Sequencing of the smallest Apicomplexan genome from the human pathogen Babesia microti

We have sequenced the genome of the emerging human pathogen Babesia microti and compared it with that of other protozoa. B. microti has the smallest nuclear genome among all Apicomplexan parasites sequenced to date with three chromosomes encoding ∼3500 polypeptides, several of which are species specific. Genome-wide phylogenetic analyses indicate that B. microti is significantly distant from all species of Babesidae and Theileridae and defines a new clade in the phylum Apicomplexa. Furthermore, unlike all other Apicomplexa, its mitochondrial genome is circular. Genome-scale reconstruction of functional networks revealed that B. microti has the minimal metabolic requirement for intraerythrocytic protozoan parasitism. B. microti multigene families differ from those of other protozoa in both the copy number and organization. Two lateral transfer events with significant metabolic implications occurred during the evolution of this parasite. The genomic sequencing of B. microti identified several targets suitable for the development of diagnostic assays and novel therapies for human babesiosis.     

Induction of IL-10-Producing CD1dhighCD5+ Regulatory B Cells following Babesia microti-Infection

Background

Understanding the induction of immune regulatory cells upon helminth infection is important for understanding the control of autoimmunity and allergic inflammation in helminth infection. Babesia microti, an intraerythrocytic protozoan of the genus Babesia, is a major cause of the emerging human disease babesiosis, an asymptomatic malaria-like disease. We examined the influence of acute B. microti infection on the development of regulatory B cells together with regulatory T cells.

Principal Findings

Our data demonstrate that B cells stimulated in vitro with B. microti produce interleukin (IL)-10. This cytokine is also secreted by B cells isolated from B. microti-infected mice in response to lipopolysaccharide stimulation. In addition, high levels of IL-10 were detected in the serum of mice after infection with B. microti. The frequency of IL-10-producing CD1d^highCD5⁺ regulatory B cells (Bregs) and CD4⁺CD25⁺FoxP3⁺ T cells increased during the course of B. microti infection. Furthermore, adoptive transfer of IL-10-producing B cells induced by B. microti infection led to increased susceptibility of recipient mice to infection with B. microti. In contrast, experiments with B cell-deficient (µMT) mice demonstrated that lack of B cells enhances susceptibility to B. microti infection.

Conclusions

This study is the first demonstration of the expansion of Bregs following infection by an intraerythrocytic protozoan parasite. These data suggest that B. microti infection in mice provides an excellent model for studying Breg-mediated immune responses and begins to elucidate the mechanism by which helminth infection regulates autoimmunity and allergic inflammation.     

Investigating disease severity in an animal model of concurrent babesiosis and Lyme disease

The incidence of babesiosis, Lyme disease and other tick-borne diseases has increased steadily in Europe and North America during the last five decades. Babesia microti is transmitted by species of Ixodes, the same ticks that transmit the Lyme disease-causing spirochete, Borrelia burgdorferi. B. microti can also be transmitted through transfusion of blood products and is the most common transfusion-transmitted infection in the U.S.A. Ixodes ticks are commonly infected with both B. microti and B. burgdorferi, and are competent vectors for transmitting them together into hosts. Few studies have examined the effects of coinfections on humans and they had somewhat contradictory results. One study linked coinfection with B. microti to a greater number of symptoms of overall disease in patients, while another report indicated that B. burgdorferi infection either did not affect babesiosis symptoms or decreased its severity. Mouse models of infection that manifest pathological effects similar to those observed in human babesiosis and Lyme disease offer a unique opportunity to thoroughly investigate the effects of coinfection on the host. Lyme disease has been studied using the susceptible C3H mouse infection model, which can also be used to examine B. microti infection to understand pathological mechanisms of human diseases, both during a single infection and during coinfections. We observed that high B. microti parasitaemia leads to low haemoglobin levels in infected mice, reflecting the anaemia observed in human babesiosis. Similar to humans, B. microti coinfection appears to enhance the severity of Lyme disease-like symptoms in mice. Coinfected mice have lower peak B. microti parasitaemia compared to mice infected with B. microti alone, which may reflect attenuation of babesiosis symptoms reported in some human coinfections. These findings suggest that B. burgdorferi coinfection attenuates parasite growth while B. microti presence exacerbates Lyme disease-like symptoms in mice.     

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.     

