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.

Serological Assays Estimate Highly Variable SARS-CoV-2 Neutralizing Antibody Activity in Recovered COVID-19 Patients.

The development of neutralizing antibodies (NAbs) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) following infection or vaccination is likely to be critical for the development of sufficient population immunity to drive cessation of the coronavirus disease of 2019 (COVID-19) pandemic. A large number of serologic tests, platforms, and methodologies are being employed to determine seroprevalence in populations to select convalescent plasma samples for therapeutic trials and to guide policies about reopening. However, the tests have substantial variations in sensitivity and specificity, and their ability to quantitatively predict levels of NAbs is unknown. We collected 370 unique donors enrolled in the New York Blood Center Convalescent Plasma Program between April and May of 2020. We measured levels of antibodies in convalescent plasma samples using commercially available SARS-CoV-2 detection tests and in-house enzyme-linked immunosorbent assays (ELISAs) and correlated serological measurements with NAb activity measured using pseudotyped virus particles, which offer the most informative assessment of antiviral activity of patient sera against viral infection. Our data show that a large proportion of convalescent plasma samples have modest antibody levels and that commercially available tests have various degrees of accuracy in predicting NAb activity. We found that the Ortho anti-SARS-CoV-2 total Ig and IgG high-throughput serological assays (HTSAs) and the Abbott SARS-CoV-2 IgG assay quantify levels of antibodies that strongly correlate with the results of NAb assays and are consistent with gold standard ELISA results. These findings provide immediate clinical relevance to serology results that can be equated to NAb activity and could serve as a valuable roadmap to guide the choice and interpretation of serological tests for SARS-CoV-2.

Human babesiosis: recent advances and future challenges.

PURPOSE OF REVIEW: As human babesiosis caused by apicomplexan parasites of the Babesia genus is associated with transfusion-transmitted illness and relapsing disease in immunosuppressed populations, it is important to report novel findings relating to parasite biology that may be responsible for such pathology. Blood screening tools recently licensed by the FDA are also described to allow understanding of their impact on keeping the blood supply well tolerated. RECENT FINDINGS: Reports of tick-borne cases within new geographical regions such as the Pacific Northwest of the USA, through Eastern Europe and into China are also on the rise. Novel features of the parasite lifecycle that underlie the basis of parasite persistence have recently been characterized. These merit consideration in deployment of both detection, treatment and mitigation tools such as pathogen inactivation technology. The impact of new blood donor screening tests in reducing transfusion transmitted babesiosis is discussed. SUMMARY: New Babesia species have been identified globally, suggesting that the epidemiology of this disease is rapidly changing, making it clear that human babesiosis is a serious public health concern that requires close monitoring and effective intervention measures. Unlike other erythrocytic parasites, Babesia exploits unconventional lifecycle strategies that permit host cycles of different lengths to ensure survival in hostile environments. With the licensure of new blood screening tests, incidence of transfusion transmission babesiosis has decreased.

Serological Assays Estimate Highly Variable SARS-CoV-2 Neutralizing Antibody Activity in Recovered COVID19 Patients.

The development of neutralizing antibodies (nAb) against SARS-CoV-2, following infection or vaccination, is likely to be critical for the development of sufficient population immunity to drive cessation of the COVID19 pandemic. A large number of serologic tests, platforms and methodologies are being employed to determine seroprevalence in populations to select convalescent plasmas for therapeutic trials, and to guide policies about reopening. However, tests have substantial variability in sensitivity and specificity, and their ability to quantitatively predict levels of nAb is unknown. We collected 370 unique donors enrolled in the New York Blood Center Convalescent Plasma Program between April and May of 2020. We measured levels of antibodies in convalescent plasma using commercially available SARS-CoV- 2 detection tests and in-house ELISA assays and correlated serological measurements with nAb activity measured using pseudotyped virus particles, which offer the most informative assessment of antiviral activity of patient sera against viral infection. Our data show that a large proportion of convalescent plasma samples have modest antibody levels and that commercially available tests have varying degrees of accuracy in predicting nAb activity. We found the Ortho Anti-SARS-CoV-2 Total Ig and IgG high throughput serological assays (HTSAs), as well as the Abbott SARS-CoV-2 IgG assay, quantify levels of antibodies that strongly correlate with nAb assays and are consistent with gold-standard ELISA assay results. These findings provide immediate clinical relevance to serology results that can be equated to nAb activity and could serve as a valuable 'roadmap' to guide the choice and interpretation of serological tests for SARS-CoV-2.

