A divergent protein kinase A regulatory subunit essential for morphogenesis of the human pathogen Leishmania.

Parasitic protozoa of the genus Leishmania cycle between the phagolysosome of mammalian macrophages, where they reside as rounded intracellular amastigotes, and the midgut of female sand flies, which they colonize as elongated extracellular promastigotes. Previous studies indicated that protein kinase A (PKA) plays an important role in the initial steps of promastigote differentiation into amastigotes. Here, we describe a novel regulatory subunit of PKA (which we have named PKAR3) that is unique to Leishmania and most (but not all) other Kinetoplastidae. PKAR3 is localized to subpellicular microtubules (SPMT) in the cell cortex, where it recruits a specific catalytic subunit (PKAC3). Promastigotes of pkar3 or pkac3 null mutants lose their elongated shape and become rounded but remain flagellated. Truncation of an N-terminal formin homology (FH)-like domain of PKAR3 results in its detachment from the SPMT, also leading to rounded promastigotes. Thus, the tethering of PKAC3 via PKAR3 at the cell cortex is essential for maintenance of the elongated shape of promastigotes. This role of PKAR3 is reminiscent of PKARIß and PKARIIß binding to microtubules of mammalian neurons, which is essential for the elongation of dendrites and axons, respectively. Interestingly, PKAR3 binds nucleoside analogs, but not cAMP, with a high affinity similar to the PKAR1 isoform of Trypanosoma. We propose that these early-diverged protists have re-purposed PKA for a novel signaling pathway that spatiotemporally controls microtubule remodeling and cell shape.

Morphogenesis Dynamics in Leishmania Differentiation.

Leishmania, the causative agent of leishmaniasis, is an obligatory intracellular parasite that cycles between phagolysosome of mammalian macrophages, where it resides as round intracellular amastigotes, and the midgut of female sandflies, where it resides as extracellular elongated promastigotes. This protozoan parasite cytoskeleton is composed of stable and abundant subpellicular microtubules (SPMT). This study aims to determine the kinetics of developmental morphogenesis and assess whether microtubules remodelling is involved in this process. Using image-streaming technology, we observed that rounding of promastigotes during differentiation into amastigotes was initiated promptly after exposure to the differentiation signal. Stabilizing microtubules with taxol sped rounding, but later killed differentiating parasites if taxol was not removed. Microtubule destabilizers such as vinblastine had no effect on the rate of rounding, nor on the viability of differentiating parasites. In the reverse process, elongation is initiated after a delay of 7.5 and completed 72 h after exposure to the amastigote to the promastigote differentiation signal. During the delay, parasites became highly sensitive to treatment with microtubule destabilizers. The addition of vinblastine during the first 7.5 h halted differentiation and killed parasites. Between 8 and 24 h, parasites gradually became resistant to vinblastine and, in parallel, started to elongate. In contrast, taxol had no effect on parasite elongation, nor on the viability of these cells. In a parallel study, we showed that the Leishmania-specific protein kinase A (PKA) holoenzyme containing the LdPKAR3-C3 complex is essential for promastigote elongation. Mutant promastigotes lacking either of these proteins are round, but maintain their flagella. Here, we observed that during differentiation into amastigotes, these mutants round at the same rate as the wild type, but never exceed the WT density of round amastigotes. In the reverse process, these mutants undergo the same initial delay and then elongate at the same rate as the WT. They stop elongating when they reach 20% of elongated cells in mature promastigotes. Our analysis indicates that while promastigote rounding into amastigotes did not require microtubule remodelling, morphogenesis of round amastigotes into elongated promastigotes required microtubule rearrangement before elongation was initiated. This is the first study that investigates the dynamics of microtubules during parasite development.

The arginine sensing and transport binding sites are distinct in the human pathogen Leishmania.

The intracellular protozoan parasite Leishmania donovani causes human visceral leishmaniasis. Intracellular L. donovani that proliferate inside macrophage phagolysosomes compete with the host for arginine, creating a situation that endangers parasite survival. Parasites have a sensor that upon arginine deficiency activates an Arginine Deprivation Response (ADR). L. donovani transport arginine via a high-affinity transporter (LdAAP3) that is rapidly up-regulated by ADR in intracellular amastigotes. To date, the sensor and its ligand have not been identified. Here, we show that the conserved amidino group at the distal cap of the arginine side chain is the ligand that activates ADR, in both promastigotes and intracellular amastigotes, and that arginine sensing and transport binding sites are distinct in L. donovani. Finally, upon addition of arginine and analogues to deprived cells, the amidino ligand activates rapid degradation of LdAAP3. This study provides the first identification of an intra-molecular ligand of a sensor that acts during infection.