Elevated NAD+ drives Sir2A-mediated GCβ deacetylation and OES localization for Plasmodium ookinete gliding and mosquito infection

cGMP signal-activated ookinete gliding is essential for mosquito midgut infection of Plasmodium in malaria transmission. During ookinete development, cGMP synthesizer GC{beta} polarizes to a unique localization <ookinete extrados site= (OES) until ookinete maturation and activates cGMP signaling for initiating parasite motility. However, the mechanism underlying GC{beta} translocation from cytosol to OES remains elusive. Here, we used protein proximity labeling to search the GC{beta}-interacting proteins in ookinetes of the rodent malaria parasite P. yoelii, and found the top hit Sir2A, a NAD+-dependent sirtuin family deacetylase. Sir2A interacts with GC{beta} throughout ookinete development. In mature ookinetes, Sir2A co-localizes with GC{beta} at OES in a mutually dependent manner. Parasites lacking Sir2A lose GC{beta} localization at OES, ookinete gliding, and mosquito infection, phenocopying GC{beta} deficiency. GC{beta} is acetylated at gametocytes but is deacetylated by Sir2A for OES localization at mature ookinetes. We further demonstrated that the level of NAD+, an essential co-substrate for sirtuin, increases during the ookinete development. The NAD+ at its maximal level until ookinete maturation promotes Sir2A-catalyzed GC{beta} deacetylation, ensuring GC{beta} localization at OES. This study highlights the spatiotemporal coordination of cytosolic NAD+ level and NAD+-dependent Sir2A in regulating GC{beta} deacetylation and dynamic localization for Plasmodium ookinete gliding.