Quantitative proteomics and phosphoproteomic analyses of mouse livers after tick-borne Babesia microti infection

Babesia microti is a tick-borne protozoan parasite that infects the red blood cells of mice, humans, and other mammals. The liver tissues of BALB/c mice infected with B. microti exhibit severe injury. To further investigate the molecular mechanisms underlying liver injury and liver self-repair after B. microti infection, data-independent acquisition (DIA) quantitative proteomics was used to analyse changes in the expression and phosphorylation of proteins in liver tissues of BALB/c mice during a B. microti infection period and a recovery period. The expression of FABP1 and ACBP, which are related to fatty acid transport in the liver, was downregulated after infection with B. microti, as was the expression of Acox1, Ehhadh and Acaa1a, which are crucial rate-limiting enzymes in the process of fatty acid β oxidation. The phosphorylation levels of AMP-activated protein kinase (AMPK) and Hormone-sensitive lipase (HSL) were also downregulated. In addition, the expression of PSMB9, CTSC, and other immune-related proteins was increased, reflecting an active immune regulation mechanism in the mice. The weights of mice infected with B. microti were significantly reduced, and the phosphorylation levels of IRS-1, c-Raf, mTOR, and other proteins related to growth and development were downregulated.     

The proliferative response of mouse lymphoid tissues during infections with Babesia microti or Babesia rodhaini

Inchley C. J., Grieve E. M. and Preston P. M. 1987. The proliferative response of mouse lymphoid tissues during infections with Babesia microti or Babesia rodhaini. International Journal for Parasitology17: 945–950. Proliferative responses induced by the lethal protozoan parasite, Babesia rodhaini, or the non-lethal species, B. microti were measured in the lymphoid tissues of infected mice. Both stimulated equally rapid DNA synthesis in the spleen, but spleen enlargement during the first week was more pronounced after infection with B. rodhaini, suggesting an earlier influx of cirulating cells than during B. microti infections. Termination of B. rodhaini infections by chemotherapy revealed that the recruitment of cells, but not the proliferative response, was dependent on the presence of live parasites. The spleen enlargement typical of B. microti-infected mice developed during the second week, but up to half of this response could be attributed to compensatory erythropoiesis. Babesia microti, but not B. rodhaini, induced a proliferative response in peripheral lymph nodes, and transiently depressed cell division in the thymus.     

Vaccination against babesiosis using recombinant GPI-anchored proteins

The increase in human babesiosis is of major concern to health authorities. In the USA, most of these cases are due to infections with Babesia microti, whereas in Europe B. divergens is the major cause of clinical disease in humans. Here we review the immunological and biological literature of glycosylphosphatidylinositol (GPI)-anchored merozoite proteins of human Babesia parasites with emphasis on their role in immunity, and provide some new bioinformatical information on B. microti GPI-Anchored Proteins (GPI-AP). Cattle can be vaccinated with soluble parasite antigens (SPA) of Babesia divergens that are released by the parasite during proliferation. The major component in SPA preparations appeared to be a 37?kDa merozoite surface protein that is anchored in the merozoite membrane by a GPI anchor. Animals could be protected by vaccination with the recombinant 37?kDa protein expressed in Escherichia coli, provided the protein had a hydrophobic terminal sequence. Based on this knowledge, a recombinant vaccine was developed against Babesia canis infection in dogs, successfully. In order to identify similar GPI-AP in B. microti, the genome was analysed. Here it is shown that B. microti encodes all proteins necessary for GPI assembly and its subsequent protein transfer. In addition, in total 21 genes encoding for GPI-AP were detected, some of which reacted particularly strongly with sera from B. microti-infected human patients. Reactivity of antibodies with GPI-anchored merozoite proteins appears to be dependent on the structural conformation of the molecule. It is suggested that the three-dimensional structure of the protein that is anchored in the membrane is different from that of the protein that has been shed from the merozoite surface. The significance of this protein’s dynamics in parasite biology and immune evasion is discussed. Finally, we discuss developments in tick and Babesia vaccine research, and the role such vaccines could play in the control of human babesiosis.     

