Morphological events during the cell cycle of Leishmania major.

The morphological events involved in the Leishmania major promastigote cell cycle have been investigated in order to provide a detailed description of the chronological processes by which the parasite replicates its set of single-copy organelles and generates a daughter cell. Immunofluorescence labeling of ß-tubulin was used to follow the dynamics of the subcellular cytoskeleton and to monitor the division of the nucleus via visualization of the mitotic spindle, while RAB11 was found to be a useful marker to track flagellar pocket division and to follow mitochondrial DNA (kinetoplast) segregation. Classification and quantification of these morphological events were used to determine the durations of phases of the cell cycle. Our results demonstrate that in L. major promastigotes, the extrusion of the daughter flagellum precedes the onset of mitosis, which in turn ends after kinetoplast segregation, and that significant remodelling of cell shape accompanies mitosis and cytokinesis. These findings contribute to a more complete foundation for future studies of cell cycle control in Leishmania.

The leishmania ARL-1 and Golgi traffic.

We present here the characterisation of the Leishmania small G protein ADP-Ribosylation Factor-Like protein 1 (ARL-1). The ARL-1 gene is present in one copy per haploid genome and conserved among trypanosomatids. It encodes a protein of 20 kDa, which is equally expressed in the insect promastigote and mammalian amastigote forms of the parasite. ARL-1 localises to the Trans-Golgi Network (TGN); N-terminal myristoylation is essential for TGN localisation. In vivo expression of the LdARL-1/Q74L and LdARL-1/T51N mutants (GTP- and GDP-bound blocked forms respectively) shows that GDP/GTP cycling occurs entirely within the TGN. This is contrary to previous reports in yeast and mammals, where the mutant empty form devoid of nucleotide has been considered as the GDP-blocked form. The dominant-negative empty form mutant LdARL-1/T34N inhibits endocytosis and intracellular trafficking from the TGN to the Lysosome/Multivesicular Tubule and to the acidocalcisomes; these defects are probably related to a mislocalisation of the GRIP domain-containing vesicle tethering factors which cannot be recruited to the TGN by the cytoplasmic LdARL-1/T34N. Thus, besides the functional characterization of a new mutant and a better understanding of ARL-1 GDP/GTP cycling, this work shows that Leishmania ARL-1 is a key component of an essential pathway worth future study.

Studies on the CPA cysteine peptidase in the Leishmania infantum genome strain JPCM5.

BACKGROUND: Visceral leishmaniasis caused by members of the Leishmania donovani complex is often fatal in the absence of treatment. Research has been hampered by the lack of good laboratory models and tools for genetic manipulation. In this study, we have characterised a L. infantum line (JPCM5) that was isolated from a naturally infected dog and then cloned. We found that JPCM5 has attributes that make it an excellent laboratory model; different stages of the parasite life cycle can be studied in vitro, it is accessible to genetic manipulation and it has retained its virulence. Furthermore, the L. infantum JPCM5 genome has now been fully sequenced. RESULTS: We have further focused our studies on LiCPA, the L. infantum homologue to L. mexicana cysteine peptidase CPA. LiCPA was found to share a high percentage of amino acid identity with CPA proteins of other Leishmania species. Two independent LiCPA-deficient promastigote clones (DeltaLicpa) were generated and their phenotype characterised. In contrast to L. mexicana CPA-deficient mutants, both clones of DeltaLicpa were found to have significantly reduced virulence in vitro and in vivo. Re-expression of just one LiCPA allele (giving DeltaLicpa::CPA) was sufficient to complement the reduced infectivity of both DeltaLicpa mutants for human macrophages, which confirms the importance of LiCPA for L. infantum virulence. In contrast, in vivo experiments did not show any virulence recovery of the re-expressor clone DeltaLicpaC1::CPA compared with the CPA-deficient mutant DeltaLicpaC1. CONCLUSION: The data suggest that CPA is not essential for replication of L. infantum promastigotes, but is important for the host-parasite interaction. Further studies will be necessary to elucidate the precise roles that LiCPA plays and why the re-expression of LiCPA in the DeltaLicpa mutants complemented the gene deletion phenotype only in in vitro and not in in vivo infection of hamsters.

