Identification of a Rabenosyn-5 like protein and Rab5b in host cell cytosol uptake reveals conservation of endosomal transport in malaria parasites

Abstract

Vesicular trafficking, including secretion and endocytosis, plays fundamental roles in the unique biology of P. falciparum blood-stage parasites. Endocytosis of host cell cytosol (HCC) provides nutrients and room for parasite growth and is critical for the action of antimalarial drugs and parasite drug resistance. Previous work showed that PfVPS45 functions in endosomal transport of HCC to the parasite's food vacuole, raising the possibility that malaria parasites possess a canonical endolysosomal system. However, the seeming absence of VPS45-typical functional interactors such as rabenosyn 5 (Rbsn5) and the re-purposing of Rab5 isoforms and other endolysosomal proteins for secretion in apicomplexans question this idea. Here we identified the likely parasite Rbsn5 and show that it functions with VPS45 in the endosomal transport of HCC. We also show that PfRab5b but not PfRab5a is involved in the same process. Inactivation of PfRbsn5 resulted in PI3P and PfRab5b decorated HCC-filled vesicles, typical for endosomal compartments. Overall this indicates that despite the low sequence conservation of PfRbsn5 and the unusual N-terminal modification of PfRab5b, principles of endosomal transport in malaria parasite are similar to that of model organisms. Using a conditional double protein inactivation system, we further provide evidence that the PfKelch13 compartment, an unusual apicomplexa-specific endocytosis structure at the parasite plasma membrane, is connected upstream of the Rbsn5/VPS45/Rab5b-dependent endosomal route. Altogether, this work indicates that HCC-uptake consists of a highly parasite-specific part that feeds endocytosed material into a more canonical endosomal system, leading to the delivery of HCC to the food vacuole.

The Kelch13 compartment contains highly divergent vesicle trafficking proteins in malaria parasites

“…Inspection of the MyoF-2xFKBP-GFP-2xFKBP endo , compared to 3D7 wild type-trophozoites, revealed an increased number of vesicles in the parasite cytoplasm ( Fig 1H ), resembling the phenotype observed after inactivation of VPS45 or Rbsn5, two proteins involved in the transport of host cell cytosol to the parasite’s food vacuole [ 54 , 55 ]. This was even more pronounced upon conditional inactivation of MyoF by knock-sideways ( Fig 1H ), suggesting this is due to a reduced function of MyoF.…”

“…In order to generate the plasmids for co-localisation experiments while simultaneously carrying out knock sideways, ama1 AMA1mCherry, ama1 AROmCherry and ama1 IMC1cmCherry were amplified from pARL ama1 AMA1mCherry, pARL ama1 AROmCherry or pARL ama1 IMC1cmCherry [ 65 ] by PCR and cloned into ama1 -p40-mSca_ nmd3 ’-NLS-FRB-T2A-yDHODH, which was generated by inserting the ama1 promoter using the NotI/KpnI restriction sites into sf3a2 -p40-mSca_ nmd3 ’-NLS-FRB-T2A-yDHODH [ 55 ], using the KpnI/XmaI restriction sites, resulting in ama1 AROmCherry_ nmd3 ’-NLS-FRB-T2A-yDHODH, ama1 AMA1mCherry_ nmd3 ’-NLS-FRB-T2A-yDHODH and ama1 IMC1cmCherry_ nmd3 ’-NLS-FRB-T2A-yDHODH.…”

“…The molecular effectors involved in this process remain poorly characterized. So far VPS45 [ 54 ], Rbsn5 [ 55 ], Rab5b [ 55 ], the phosphoinositide-binding protein PX1 [ 56 ], the host enzyme peroxiredoxin 6 [ 57 ] and K13 and some of its compartment proteins (Eps15, AP2μ, KIC7, UBP1) [ 29 ] have been reported to act at different steps in the endocytic uptake pathway of hemoglobin. While inactivation of VPS45, Rbsn5, Rab5b, PX1 or actin resulted in an accumulation of hemoglobin filled vesicles [ 53 – 56 ], indicative of a block during endosomal transport (late steps in endocytosis), no such vesicles were observed upon inactivation of K13 and its compartment proteins [ 29 ], suggesting a role of these proteins during initiation of endocytosis (early steps in endocytosis).…”