Babesia hylomysci and Babesia microti: Dexamethasone treatment of infected mice

The effect of dexamethason on Babesia hylomysci and B. microti was investigated in LACA mice. The drug enhanced both infections by depressing the immune mechanisms of the host when treatment was initiated before parasite inoculation, but had no effects on established and subpatent infections. The degree of parasitemia in the treated mice seemed to depend on the tropism of either parasite toward mature erythrocytes or reticulocytes. B. hylomysci, which favors mature erythrocytes, produced fulminating infections in treated mice. B. microti, which prefers reticulocytes, produced similar parasitemia patterns in treated and untreated mice, but only the treated mice succumbed to the infection. The drug, which suppressed cellular proliferation in the spleens of infected animals, together with its direct lympholytic effects, drastically changed the architecture of the organ.     

Nippostrongylus brasiliensis: mast cells and histamine levels in tissues of infected and normal rats.

The role of the spleen during Babesia microti and B. hylomysci infection was investigated by examining the course of infection in both intact and splenectomized mice. The presence of the spleen was critical during the early stages of infection to control excessive multiplication of either parasite, a role taken over by other lymphoid sites as the infection progressed. Mice splenectomized prior to or within 1 week of B. microti inoculation developed extended infections with some deaths, and others were unable to check their parasitemias. All intact mice, and those splenectomized 1 week after infection with B. microti, recovered completely with subsequent development of sterile immunity. Mice splenectomized prior to or within 1 week of B. hylomysci inoculation succumbed to hyperacute infections: Some of the intact mice, and those splenectomized 12 days after infection, recovered but continued to harbor a low-grade infection with periodical recrudescences. Erythrophagocytosis of infected and uninfected erythrocytes was detected in saline preparations and impression smears of spleen and bone marrow and rarely in blood smears of infected mice. This coincided with anemia, splenomegaly, and relatively high levels of opsonizing antibodies, especially during B. microti infection. The colloidal carbon clearance method was used to investigate the phagocytic activity of the reticuloendothelial system. Carbon clearance rates increased rapidly during both infections, but peak B. hylomysci parasitemia coincided with reticuloendothelial phagocytic depression and death of the host. Babesia microti stimulated a consistently higher reticuloendothelial phagocytic activity with higher erythrophagocytosis both in the spleen and bone marrow than did B. hylomysci.     

Borrelia burgdorferi Promotes the Establishment of Babesia microti in the Northeastern United States

Babesia microti and Borrelia burgdorferi, the respective causative agents of human babesiosis and Lyme disease, are maintained in their enzootic cycles by the blacklegged tick (Ixodes scapularis) and use the white-footed mouse (Peromyscus leucopus) as primary reservoir host. The geographic range of both pathogens has expanded in the United States, but the spread of babesiosis has lagged behind that of Lyme disease. Several studies have estimated the basic reproduction number (R ₀) for B. microti to be below the threshold for persistence (<1), a finding that is inconsistent with the persistence and geographic expansion of this pathogen. We tested the hypothesis that host coinfection with B. burgdorferi increases the likelihood of B. microti transmission and establishment in new areas. We fed I. scapularis larva on P. leucopus mice that had been infected in the laboratory with B. microti and/or B. burgdorferi. We observed that coinfection in mice increases the frequency of B. microti infected ticks. To identify the ecological variables that would increase the probability of B. microti establishment in the field, we integrated our laboratory data with field data on tick burden and feeding activity in an R ₀ model. Our model predicts that high prevalence of B. burgdorferi infected mice lowers the ecological threshold for B. microti establishment, especially at sites where larval burden on P. leucopus is lower and where larvae feed simultaneously or soon after nymphs infect mice, when most of the transmission enhancement due to coinfection occurs. Our studies suggest that B. burgdorferi contributes to the emergence and expansion of B. microti and provides a model to predict the ecological factors that are sufficient for emergence of B. microti in the wild.     

Physiological,but not fitness,effects of two interacting haemoparasitic infections in a wild rodent

In contrast to the conditions in most laboratory studies, wild animals are routinely challenged by multiple infections simultaneously, and these infections can interact in complex ways. This means that the impact of a parasite on its host’s physiology and fitness cannot be fully assessed in isolation, and requires consideration of the interactions with other co-infections. Here we examine the impact of two common blood parasites in the field vole (Microtus agrestis): Babesia microti and Bartonella spp., both of which have zoonotic potential. We collected longitudinal and cross-sectional data from four populations of individually tagged wild field voles. This included data on biometrics, life history, ectoparasite counts, presence/absence of microparasites, immune markers and, for a subset of voles, more detailed physiological and immunological measurements. This allowed us to monitor infections over time and to estimate components of survival and fecundity. We confirm, as reported previously, that B. microti has a preventative effect on infection with Bartonella spp., but that the reverse is not true. We observed gross splenomegaly following B. microti infection, and an increase in IL-10 production together with some weight loss following Bartonella spp. infection. However, these animals appeared otherwise healthy and we detected no impact of infection on survival or fecundity due to the two haemoparasite taxa. This is particularly remarkable in the case of B. microti which induces apparently drastic long-term changes to spleen sizes, but without major adverse effects. Our work sheds light on the ecologies of these important zoonotic agents, and more generally on the influence that interactions among multiple parasites have on their hosts in the wild.     

