LdARL-3A, a Leishmania promastigote-specific ADP-ribosylation factor-like protein, is essential for flagellum integrity

The small G protein-encoding LdARL-3A gene, a homologue of the human ARL-3 gene, was isolated from Leishmania donovani, and its protein product charac terised, It is unique in the Leishmania genome and expressed only in the ex tracellular promastigote insect form, but not in the intracellular amastigo te mammalian form, as shown by northern blots and western blots developed w ith a specific anti-C terminus immune serum, Indirect immunofluorescence mi croscopy revealed distinct labelled spots regularly distributed on the plas ma membrane, including the part lining the flagellum and the flagellar pock et. By transfection experiments, it was found that wild-type LdA RL-3A-over expressing promastigotes reached higher densities in culture, but released significantly less secreted acid phosphatase in the extracellular medium th an the parental strain. When LdARL-3A blocked under the GDP-bound 'inactive ' form or with an inactivated potential myristoylation site was overexpress ed, the cells displayed an apparent wild-type phenotype, but died earlier i n the stationary phase; in contrast to parental cells, they showed a diffus e pattern of fluorescence labelling in the cytoplasm and on the cell membra ne, Strikingly, when a constitutively 'active' form of LdARL-3A (blocked un der the GTP-bound form) was overexpressed, the promastigotes were immobile with a very short flagellum, a slow growth rate and a low level of acid pho sphatase secretion; the length of the flagellum was inversely proportional to mutant protein expression. We concluded that LdARL-3A could be an essent ial gene involved in flagellum biogenesis; it may provide new approaches fo r control of the parasite at the insect stage.