Direct Diagnostic Tests for Lyme Disease.

Borrelia burgdorferi was discovered to be the cause of Lyme disease in 1983, leading to seroassays. The 1994 serodiagnostic testing guidelines predated a full understanding of key B. burgdorferi antigens and have a number of shortcomings. These serologic tests cannot distinguish active infection, past infection, or reinfection. Reliable direct-detection methods for active B. burgdorferi infection have been lacking in the past but are needed and appear achievable. New approaches have effectively been applied to other emerging infections and show promise in direct detection of B. burgdorferi infections.

Advances in Serodiagnostic Testing for Lyme Disease Are at Hand.

The cause of Lyme disease, Borrelia burgdorferi, was discovered in 1983. A 2-tiered testing protocol was established for serodiagnosis in 1994, involving an enzyme immunoassay (EIA) or indirect fluorescence antibody, followed (if reactive) by immunoglobulin M and immunoglobulin G Western immunoblots. These assays were prepared from whole-cell cultured B. burgdorferi, lacking key in vivo expressed antigens and expressing antigens that can bind non-Borrelia antibodies. Additional drawbacks, particular to the Western immunoblot component, include low sensitivity in early infection, technical complexity, and subjective interpretation when scored by visual examination. Nevertheless, 2-tiered testing with immunoblotting remains the benchmark for evaluation of new methods or approaches. Next-generation serologic assays, prepared with recombinant proteins or synthetic peptides, and alternative testing protocols, can now overcome or circumvent many of these past drawbacks. This article describes next-generation serodiagnostic testing for Lyme disease, focusing on methods that are currently available or near-at-hand.

Radiation-induced cellular and molecular alterations in asexual intraerythrocytic Plasmodium falciparum.

BACKGROUND: γ-irradiation is commonly used to create attenuation in Plasmodium parasites. However, there are no systematic studies on the survival, reversion of virulence, and molecular basis for γ-radiation-induced cell death in malaria parasites. METHODS: The effect of γ-irradiation on the growth of asexual Plasmodium falciparum was studied in erythrocyte cultures. Cellular and ultrastructural changes within the parasite were studied by fluorescence and electron microscopy, and genome-wide transcriptional profiling was performed to identify parasite biomarkers of attenuation and cell death. RESULTS: γ-radiation induced the death of P. falciparum in a dose-dependent manner. These parasites had defective mitosis, sparse cytoplasm, fewer ribosomes, disorganized and clumped organelles, and large vacuoles-observations consistent with "distressed" or dying parasites. A total of 185 parasite genes were transcriptionally altered in response to γ-irradiation (45.9% upregulated, 54.1% downregulated). Loss of parasite survival was correlated with the downregulation of genes encoding translation factors and with upregulation of genes associated with messenger RNA-sequestering stress granules. Genes pertaining to cell-surface interactions, host-cell remodeling, and secreted proteins were also altered. CONCLUSIONS: These studies provide a framework to assess the safety of γ-irradiation attenuation and promising targets for genetic deletion to produce whole parasite-based attenuated vaccines.

Protection from experimental cerebral malaria with a single dose of radiation-attenuated, blood-stage Plasmodium berghei parasites.

BACKGROUND: Whole malaria parasites are highly effective in inducing immunity against malaria. Due to the limited success of subunit based vaccines in clinical studies, there has been a renewed interest in whole parasite-based malaria vaccines. Apart from attenuated sporozoites, there have also been efforts to use live asexual stage parasites as vaccine immunogens. METHODOLOGY AND RESULTS: We used radiation exposure to attenuate the highly virulent asexual blood stages of the murine malaria parasite P. berghei to a non-replicable, avirulent form. We tested the ability of the attenuated blood stage parasites to induce immunity to parasitemia and the symptoms of severe malaria disease. Depending on the mouse genetic background, a single high dose immunization without adjuvant protected mice from parasitemia and severe disease (CD1 mice) or from experimental cerebral malaria (ECM) (C57BL/6 mice). A low dose immunization did not protect against parasitemia or severe disease in either model after one or two immunizations. The protection from ECM was associated with a parasite specific antibody response and also with a lower level of splenic parasite-specific IFN-γ production, which is a mediator of ECM pathology in C57BL/6 mice. Surprisingly, there was no difference in the sequestration of CD8+ T cells and CD45+ CD11b+ macrophages in the brains of immunized, ECM-protected mice. CONCLUSIONS: This report further demonstrates the effectiveness of a whole parasite blood-stage vaccine in inducing immunity to malaria and explicitly demonstrates its effectiveness against ECM, the most pathogenic consequence of malaria infection. This experimental model will be important to explore the formulation of whole parasite blood-stage vaccines against malaria and to investigate the immune mechanisms that mediate protection against parasitemia and cerebral malaria.

