RESUMO
The adhesion of infected red blood cells (iRBCs) to human endothelium is considered a key event in the pathogenesis of cerebral malaria and other life-threatening complications caused by the most prevalent malaria parasite Plasmodium falciparum. In the past 30 years, 14 endothelial receptors for iRBCs have been identified. Exposing 10 additional surface proteins of endothelial cells to a mixture of P. falciparum isolates from three Ghanaian malaria patients, we identified seven new iRBC receptors, all expressed in brain vessels. This finding strongly suggests that endothelial binding of P. falciparumâ iRBCs is promiscuous and may use a combination of endothelial surface moieties.
Assuntos
Adesão Celular , Células Endoteliais/fisiologia , Eritrócitos/fisiologia , Eritrócitos/parasitologia , Plasmodium falciparum/crescimento & desenvolvimento , Gana , Humanos , Plasmodium falciparum/isolamento & purificação , Plasmodium falciparum/parasitologiaRESUMO
BACKGROUND: In order to proceed through their life cycle, Leishmania parasites switch between sandflies and mammals. The flagellated promastigote cells transmitted by the insect vector are phagocytized by macrophages within the mammalian host and convert into the amastigote stage, which possesses a rudimentary flagellum only. During an earlier proteomic study of the stage differentiation of the parasite we identified a component of the outer dynein arm docking complex, a structure of the flagellar axoneme. The 70 kDa subunit of the outer dynein arm docking complex consists of three subunits altogether and is essential for the assembly of the outer dynein arm onto the doublet microtubule of the flagella. According to the nomenclature of the well-studied Chlamydomonas reinhardtii complex we named the Leishmania protein LdDC2. METHODOLOGY/PRINCIPAL FINDINGS: This study features a characterization of the protein over the life cycle of the parasite. It is synthesized exclusively in the promastigote stage and localizes to the flagellum. Gene replacement mutants of lddc2 show reduced growth rates and diminished flagellar length. Additionally, the normally spindle-shaped promastigote parasites reveal a more spherical cell shape giving them an amastigote-like appearance. The mutants lose their motility and wiggle in place. Ultrastructural analyses reveal that the outer dynein arm is missing. Furthermore, expression of the amastigote-specific A2 gene family was detected in the deletion mutants in the absence of a stage conversion stimulus. In vitro infectivity is slightly increased in the mutant cell line compared to wild-type Leishmania donovani parasites. CONCLUSIONS/SIGNIFICANCE: Our results indicate that the correct assembly of the flagellum has a great influence on the investigated characteristics of Leishmania parasites. The lack of a single flagellar protein causes an aberrant morphology, impaired growth and altered infectiousness of the parasite.
Assuntos
Dineínas/química , Dineínas/metabolismo , Flagelos/metabolismo , Leishmania donovani/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Northern Blotting , Western Blotting , DNA de Protozoário , Dineínas/genética , Ensaio de Imunoadsorção Enzimática , Flagelos/genética , Flagelos/ultraestrutura , Leishmania donovani/genética , Leishmania donovani/ultraestrutura , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas de Protozoários/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Leishmania promastigote cells transmitted by their insect vector get phagocytosed by macrophages and convert into the amastigote form. In a recently performed proteomic study, a thymidine kinase (TK) was found to be preferentially expressed in amastigotes. Western blot analysis showing a marked increase in TK protein synthesis during stage differentiation from promastigotes to amastigotes confirmed this result. After comparison of the amino acid sequence of Leishmania donovani and Leishmania major thymidine kinases with thymidine kinases of other organisms the Leishmania protein has to be classified as a type II TK. Therefore, in accordance with the nomenclature of other thymidine kinases we named the Leishmania enzymes LdTK1 and LmTK1, respectively. The LdTK1 is localised within the cytoplasm of promastigotes. In amastigotes, increased expression and a clustered distribution of the protein can be observed. Lmtk1 single allele gene replacement mutants have significantly elongated flagellum. In contrast, lmtk1 double allele gene replacement mutants show a remarkably reduced flagellar length, diminished overall size and a deformed body shape. In addition, they have a 12-fold reduced growth rate. For both mutant strains, macrophage infectivity is clearly reduced compared to a L. major wildtype infection.
