Phenotypic diversity and selection maintain Leishmania amazonensis infectivity in BALB/c mouse model.

Leishmania are protozoan parasites that show remarkable diversity, as revealed by the various clinical forms of leishmaniasis, which can range from mild skin lesions to severe metastatic cutaneous/mucosal lesions. The exact nature and extent of Leishmania phenotypic diversity in establishing infection is not fully understood. In order to try to understand some aspects of this diversity, we subcutaneously infected BALB/c mice with first and second generation subclones of a L. amazonensis strain isolated from a patient (BA125) and examined in vivo lesion growth rate and antimony susceptibility. In vivo fast-, medium- and slow-growing subclones were obtained; moreover, fast-growing subclones could generate slow-growing subclones and inversely, revealing the continuous generation of diversity after passage into mice. No antimony-resistant subclone appeared, probably a rare occurrence. By tagging subclone cells with a L. amazonensis genomic cosmid library, we found that only a very small number of founding cells could produce lesions. Leishmania clones transfected with in vivo selected individual cosmids were also diverse in terms of lesion growth rate, revealing the cosmid-independent intrinsic characteristics of each clone. Our results suggest that only a few of the infecting parasites are able to grow and produce lesions; later, within the cell mixture of each lesion, there coexist several parasite populations with different potentialities to grow lesions during the next infection round. This may reflect a sort of programmed heterogeneity of individual parasites, favoring the survival of some individuals in various environmental conditions.

Phenotypic diversity and selection maintain Leishmania amazonensis infectivity in BALB/c mouse model

Leishmania are protozoan parasites that show remarkable diversity, as revealed by the various clinical forms of leishmaniasis, which can range from mild skin lesions to severe metastatic cutaneous/mucosal lesions. The exact nature and extent of Leishmania phenotypic diversity in establishing infection is not fully understood. In order to try to understand some aspects of this diversity, we subcutaneously infected BALB/c mice with first and second generation subclones of a L. amazonensis strain isolated from a patient (BA125) and examined in vivo lesion growth rate and antimony susceptibility. In vivo fast-, medium- and slow-growing subclones were obtained; moreover, fast-growing subclones could generate slow-growing subclones and inversely, revealing the continuous generation of diversity after passage into mice. No antimony-resistant subclone appeared, probably a rare occurrence. By tagging subclone cells with a L. amazonensis genomic cosmid library, we found that only a very small number of founding cells could produce lesions. Leishmania clones transfected with in vivo selected individual cosmids were also diverse in terms of lesion growth rate, revealing the cosmid-independent intrinsic characteristics of each clone. Our results suggest that only a few of the infecting parasites are able to grow and produce lesions; later, within the cell mixture of each lesion, there coexist several parasite populations with different potentialities to grow lesions during the next infection round. This may reflect a sort of programmed heterogeneity of individual parasites, favoring the survival of some individuals in various environmental conditions.

TbFlabarin, a flagellar protein of Trypanosoma brucei, highlights differences between Leishmania and Trypanosoma flagellar-targeting signals.

TbFlabarin is the Trypanosoma brucei orthologue of the Leishmania flagellar protein LdFlabarin but its sequence is 33% shorter than LdFlabarin, as it lacks a C-terminal domain that is indispensable for LdFlabarin to localize to the Leishmania flagellum. TbFlabarin is mainly expressed in the procyclic forms of the parasite and localized to the flagellum, but only when two palmitoylable cysteines at positions 3 and 4 are present. TbFlabarin is more strongly attached to the membrane fraction than its Leishmania counterpart, as it resists complete solubilization with as much as 0.5% NP-40. Expression ablation by RNA interference did not change parasite growth in culture, its morphology or apparent motility. Heterologous expression showed that neither TbFlabarin in L. amazonensis nor LdFlabarin in T. brucei localized to the flagellum, revealing non-cross-reacting targeting signals between the two species.

Constitutive mosaic aneuploidy is a unique genetic feature widespread in the Leishmania genus.