Clinical and molecular aspects of malaria fever.

Although clinically benign, malaria fever is thought to have significant relevance in terms of parasite growth and survival and its virulence which in turn may alter the clinical course of illness. In this article, the historical literature is reviewed, providing some evolutionary perspective on the genesis and biological relevance of malaria fever, and the available molecular data on the febrile-temperature-inducible parasite factors that may contribute towards the regulation of parasite density and alteration of virulence in the host is also discussed. The potential molecular mechanisms that could be responsible for the induction and regulation of cyclical malaria fevers caused by different species of Plasmodium are also discussed.

Mitosis in the human malaria parasite Plasmodium falciparum.

Malaria is caused by intraerythrocytic protozoan parasites belonging to Plasmodium spp. (phylum Apicomplexa) that produce significant morbidity and mortality, mostly in developing countries. Plasmodium parasites have a complex life cycle that includes multiple stages in anopheline mosquito vectors and vertebrate hosts. During the life cycle, the parasites undergo several cycles of extreme population growth within a brief span, and this is critical for their continued transmission and a contributing factor for their pathogenesis in the host. As with other eukaryotes, successful mitosis is an essential requirement for Plasmodium reproduction; however, some aspects of Plasmodium mitosis are quite distinct and not fully understood. In this review, we will discuss the current understanding of the architecture and key events of mitosis in Plasmodium falciparum and related parasites and compare them with the traditional mitotic events described for other eukaryotes.

Pathogenic roles of CD14, galectin-3, and OX40 during experimental cerebral malaria in mice.

An in-depth knowledge of the host molecules and biological pathways that contribute towards the pathogenesis of cerebral malaria would help guide the development of novel prognostics and therapeutics. Genome-wide transcriptional profiling of the brain tissue during experimental cerebral malaria (ECM ) caused by Plasmodium berghei ANKA parasites in mice, a well established surrogate of human cerebral malaria, has been useful in predicting the functional classes of genes involved and pathways altered during the course of disease. To further understand the contribution of individual genes to the pathogenesis of ECM, we examined the biological relevance of three molecules -- CD14, galectin-3, and OX40 that were previously shown to be overexpressed during ECM. We find that CD14 plays a predominant role in the induction of ECM and regulation of parasite density; deletion of the CD14 gene not only prevented the onset of disease in a majority of susceptible mice (only 21% of CD14-deficient compared to 80% of wildtype mice developed ECM, p<0.0004) but also had an ameliorating effect on parasitemia (a 2 fold reduction during the cerebral phase). Furthermore, deletion of the galectin-3 gene in susceptible C57BL/6 mice resulted in partial protection from ECM (47% of galectin-3-deficient versus 93% of wildtype mice developed ECM, p<0.0073). Subsequent adherence assays suggest that galectin-3 induced pathogenesis of ECM is not mediated by the recognition and binding of galectin-3 to P. berghei ANKA parasites. A previous study of ECM has demonstrated that brain infiltrating T cells are strongly activated and are CD44(+)CD62L(-) differentiated memory T cells [1]. We find that OX40, a marker of both T cell activation and memory, is selectively upregulated in the brain during ECM and its distribution among CD4(+) and CD8(+) T cells accumulated in the brain vasculature is approximately equal.

Centrins, cell cycle regulation proteins in human malaria parasite Plasmodium falciparum.