Artesunate,a potential drug for treatment of Babesia infection

The effects of artesunate, a water-soluble artemisinin derivative, against Babesia species, including Babesia bovis, Babesia gibsoni and Babesia microti were studied. Cultures of B. bovis and B. gibsoni were treated with 0.26, 2.6, 26 and 260 μM artesunate, showing inhibition of parasite growth at concentrations equal to and greater than 2.6 μM artesunate by days 3 post-treatment for B. gibsoni and B. bovis in a dose-dependent manner. Consistent with in vitro experiments, artesunate was effective in the treatment of mice infected with B. microti at doses equal to and greater than 10 mg/kg of body weight on days 8–10 post-infection. Taken together, these results suggest that artesunate could be a potential drug against Babesia infection.     

Vertical Transmission of Babesia microti in BALB/c Mice: Preliminary Report

Babesia spp. (Apicomplexa, Piroplasmida) are obligate parasites of many species of mammals, causing a malaria-like infection- babesiosis. Three routes of Babesia infection have been recognized to date. The main route is by a tick bite, the second is via blood transfusion. The third, vertical route of infection is poorly recognized and understood. Our study focused on vertical transmission of B. microti in a well-established mouse model. We assessed the success of this route of infection in BALB/c mice with acute and chronic infections of B. microti. In experimental groups, females were mated on the 1^st day of Babesia infection (Group G0); on the 28^th day post infection (dpi) in the post- acute phase of the parasite infection (G28); and on the 90^th and 150^th dpi (G90 and G150 group, respectively), in the chronic phase of the parasite infection. Pups were obtained from 58% of females mated in the post-acute phase (G28) and from 33% of females in groups G90 and G150. Mice mated in the pre-acute phase of infection (G0) did not deliver pups. Congenital B. microti infections were detected by PCR amplification of Babesia 18S rDNA in almost all pups (96%) from the experimental groups G28, G90 and G150. Parasitaemia in the F1 generation was low and varied between 0.01–0.001%. Vertical transmission of B. microti was demonstrated for the first time in BALB/c mice.     

A Fluorescent Recombinase Aided Amplification Assay for Detection of Babesia microti

Babesia microti is one of the most common causative agents of babesiosis. A sensitive and rapid detection is necessary for screening potentially infected individuals. In this study, B. microti cytochrome c oxidase subunit I (cox1) was selected as the target gene, multiple primers were designed, and optimized by a recombinase-aided amplification (RAA) assay. The optimal primers and probe were labeled with fluorescein. The sensitivity of fluorescent RAA (fRAA) was evaluated using gradient diluents of the cox1 recombinant plasmid and genomic DNA extracted from whole blood of B. microti infected mice. The specificity of fRAA was assessed by other transfusion transmitted parasites. The analytical sensitivity of the fRAA assay was 10 copies of recombinant plasmid per reaction and 10 fg/μl B. microti genomic DNA. No cross-reaction with any other blood-transmitted parasites was observed. Our results demonstrated that the fRAA assay would be rapid, sensitive, and specific for the detection of B. microti.     

Further studies on the uptake of tritiated nucleic acid precursors by Babesia spp. of cattle and mice

Irvin A. D. and Young E. R. 1979. Further studies on the uptake of tritiated nucleic acid precursors by Babesia spp. of cattle and mice. International Journal for Parasitology9: 109–114. An in vitro culture technique developed earlier was used to study the metabolism of nucleic acid precursors by Babesia microti and B. rodhaini of mice and by B. divergens and B. major of cattle. [³H]Hypoxanthine was readily incorporated by all species of parasite, and the presence of leucocytes did not affect this uptake. When parasites were maintained in culture their ability to incorporate [³H]hypoxanthine fell rapidly after 24 h, but when B. major was maintained at 4°C its subsequent ability to incorporate [³H]hypoxanthine persisted for at least 3 days. This finding could be of practical value in assessing infectivity of stored blood in vitro.On autoradiography, [³H]hypoxanthine appeared to be incorporated into both DNA and RNA of parasites. Salvage pathways for purine metabolism appeared to be important in all species of Babesia whereas for pyrimidine metabolism salvage pathways were more important for murine babesias and the de novo pathway more important for bovine species. This difference may relate to different permeabilities of bovine and murine erythrocyte membranes or may be a more fundamental species difference.     