Using fluorescence in situ hybridization, we determined the ploidy of four species of Leishmania: Leishmania infantum, Leishmania donovani, Leishmania tropica and Leishmania amazonensis. We found that each cell in a strain possesses a combination of mono-, di- and trisomies for all chromosomes; ploidy patterns were different among all strains/species. These results extend those we previously described in Leishmania major, demonstrating that mosaic aneuploidy is a genetic feature widespread to the Leishmania genus. In addition to the genetic consequences induced by this mosaicism, the apparent absence of alternation between haploid/diploid stages questions the modality of genetic exchange in Leishmania sp.

LdFlabarin, a new BAR domain membrane protein of Leishmania flagellum.

During the Leishmania life cycle, the flagellum undergoes successive assembly and disassembly of hundreds of proteins. Understanding these processes necessitates the study of individual components. Here, we investigated LdFlabarin, an uncharacterized L. donovani flagellar protein. The gene is conserved within the Leishmania genus and orthologous genes only exist in the Trypanosoma genus. LdFlabarin associates with the flagellar plasma membrane, extending from the base to the tip of the flagellum as a helicoidal structure. Site-directed mutagenesis, deletions and chimera constructs showed that LdFlabarin flagellar addressing necessitates three determinants: an N-terminal potential acylation site and a central BAR domain for membrane targeting and the C-terminal domain for flagellar specificity. In vitro, the protein spontaneously associates with liposomes, triggering tubule formation, which suggests a structural/morphogenetic function. LdFlabarin is the first characterized Leishmania BAR domain protein, and the first flagellum-specific BAR domain protein.

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.

Flagellar length depends on LdARL-3A GTP/GDP unaltered cycling in Leishmania amazonensis.

We have shown previously that expression of the GTP-blocked form of the small G protein LdARL-3A/Q70L led to a marked shortening of Leishmania promastigotes flagella. In contrast, there was no effect with the T30N mutant, thought to represent the GDP-blocked form. However, recent data, obtained with human ARF-6, a member of the same family of G proteins, revealed that the corresponding mutant T27N was nucleotide-free and that the GDP-blocked form was the T44N mutant. When expressed in Leishmania, the corresponding new mutant, LdARL-3A/T47N, provoked also flagellum shortening. Then, it is the interruption of the cycling of LdARL-3A between a GDP- and a GTP-bound form which leads to the reduction of the flagellar length. This findings change significantly the understanding and the approaches for studying the mode of action and the role of LdARL-3A.

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.

LdARL-1 His-tagged recombinant protein: purification by immobilized metal affinity expanded bed adsorption.

Previously we have cloned three ADP-ribosylation factor-like (ARL) genes from the parasitic protozoan Leishmania donovani: LdARL-3A and 3B, LdARL-1. LdARL-3A was previously purified as an active native form, which was able to bind GTP in vitro. In this paper, we have performed the production and the purification of Histidine-tagged (His-tagged) LdARL-1 recombinant protein by immobilized metal affinity chromatography (IMAC) using expanded bed adsorption (EBA) technology. This protein was purified with more than 95% purity and could be successfully used for GTP-binding assay.

Trypanosomatid flagellum biogenesis: ARL-3A is involved in several species.

Overexpression in Leishmania amazonensis promastigotes of the GTPase-deficient small G protein LdARL-3A-Q70L specifically provokes the loss of the flagella without affecting cell viability and body size. However, motility is lost and, remarkably, cells do not survive in the insect vector Lutzomyia longipalpis gut, leading to interruption of parasite transmission. We report here that overexpression of the same protein in Leishmania major, Leishmania donovani, and Crithidia fasciculata also led to significant alterations of the flagella. Surprisingly, ablation of TbARL-3A expression by RNAi in Trypanosoma brucei brucei also provoked flagella shortening, revealing that overexpression of the GTPase-deficient protein seems functionally equivalent to a drastic reduction in its native counterpart abundance. This renders possible complementary studies of an essential pathway in related organisms. Potential significance for the protein function is discussed as well as future strategies for stopping the transmission of several neglected parasitic diseases.

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.