Molecules and cellular mechanisms that regulate the process of cell division in malaria parasites remain poorly understood. In this study we isolate and characterize the four Plasmodium falciparum centrins (PfCENs) and, by growth complementation studies, provide evidence for their involvement in cell division. Centrins are cytoskeleton proteins with key roles in cell division, including centrosome duplication, and possess four Ca(2+)-binding EF hand domains. By means of phylogenetic analysis, we were able to decipher the evolutionary history of centrins in eukaryotes with particular emphasis on the situation in apicomplexans and other alveolates. Plasmodium possesses orthologs of four distinct centrin paralogs traceable to the ancestral alveolate, including two that are unique to alveolates. By real time PCR and/or immunofluorescence, we determined the expression of PfCEN mRNA or protein in sporozoites, asexual blood forms, gametocytes, and in the oocysts developing inside mosquito mid-gut. Immunoelectron microscopy studies showed that centrin is expressed in close proximity with the nucleus of sporozoites and asexual schizonts. Furthermore, confocal and widefield microscopy using the double staining with alpha-tubulin and centrin antibodies strongly suggested that centrin is associated with the parasite centrosome. Following the episomal expression of the four PfCENs in a centrin knock-out Leishmania donovani parasite line that exhibited a severe growth defect, one of the PfCENs was able to partially restore Leishmania growth rate and overcome the defect in cytokinesis in such mutant cell line. To our knowledge, this study is the first characterization of a Plasmodium molecule that is involved in the process of cell division. These results provide the opportunity to further explore the role of centrins in cell division in malaria parasites and suggest novel targets to construct genetically modified, live attenuated malaria vaccines.

Molecular characterization and expression of a novel kinesin which localizes with the kinetoplast in the human pathogen, Leishmania donovani.

Using a variety of molecular and cell biological approaches, we identified, characterized and expressed a novel kinesin, LdK39B in the protozoan pathogen Leishmania donovani. Results of RT-PCR revealed two distinct LdK39B products with different splice leader mini-exon sites, indicative of two potentially mature mRNA transcripts. Analyses indicated that LdK39B had a calculated molecular mass of >261, 327 Da and contained multiple amino acid repeat units. Several GFP-LdK39B fusion constructs were generated and used for episomal-expression in these parasites. Results of confocal and immunoelectron microscopy indicated that the GFP-LdK39B-fusion proteins localized to a region adjacent to the flagellar pocket and the kinetoplast i.e. the mitochondrial-DNA containing organelle that is physically tethered to the flagellar basal bodies. Sub-cellular fractionation results showed that GFP-LdK39B proteins were insoluble in nature and remained tightly associated with purified flagella/kinetoplasts following their extraction with detergent and high salts. Our cumulative results suggest that the LdK39B may play a scaffold-like role in facilitating and maintaining the unique spatial/structural association between the flagellum-basal body-kinetoplast complex in these parasites.

Molecular dissection and expression of the LdK39 kinesin in the human pathogen, Leishmania donovani.

In this study we show for the first time the intracellular distribution of a K39 kinesin homologue in Leishmania donovani, a medically important parasite of humans. Further, we demonstrated that this motor protein is expressed in both the insect and mammalian developmental forms (i.e. promastigote and amastigotes) of this organism. Moreover, in both of these parasite developmental stages, immunofluorescence indicated that the LdK39 kinesin accumulated at anterior and posterior cell poles and that it displayed a peripheral localization consistent with the cortical cytoskeleton. Using a molecular approach, we identified, cloned and characterized the first complete open reading frame for the gene (LdK39) encoding this large (> 358 kDa) motor protein in L. donovani. Based on these observations, we subsequently used a homologous episomal expression system to dissect and express the functional domains that constitute the native molecule. Cell fractionation experiments demonstrated that LdK39 was soluble and that it bound to detergent-extracted cytoskeletons of these parasites in an ATP-dependent manner. The cumulative results of these experiments are consistent with LdK39 functioning as an ATP-dependent kinesin which binds to and travels along the cortical cytoskeleton of this important human pathogen.

Native nonmuscle myosin II stability and light chain binding in Drosophila melanogaster.

Native nonmuscle myosin IIs play essential roles in cellular and developmental processes throughout phylogeny. Individual motor molecules consist of a heterohexameric complex of three polypeptides which, when properly assembled, are capable of force generation. Here, we more completely characterize the properties, relationships and associations that each subunit has with one another in Drosophila melanogaster. All three native nonmuscle myosin II polypeptide subunits are expressed in close to constant stoichiometry to each other throughout development. We find that the stability of two subunits, the heavy chain and the regulatory light chain, depend on one another whereas the stability of the third subunit, the essential light chain, does not depend on either the heavy chain or regulatory light chain. We demonstrate that heavy chain aggregates, which form when regulatory light chain is lacking, associate with the essential light chain in vivo-thus showing that regulatory light chain association is required for heavy chain solubility. By immunodepletion we find that the majority of both light chains are associated with the nonmuscle myosin II heavy chain but pools of free light chain and/or light chain bound to other proteins are present. We identify four myosins (myosin II, myosin V, myosin VI and myosin VIIA) and a microtubule-associated protein (asp/Abnormal spindle) as binding partners for the essential light chain (but not the regulatory light chain) through mass spectrometry and co-precipitation. Using an in silico approach we identify six previously uncharacterized genes that contain IQ-motifs and may be essential light chain binding partners.