Bloodstream form Trypanosoma brucei depend upon multiple metacaspases associated with RAB11-positive endosomes.

Trypanosoma brucei possesses five metacaspase genes. Of these, MCA2 and MCA3 are expressed only in the mammalian bloodstream form of the parasite, whereas MCA5 is expressed also in the insect procyclic form. Triple RNAi analysis showed MCA2, MCA3 and MCA5 to be essential in the bloodstream form, with parasites accumulating pre-cytokinesis. Nevertheless, triple null mutants (deltamca2/3deltamca5) could be isolated after sequential gene deletion. Thereafter, deltamca2/3deltamca5 mutants were found to grow well both in vitro in culture and in vivo in mice. We hypothesise that metacaspases are essential for bloodstream form parasites, but they have overlapping functions and their progressive loss can be compensated for by activation of alternative biochemical pathways. Analysis of deltamca2/3deltamca5 revealed no greater or lesser susceptibility to stresses reported to initiate programmed cell death, such as treatment with prostaglandin D2. The metacaspases were found to colocalise with RAB11, a marker for recycling endosomes. However, variant surface glycoprotein (VSG) recycling processes and the degradation of internalised anti-VSG antibody were found to occur similarly in wild type, deltamca2/3deltamca5 and triple RNAi induced parasites. Thus, the data provide no support for the direct involvement of T. brucei metacaspases in programmed cell death and suggest that the proteins have a function associated with RAB11 vesicles that is independent of known recycling processes of RAB11-positive endosomes.

A soluble pyrophosphatase, a key enzyme for polyphosphate metabolism in Leishmania.

We report the functional characterization in Leishmania amazonensis of a soluble pyrophosphatase (LaVSP1) that localizes in acidocalcisomes, a vesicular acidic compartment. LaVSP1 is preferentially expressed in metacyclic forms. Experiments with dominant negative mutants show the requirement of LaVSP1 functional expression for metacyclogenesis and virulence in mice. Depending on the pH and the cofactors Mg2+ or Zn2+, both present in acidocalcisomes, LaVSP1 hydrolyzes either inorganic pyrophosphate (Km = 92 microM, kcat = 125 s(-1)), tripolyphosphate (Km = 1153 microM, kcat = 131 s(-1)), or polyphosphate of 28 residues (Km = 123 microM, kcat = 8 s(-1)). Predicted structural analysis suggests that the structural orientation of the residue Lys78 in LaVSP1 accounts for the observed increase in Km compared with the yeast pyrophosphatase and for the ability of trypanosomatid VSP1 enzymes to hydrolyze polyphosphate. These results make the VSP1 enzyme an attractive drug target against trypanosomatid parasites.

Abortive infection of Lutzomyia longipalpis insect vectors by aflagellated LdARL-3A-Q70L overexpressing Leishmania amazonensis parasites.

Leishmania donovani ADP-ribosylation factor-like protein 3A (LdARL-3A) is a small G protein isolated from the protozoan parasite L. donovani with no defined physiological function. Previously [Cuvillier, A., Redon, F., Antoine, J.-C., Chardin, P., DeVos, T., and Merlin, G. (2000) J Cell Sci 113: 2065-2074] we have shown that overexpression in L. amazonensis promastigotes of the mutated protein LdARL-3A-Q70L, which remains constitutively associated with GTP, leads to the disappearance of the flagellum but does not impair cell viability or growth. Here we report that parasites overexpressing LdARL-3A-Q70L can invade in vitro cultivated macrophages to the same extent as control cells, demonstrating that the flagellum is not necessary for attachment to or engulfment into macrophages. These infections are productive because amastigotes differentiate and multiply. However, aflagellated LdARL-3A-Q70L-overexpressing Leishmania promastigotes could not survive in experimentally infected Lutzomyia longipalpis insect vectors, in contrast to untransfected or native LdARL-3A-overexpressing cells. Overexpression of the native and mutated proteins did not modify in vitro procyclic to metacyclic lipophosphoglycan maturation or differentiation from procyclic to metacyclic promastigotes, nevertheless there is a block in transmission of Leishmania. Better understanding of LdARL-3A pathways, notably those regarding flagellum biogenesis, may lead to the future development of Leishmania-specific drugs, which may stop parasite transmission in nature without affecting other species.