Babesia microti: Molecular and antigenic characterizations of a novel 94-kDa protein (BmP94)

A novel gene, BmP94, encoding 94-kDa protein of Babesia microti was identified by immunoscreening of the cDNA expression library. The full-length of BmP94 was expressed in Escherichia coli (rBmP94), which resulted in insoluble form with low yield, and the truncated hydrophilic C-terminus region of the gene was expressed as a soluble protein (rBmP94/CT) with improved productivity. Antiserum raised against rBmP94/CT recognized the 94-kDa native protein in the parasite extract by Western blot analysis. Next, an ELISA using rBmP94/CT was evaluated for diagnostic use, and it demonstrated high sensitivity and specificity when tested with the sera from mice experimentally infected with B. microti and closely related parasites. Moreover, the immunoprotective property of rBmP94/CT as a subunit vaccine was evaluated in BALB/c mice against a B. microti challenge, but no significant protection was observed. Our data suggest that the immunodominant antigen BmP94 could be a promising candidate for diagnostic use for human babesiosis.     

The role of Ixodes trianguliceps tick larvae in circulation of Babesia microti in the Middle Urals

The nested PCR method with primers flanking a conserved fragment of the Babesia microti ss-rDNA gene was used to examine 834 larvae of Ixodes trianguliceps ticks engorged to a varying degree, taken off 237 hosts of 12 species (rodents and insectivores). The hosts were collected in southern taiga forests in the lowmountain area of the Middle Urals (Chusovoi District, Perm Province) in 2003–2010. Babesia DNA was detected in 89 (10.7%) larvae from 8 species of small mammals. According to the data obtained by PCR and microscopic methods, either B. microti DNA or the parasites themselves were found in the blood of 45.2% of the mammals. The nucleotide sequences of 15 amplicons of Babesia DNA obtained from larvae of I. trianguliceps ticks and their hosts were identical to those of B. microti available in GenBank. In 13 cases, they were similar to B. microti US-type (a human pathogen) and in two cases (those from I. trianguliceps and from the vole Clethrionomys rufocanus from which it was removed), to B. microti of the Munich strain which is not pathogenic to humans. The duration of feeding on small mammals seems to exert the main influence on the infection rate of I. trianguliceps larvae. The fully engorged larvae contained B. microti DNA more often and usually in greater amounts than those collected during the first days of blood-sucking. The latter usually revealed Babesia DNA in the minimum quantity (< 0.064 ng/μl). According to the data obtained, transovarial transmission of Babesia in I. trianguliceps is unlikely. The processes of horizontal and transstadial transmission appear to be of crucial importance for the functioning of the natural foci of babesiosis.     

Non‐canonical metabolic pathways in the malaria parasite detected by isotope‐tracing metabolomics

The malaria parasite, Plasmodium falciparum, proliferates rapidly in human erythrocytes by actively scavenging multiple carbon sources and essential nutrients from its host cell. However, a global overview of the metabolic capacity of intraerythrocytic stages is missing. Using multiplex ¹³C‐labelling coupled with untargeted mass spectrometry and unsupervised isotopologue grouping, we have generated a draft metabolome of P. falciparum and its host erythrocyte consisting of 911 and 577 metabolites, respectively, corresponding to 41% of metabolites and over 70% of the metabolic reaction predicted from the parasite genome. An additional 89 metabolites and 92 reactions were identified that were not predicted from genomic reconstructions, with the largest group being associated with metabolite damage‐repair systems. Validation of the draft metabolome revealed four previously uncharacterised enzymes which impact isoprenoid biosynthesis, lipid homeostasis and mitochondrial metabolism and are necessary for parasite development and proliferation. This study defines the metabolic fate of multiple carbon sources in P. falciparum, and highlights the activity of metabolite repair pathways in these rapidly growing parasite stages, opening new avenues for drug discovery.