Babesia divergens: A Drive to Survive.

Babesia divergens is an obligate intracellular protozoan parasite that causes zoonotic disease. Central to its pathogenesis is the ability of the parasite to invade host red blood cells of diverse species, and, once in the host blood stream, to manipulate the composition of its population to allow it to endure unfavorable conditions. Here we will review key in vitro studies relating to the survival strategies that B. divergens adopts during its intraerythrocytic development to persist and how proliferation is restored in the parasite population once optimum conditions return.

A library of recombinant Babesia microti cell surface and secreted proteins for diagnostics discovery and reverse vaccinology.

Human babesiosis is an emerging tick-borne parasitic disease and blood transfusion-transmitted infection primarily caused by the apicomplexan parasite, Babesia microti. There is no licensed vaccine for B. microti and the development of a reliable serological screening test would contribute to ensuring the safety of the donated blood supply. The recent sequencing of the B. microti genome has revealed many novel genes encoding proteins that can now be tested for their suitability as subunit vaccine candidates and diagnostic serological markers. Extracellular proteins are considered excellent vaccine candidates and serological markers because they are directly exposed to the host humoral immune system, but can be challenging to express as soluble recombinant proteins. We have recently developed an approach based on a mammalian expression system that can produce large panels of functional recombinant cell surface and secreted parasite proteins. Here, we use the B. microti genome sequence to identify 54 genes that are predicted to encode surface-displayed and secreted proteins expressed during the blood stages, and show that 41 (76%) are expressed using our method at detectable levels. We demonstrate that the proteins contain conformational, heat-labile, epitopes and use them to serologically profile the kinetics of the humoral immune responses to two strains of B. microti in a murine infection model. Using sera from validated human infections, we show a concordance in the host antibody responses to B. microti infections in mouse and human hosts. Finally, we show that BmSA1 expressed in mammalian cells can elicit high antibody titres in vaccinated mice using a human-compatible adjuvant but these antibodies did not affect the pathology of infection in vivo. Our library of recombinant B. microti cell surface and secreted antigens constitutes a valuable resource that could contribute to the development of a serological diagnostic test, vaccines, and elucidate the molecular basis of host-parasite interactions.

Robust adaptive immune response against Babesia microti infection marked by low parasitemia in a murine model of sickle cell disease.

The intraerythrocytic parasite Babesia microti is the number 1 cause of transfusion-transmitted infection and can induce serious, often life-threatening complications in immunocompromised individuals including transfusion-dependent patients with sickle cell disease (SCD). Despite the existence of strong long-lasting immunological protection against a second infection in mouse models, little is known about the cell types or the kinetics of protective adaptive immunity mounted following Babesia infection, especially in infection-prone SCD that are thought to have an impaired immune system. Here, we show, using a mouse B microti infection model, that infected wild-type (WT) mice mount a very strong adaptive immune response, characterized by (1) coordinated induction of a robust germinal center (GC) reaction; (2) development of follicular helper T (TFH) cells that comprise ∼30% of splenic CD4+ T cells at peak expansion by 10 days postinfection; and (3) high levels of effector T-cell cytokines, including interleukin 21 and interferon γ, with an increase in the secretion of antigen (Ag)-specific antibodies (Abs). Strikingly, the Townes SCD mouse model had significantly lower levels of parasitemia. Despite a highly disorganized splenic architecture before infection, these mice elicited a surprisingly robust adaptive immune response (including comparable levels of GC B cells, TFH cells, and effector cytokines as control and sickle trait mice), but higher immunoglobulin G responses against 2 Babesia-specific proteins, which may contain potential immunogenic epitopes. Together, these studies establish the robust emergence of adaptive immunity to Babesia even in immunologically compromised SCD mice. Identification of potentially immunogenic epitopes has implications to identify long-term carriers, and aid Ag-specific vaccine development.

Identification and Characterization of the Rhoptry Neck Protein 2 in Babesia divergens and B. microti.

