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A divergent protein kinase A in the human pathogen Leishmania is associated with developmental morphogenesi

ABSTRACT: Parasitic protozoa of the genus Leishmania are obligatory intracellular parasites that caus e human leishmaniasis . During their life, they cycle between the phagolysosome of mammalian macrophages ( round intracellular amastigotes ) , and the mid - gut of female sand flies ( elongated extracellular promastigotes ) . Shifting axenic promastigotes to a lysosome - like environment ( pH 5.5 and 37 ̊C , 5% C O 2 ) initiate s their development into amastigotes. Previous studies suggested a role for protein kinase A in differentiation initiation . In this study we assessed this hypothesis by describ ing a new , divergent , regulatory subunit of protein kinase A (LdPKAR3) whose phosphorylation is regulated by the differentiation signal . This protein exists exclusively in the genome s of Leishmania and T rypano soma cruzi , both obligatory intracellular parasites . W hile the C - terminal half of this protein is conserved , the N - termin al region is divergent and much longer than the consensus R subunits . We found that LdPKAR3 is bound to the subpellicular microtubules cell cortex via a formin FH2 domain at its N - terminal tip. Immunoprecipitation , fluorescence resonance energy transfer ( FRET ) , proteomics, and 3D modeling analyses indicated that LdPKAR3 associate s with the PKA catalytic subunit 3 (C3) to form a PKA holoenzyme , indicating that R3 is a true PKA regulatory subunit. In promastigotes, LdPKA R3 recruits C3 to the central region of the subpellicular microtubules , where it determines the ir elongated shape . Deleti ng either R3 or C3 resulted in significant rounding of most of the promastigote population . Adding back the full - length wild type genes restored the elongat e shape of promastigotes. This is the first time that a PKA regulatory s ubunit has been shown to play a role in Leishmania morphogenesis

INSTRUMENT(S): Q Exactive

ORGANISM(S): Leishmania Donovani

SUBMITTER: Keren Bendalak

LAB HEAD: Dan Zilberstein

PROVIDER: PXD025222 | Pride | 2024-03-21

REPOSITORIES: Pride

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Project description:Protozoa of the genus Leishmania are the causative agents of leishmaniasis in humans. These parasites cycle between promastigotes in the sand fly mid-gut and amastigotes in phagolysosome of mammalian macrophages. During infection, host up-regulate nitric oxide synthase and parasite induce host arginase expression, both of which use arginine as a substrate. These elevated activities deplete macrophage arginine pools, a situation that invading Leishmania must overcome since it is an essential amino acid. Leishmania donovani imports exogenous arginine via a mono-specific amino acid transporter (AAP3) and utilizes it primarily through the polyamine pathway to provide precursors for trypanothione biosynthesis. Here we report the discovery of a pathway whereby promastigote and amastigote forms of the Leishmania sense the lack of environmental arginine and respond with rapid up-regulation in AAP3 expression and activity, as well as several other transporters. Significantly, this arginine deprivation response is also activated in parasites during macrophage infection. Phosphoproteomic analyses of L. donovani promastigotes have implicated a Mitogen-Activated Protein Kinase 2 (MPK2)-mediated signaling cascade in this response and L. mexicana mutants lacking MPK2 are unable to respond to arginine deprivation. In this study, we established that Leishmania cells sense the absence of arginine in their environment; both in culture (axenic promastigotes and amastigotes) and in macrophages during infection (amastigotes). This study describes the first amino acid deprivation sensing mechanism and the pathway that transduce this response, and reveals a novel host-pathogen metabolic interplay. Total RNA from Ten Leishmania donovani samples were analyzed using RNA-Seq. Cells from two life stages (promastigotes and amastigotes) were grown in axenic culture in the presence and absense of arginine. For each condition two biological replicates were grown and analyzed. In addition two macrophage grown amastigotes were analyzed.

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A divergent protein kinase A in the human pathogen Leishmania is associated with developmental morphogenesi

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