A divergent protein kinase A in the human pathogenLeishmaniais associated with cell cortex microtubules and controls cell shape

Abstract

Parasitic protozoa of the genus Leishmania cause human leishmaniasis. They cycle between the phagolysosome of mammalian macrophages, where they reside as round intracellular amastigotes, and the mid-gut of female sand flies, which they colonize as elongated extracellular promastigotes. Shifting promastigotes to a lysosome-like environment (pH 5.5 and 37°C, 5% CO2) initiates their development into amastigotes. Previous studies suggested a role for protein kinase A (PKA) in this differentiation process. Here, we describe a new, divergent, regulatory subunit (PKAR3) found only in a limited member of the family Kinetoplastidae. In L. donovani, phosphorylation of PKAR3 was regulated by the differentiation signal and coincided with parasite morphogenesis during stage development. LdPKAR3 was bound to the subpellicular microtubules cell cortex via a formin homology (FH2)-like domain at the tip of a large and divergent N-terminal domain. Immunoprecipitation, fluorescence resonance energy transfer (FRET), proteomics analyses, and structural modeling showed that PKAR3 selectively binds the C3 isoform of the PKA catalytic subunit in a holoenzyme complex. In promastigotes, PKAR3 recruited PKAC3 to the subpellicular microtubules at the central cortex. After exposure to a differentiation signal, PKAR3 homogenously distributed across the entire cortex, in concert with cell rounding. Deleting the genes encoding either the R3 or C3 subunit resulted in premature rounding of the promastigote population, indicating that PKA determines the normal elongated shape. Regulation of Leishmania developmental morphogenesis by interaction with the subpellicular microtubule corset is a novel function for a unique PKA complex not present in the host cell.

Divergent Protein Kinase A contributes to the regulation of flagellar waveform of the motile flagellum ofLeishmania mexicana

“…Here, we used a genetic approach combined with fluorescence- and video-microscopy, to dissect the localisations and paring preferences of the three L. mexicana PKAC and two PKAR proteins and studied how gene deletions impacted cell swimming and generation of flagellar waveforms. Our results corroborate recent reports of PKAC/R pairings in Leishmania (55) and extend previous analyses on PKA function by providing multiple lines of evidence for a vital function for Lmx PKAC1 in flagellar beat regulation. We found that there are two regions of PKA concentration in promastigote forms: Lmx PKAC1, Lmx PKAC2 and Lmx PKAR1 showed enrichment in the flagellum, while a strong cell body cortex signal was detected for Lmx PKAC3 and Lmx PKAR3.…”

“…Several reports have highlighted the existence of a second PKAR-like protein within the trypanosomatids (53,54), which was recently given the name PKAR3 in L. donovani (55); for consistency, we adopt this terminology here . L. mexicana has a syntenic orthologue of PKAR3 (Table 1) (Supplementary Figure 1D).…”

“…It remains an open question whether Lmx PKAC3 has bona-fide phosphorylation targets within the flagellum, or whether the increased amount of soluble catalytic subunit in the Lmx PKAR3 deletion mutant causes off-target phosphorylation events in the axoneme that affect motility, secondary to its main role elsewhere. A recent report linked Lmx PKAR3 and Lmx PKAC3 to processes involved in cell morphology in L. donovani (55). This may explain the shortened flagella in our ΔLmxPKAC3 cells but we did not observe any morphological changes in ΔLmxPKAR3 .…”

Lysosome Sensing Is a Key Mechanism in Leishmania Intracellular Development

“…In addition to R1, Leishmania genome encodes LdPKAR3 (LinJ.34.2680) that similar to R1 form heterodimers with LdPKAC subunits and is cAMP-independent. In LdPKAR3, the C-terminus half is conserved with higher Eukaryotes while the N-terminus half is divergent and most likely unstructured (Fischer-Weinberger et al, 2021).…”

“…Recent experiments indicate that LdPKAR3 is covalently bound to the subpellicular microtubules at the cell cortex. R3 associates with C3, and this association is important for the elongated shape of promastigotes (Fischer-Weinberger et al, 2021).…”