Apicomplexan parasites include those of the genera Plasmodium, Cryptosporidium, and Toxoplasma and those of the relatively understudied zoonotic genus Babesia In humans, babesiosis, particularly transfusion-transmitted babesiosis, has been emerging as a major threat to public health. Like malaria, the disease pathology is a consequence of the parasitemia which develops through cyclical replication of Babesia parasites in host erythrocytes. However, there are no exoerythrocytic stages in Babesia, so targeting of the blood stage and associated proteins to directly prevent parasite invasion is the most desirable option for effective disease control. Especially promising among such molecules are the rhoptry neck proteins (RONs), whose homologs have been identified in many apicomplexan parasites. RONs are involved in the formation of the moving junction, along with AMA1, but no RON has been identified and characterized in any Babesia spp. Here we identify the RON2 proteins of Babesia divergens (BdRON2) and B. microti (BmRON2) and show that they are localized apically and that anti-BdRON2 antibodies are significant inhibitors of parasite invasion in vitro Neither protein is immunodominant, as both proteins react only marginally with sera from infected animals. Further characterization of the direct role of both BdRON2 and BmRON2 in parasite invasion is required, but knowledge of the level of conformity of RON2 proteins within the apicomplexan phylum, particularly that of the AMA1-RON2 complex at the moving junction, along with the availability of an animal model for B. microti studies, provides a key to target this complex with a goal of preventing the erythrocytic invasion of these parasites and to further our understanding of the role of these conserved ligands in invasion.

Babesia divergens builds a complex population structure composed of specific ratios of infected cells to ensure a prompt response to changing environmental conditions.

Babesia parasites cause a malaria-like febrile illness by infection of red blood cells (RBCs). Despite the growing importance of this tick-borne infection, its basic biology has been neglected. Using novel synchronization tools, the sequence of intra-erythrocytic events was followed from invasion through development and differentiation to egress. The dynamics of the parasite population were studied in culture, revealing for the first time, the complete array of morphological forms in a precursor-product relationship. Important chronological constants including Babesia's highly unusual variable intra-erythrocytic life cycle, the life span of each population of infected cells and the time required for the genesis of the different parasite stages were elucidated. Importantly, the maintenance of specific ratios of the infected RBC populations was shown to be responsible for the parasites' choice of developmental pathways, enabling swift responses to changing environmental conditions like availability of RBCs and nutrition. These results could impact the control of parasite proliferation and therefore disease.

Malaria invasion ligand RH5 and its prime candidacy in blood-stage malaria vaccine design.

With drug resistance to available therapeutics continuing to develop against Plasmodium falciparum malaria, the development of an effective vaccine candidate remains a major research goal. Successful interruption of invasion of parasites into erythrocytes during the blood stage of infection will prevent the severe clinical symptoms and complications associated with malaria. Previously studied blood stage antigens have highlighted the hurdles that are inherent to this life-cycle stage, namely that highly immunogenic antigens are also globally diverse, resulting in protection only against the vaccine strain, or that naturally acquired immunity to blood stage antigens do not always correlate with actual protection. The blood stage antigen reticulocyte binding homolog RH5 is essential for parasite viability, has globally limited diversity, and is associated with protection from disease. Here we summarize available information on this invasion ligand and recent findings that highlight its candidacy for inclusion in a blood-stage malaria vaccine.

Identification and characterization of the RouenBd1987 Babesia divergens Rhopty-Associated Protein 1.

Human babesiosis is caused by one of several babesial species transmitted by ixodid ticks that have distinct geographical distributions based on the presence of competent animal hosts. The pathology of babesiosis, like malaria, is a consequence of the parasitaemia which develops through the cyclical replication of Babesia parasites in a patient's red blood cells, though symptoms typically are nonspecific. We have identified the gene encoding Rhoptry-Associated Protein -1 (RAP-1) from a human isolate of B. divergens, Rouen1987 and characterized its protein product at the molecular and cellular level. Consistent with other Babesia RAP-1 homologues, BdRAP-1 is expressed as a 46 kDa protein in the parasite rhoptries, suggesting a possible role in red cell invasion. Native BdRAP-1 binds to an unidentified red cell receptor(s) that appears to be non-sialylated and non-proteinacious in nature, but we do not find significant reduction in growth with anti-rRAP1 antibodies in vitro, highlighting the possibility the B. divergens is able to use alternative pathways for invasion, or there is an alternative, complementary, role for BdRAP-1 during the invasion process. As it is the parasite's ability to recognize and then invade host cells which is central to clinical disease, characterising and understanding the role of Babesia-derived proteins involved in these steps are of great interest for the development of an effective prophylaxis.

A malaria vaccine candidate based on an epitope of the Plasmodium falciparum RH5 protein.