Rab5b localization to early endosomes in the protozoan human pathogen Leishmania donovani.

Leishmania donovani is a primitive trypanosomatid pathogen of humans. This protozoan is apically polarized such that the flagellar reservoir, the exclusive site of endocytosis and exocytosis, is situated at the anterior end. Recent evidence for the existence of an endocytic pathway in Leishmania has prompted us to investigate candidate temporal markers for endocytosis. In this study we identify the L. donovani Rab5b gene, and demonstrate the localization of a Rab5b chimera to early endosomes. A full-length Rab5b protein was fused to green fluorescent protein (GFP) to generate a chimeric protein GFP::Rab5b. Transfected L. donovani promastigotes carrying this chimeric construct displayed GFP::Rab5b localization. Additionally, incubation of transfected promastigotes with the fluid-phase marker Texas Red dextran demonstrated anterior co-localization of GFP::Rab5b and dye. This suggests Rab5b may act as a marker for early endosomes in L. donovani.

LdARF1 in trafficking and structural maintenance of the trans-Golgi cisternal network in the protozoan pathogen Leishmania donovani.

Adenosine diphosphate ribosylation factors (ARFs) are small guanosine-5'-triphosphatases that are essential in vesicular trafficking and in the maintenance of the Golgi network. In this report, we identified a homolog of the mammalian ARF1 in the human pathogenic protozoan parasite, Leishmania donovani (Ld). Ld ARF1 is a 549 bp gene encoding a 183-amino acid deduced protein of approximately 20 kDa. We demonstrated by Southern blot analysis that there are at least two copies of ARF1 in the Ld genome. Moreover, Northern blot analysis revealed that Ld ARF1 is expressed on a 1.35 kb transcript in both the insect vector (promastigotes) and mammalian host (amastigotes) forms of this parasite. Fluorescent microscopy studies using Ld promastigotes episomally transfected with an ARF1::GFP (green fluorescent protein) chimeric construct showed that such chimeras appeared to localize to the Golgi region of these organisms. This observation was verified by immunoelectron microscopy using an anti-GFP antibody. Such studies also revealed that Ld ARF1::GFP chimeras localized to trans-Golgi vesicles, the flagellar pocket/reservoir and other vesicles located between the trans-Golgi network and flagellar pocket in these apically polarized cells. Fluorescence recovery after photobleaching and fluorescence loss in photobleaching experiments revealed both the dynamic binding and releasing activity of Ld ARF1 from the Golgi network in these parasites. Further, episomal expression of a constitutively active ("on") ARF1 (Q71L mutation) resulted in the aberrant swelling and distended-structure of the trans-Golgi cisternae in these cells. These results show that Ld ARF1 is transiently associated with the Golgi network and plays a role in the structural maintenance of this organelle in these important human pathogens.

The Dictyostelium LvsA protein is localized on the contractile vacuole and is required for osmoregulation.

LvsA is a Dictyostelium protein that is essential for cytokinesis and that is related to the mammalian beige/LYST family of proteins. To better understand the function of this novel protein family we tagged LvsA with GFP using recombination techniques. GFP-LvsA is primarily associated with the membranes of the contractile vacuole system and it also has a punctate distribution in the cytoplasm. Two markers of the Dictyostelium contractile vacuole, the vacuolar proton pump and calmodulin, show extensive colocalization with GFP-LvsA on contractile vacuole membranes. Interestingly, the association of LvsA with contractile vacuole membranes occurs only during the discharge phase of the vacuole. In LvsA mutants the contractile vacuole becomes disorganized and calmodulin dissociates from the contractile vacuole membranes. Consequently, the contractile vacuole is unable to function normally, it can swell but seems unable to discharge and the LvsA mutants become osmosensitive. These results demonstrate that LvsA can associate transiently with the contractile vacuole membrane compartment and that this association is necessary for the function of the contractile vacuole during osmoregulation. This transient association with specific membrane compartments may be a general property of other BEACH-domain containing proteins.