system to investigate the impact of PKAN-associated mutations on the metabolic pathways altered in this disease.

Cytochrome b Drug Resistance Mutation Decreases Babesia Fitness in the Tick Stages But Not the Mammalian Erythrocytic Cycle.

Human babesiosis is an emerging tick-borne malaria-like illness caused by Babesia parasites following their development in erythrocytes. Here, we show that a mutation in the Babesia microti mitochondrial cytochrome b (Cytb) that confers resistance to the antibabesial drug ELQ-502 decreases parasite fitness in the arthropod vector. Interestingly, whereas the mutant allele does not affect B. microti fitness during the mammalian blood phase of the parasite life cycle and is genetically stable as parasite burden increases, ELQ-502-resistant mutant parasites developing in the tick vector are genetically unstable with a high rate of the wild-type allele emerging during the nymphal stage. Furthermore, we show that B. microti parasites with this mutation are transmitted from the tick to the host, raising the possibility that the frequency of Cytb resistance mutations may be decreased by passage through the tick vector, but could persist in the environment if present when ticks feed.

Babesia microti Immunoreactive Rhoptry-Associated Protein-1 Paralogs Are Ancestral Members of the Piroplasmid-Confined RAP-1 Family.

Babesia, Cytauxzoon and Theileria are tick-borne apicomplexan parasites of the order Piroplasmida, responsible for diseases in humans and animals. Members of the piroplasmid rhoptry-associated protein-1 (pRAP-1) family have a signature cysteine-rich domain and are important for parasite development. We propose that the closely linked B. microti genes annotated as BMR1_03g00947 and BMR1_03g00960 encode two paralogue pRAP-1-like proteins named BmIPA48 and Bm960. The two genes are tandemly arranged head to tail, highly expressed in blood stage parasites, syntenic to rap-1 genes of other piroplasmids, and share large portions of an almost identical ~225 bp sequence located in their 5' putative regulatory regions. BmIPA48 and Bm960 proteins contain a N-terminal signal peptide, share very low sequence identity (<13%) with pRAP-1 from other species, and harbor one or more transmembrane domains. Diversification of the piroplasmid-confined prap-1 family is characterized by amplification of genes, protein domains, and a high sequence polymorphism. This suggests a functional involvement of pRAP-1 at the parasite-host interface, possibly in parasite adhesion, attachment, and/or evasion of the host immune defenses. Both BmIPA48 and Bm960 are recognized by antibodies in sera from humans infected with B. microti and might be promising candidates for developing novel serodiagnosis and vaccines.

Treatment of Human Babesiosis: Then and Now.

Babesiosis is an emerging tick-borne disease caused by apicomplexan parasites of the genus Babesia. With its increasing incidence worldwide and the risk of human-to-human transmission through blood transfusion, babesiosis is becoming a rising public health concern. The current arsenal for the treatment of human babesiosis is limited and consists of combinations of atovaquone and azithromycin or clindamycin and quinine. These combination therapies were not designed based on biological criteria unique to Babesia parasites, but were rather repurposed based on their well-established efficacy against other apicomplexan parasites. However, these compounds are associated with mild or severe adverse events and a rapid emergence of drug resistance, thus highlighting the need for new therapeutic strategies that are specifically tailored to Babesia parasites. Herein, we review ongoing babesiosis therapeutic and management strategies and their limitations, and further review current efforts to develop new, effective, and safer therapies for the treatment of this disease.