BACKGROUND: The Plasmodium falciparum protein RH5 is an adhesin molecule essential for parasite invasion of erythrocytes. Recent studies show that anti-PfRH5 sera have potent invasion-inhibiting activities, supporting the idea that the PfRH5 antigen could form the basis of a vaccine. Therefore, epitopes recognized by neutralizing anti-PfRH5 antibodies could themselves be effective vaccine immunogens if presented in a sufficiently immunogenic fashion. However, the exact regions within PfRH5 that are targets of this invasion-inhibitory activity have yet to be identified. METHODS: A battery of anti-RH5 monoclonal antibodies (mAbs) were produced and screened for their potency by inhibition of invasion assays in vitro. Using an anti-RH5 mAb that completely inhibited invasion as the selecting mAb, affinity-selection using random sequence peptide libraries displayed on virus-like particles of bacteriophage MS2 (MS2 VLPs) was performed. VLPs were sequenced to identify the specific peptide epitopes they encoded and used to raise specific antisera that was in turn tested for inhibition of invasion. RESULTS: Three anti-RH5 monoclonals (0.1 mg/mL) were able to inhibit invasion in vitro by >95%. Affinity-selection with one of these mAbs yielded a VLP which yielded a peptide whose sequence is identical to a portion of PfRH5 itself. The VLP displaying the peptide binds strongly to the antibody, and in immunized animals elicits an anti-PfRH5 antibody response. The resulting antisera against the specific VLP inhibit parasite invasion of erythrocytes more than 90% in vitro. CONCLUSIONS: Here, data is presented from an anti-PfRH5 mAb that completely inhibits erythrocyte invasion by parasites in vitro, one of the few anti-malarial monoclonal antibodies reported to date that completely inhibits invasion with such potency, adding to other studies that highlight the potential of PfRH5 as a vaccine antigen. The specific neutralization sensitive epitope within RH5 has been identified, and antibodies against this epitope also elicit high anti-invasion activity, suggesting this epitope could form the basis of an effective vaccine against malaria.

Identification of binding domains on red blood cell glycophorins for Babesia divergens.

BACKGROUND: Invasion of red blood cells (RBCs) is one of the critical points in the lifecycle of Babesia. The parasite does not invade other host cells. Earlier work has shown that GPA and GPB function as putative receptors during parasite invasion. The primary focus of this study was the delineation of parasite-binding domains on GPA and GPB. STUDY DESIGN AND METHODS: The assay of choice to validate molecules that participate in invasion is an inhibition of invasion assay, in which changes in parasitemia are assessed relative to a wild-type assay (no inhibitors). Inhibition of invasion can be achieved by modification of different components of the assay or by the addition of competitors of the molecules that participate in invasion. In this study purified antibody fragments to various domains on GPA and GPB were tested for magnitude of inhibition of parasite invasion. Effects on invasion were monitored by assessment of Giemsa-stained smears every 24 hours. RESULTS: Among 10 selected antibodies directed at various epitopes on GPA and GPB, antibodies directed against GPA(M) epitopes had the most severe effect (up to 35%) on inhibition of invasion, followed by antibodies directed against GPB(S) epitope (up to 24%). CONCLUSION: This study confirms the role of RBC glycophorins A and B in Babesia divergens invasion and shows that the GPA(M) and GPB(S) epitopes are likely to play an important role in the entry process.

Babesia and red cell invasion.

PURPOSE OF REVIEW: Babesiosis is a zoonosis, a disease communicable from animals to humans and an important blood-borne human parasitic infection. Despite its public health impact, its study has largely been neglected. The objective of this review is to present up-to-date information on both parasite and red blood cell molecules that function at the host-parasite interface to facilitate successful invasion. RECENT FINDINGS: In the last few years, a number of parasite proteins have been identified from genome projects and from functional red cell-binding assays. However, their cognate receptors as well as the precise function these ligands perform in the cascade of invasive events remain umknown. There also appears to be a significant overlap in the structural and functional aspects of the invasion machinery between malaria and Babesia. SUMMARY: Recognizing that Babesia is an expanding blood safety threat, there should be rapid progress in the development of viable interventions to detect and halt transmission of these pathogens via blood transfusions. By developing a detailed mechanistic understanding of invasion, we can then exploit the participating molecules to procure much needed reagents for diagnosis, epidemiology, treatment and prevention of human babesiosis.

Targeting sialic acid dependent and independent pathways of invasion in Plasmodium falciparum.

The pathology of malaria is a consequence of the parasitaemia which develops through the cyclical asexual replication of parasites in a patient's red blood cells. Multiple parasite ligand-erythrocyte receptor interactions must occur for successful Plasmodium invasion of the human red cell. Two major malaria ligand families have been implicated in these variable ligand-receptor interactions used by Plasmodium falciparum to invade human red cells: the micronemal proteins from the Erythrocyte Binding Ligands (EBL) family and the rhoptry proteins from the Reticulocyte binding Homolog (PfRH) family. Ligands from the EBL family largely govern the sialic acid (SA) dependent pathways of invasion and the RH family ligands (except for RH1) mediate SA independent invasion. In an attempt to dissect out the invasion inhibitory effects of antibodies against ligands from both pathways, we have used EBA-175 and RH5 as model members of each pathway. Mice were immunized with either region II of EBA-175 produced in Pichia pastoris or full-length RH5 produced by the wheat germ cell-free system, or a combination of the two antigens to look for synergistic inhibitory effects of the induced antibodies. Sera obtained from these immunizations were tested for native antigen recognition and for efficacy in invasion inhibition assays. Results obtained show promise for the potential use of such hybrid vaccines to induce antibodies that can block multiple parasite ligand-red cell receptor interactions and thus inhibit parasite invasion.

Babesia divergens apical membrane antigen 1 and its interaction with the human red blood cell.

Multiple parasite ligand-erythrocyte receptor interactions must occur for successful Babesia and Plasmodium invasion of the human red cell. One such parasite ligand is the apical membrane antigen 1 (AMA1) which is a conserved apicomplexan protein present in the micronemes and then secreted onto the surface of the merozoite. Much evidence exists for a vital role for AMA1 in host cell invasion; however, its interaction with the host erythrocyte has remained controversial. In this paper, we present a detailed characterization of a Babesia divergens homolog of AMA1 (BdAMA1), and taking advantage of the relatively high amounts of native BdAMA1 available from the parasite culture system, show that proteolytic products of native BdAMA1 bind to a trypsin- and chymotrypsin-sensitive receptor on the red blood cell. Moreover, immuno-electron microscopic images of the B. divergens merozoite captured during invasion offer additional evidence of the presence of BdAMA1 on the red cell membrane. Given the importance of AMA1 in invasion and the central role invasion plays in pathogenesis, these studies have implications both for novel drug design and for the development of new vaccine approaches aimed at interfering with AMA1 function.

Mutation in Caenorhabditis elegans Krüppel-like factor, KLF-3 results in fat accumulation and alters fatty acid composition.

In vertebrates, adipose tissue stores energy in the form of fat. Fat storage is tightly controlled by and dynamically balanced with energy expenditure under physiological settings; the perturbation of fat in either excess (obese) or deficit (lipodystrophy) has devastating pathologic consequences in the fueling of homeostasis and organismal fitness. The process by which fat storage is coordinated through positive and negative feedback signals is still poorly understood. To address potential mechanisms underlying fat storage we study a Caenorhabditis elegans Krüppel-like transcription factor, Ce-klf-3 and demonstrate that klf-3 is a hitherto unrecognized key regulator of fat metabolism in C. elegans. The Ce-klf-3 is highly expressed during larval development and predominantly present in intestine: the site of fat digestion, absorption, storage, and utilization. We found a strong positive correlation between klf-3 expression and fat deposition in a worm's intestine. Significantly, a klf-3 (ok1975) loss-of-function mutation, characterized by the deletion of a 1658-bp sequence spanning the 3' end of exon 2 through to the 5' end of exon 3 of klf-3, enhanced fat deposition in the intestine and caused severe defects in worm reproduction. Although klf-3 mutants seemed very similar to wild type worms in appearance and life span, 70% of mutants became semi-sterile, each producing 40-50 viable progenies, and the remaining 30% were rendered completely sterile toward adulthood. Notably, both mutant types displayed extensive deposition of fat in the intestine. Our study also demonstrates that klf-3 is critical for maintaining normal fatty acid composition by regulating genes involved in a fatty acid desaturation pathway. Strikingly, klf-3 mutant animals with impaired fatty acid beta-oxidation pathway genes resulted in fat accumulation in the mutant worm. We present the first clear in vivo evidence supporting essential regulatory roles of KLF-3 in fat storage in C. elegans and shed light on the human equivalent in disease-gene association.