A novel leishmanial copper P-type ATPase plays a vital role in parasite infection and intracellular survival

Abstract

Copper (Cu) is essential for all life forms; however, in excess, it becomes toxic. Toxic properties of Cu are known to be utilized by host species against various pathogenic invasions. Leishmania , in both free-living and intracellular forms, exhibits appreciable tolerance toward Cu stress. While determining the mechanism of Cu-stress evasion employed by Leishmania , we identified and characterized a hitherto unknown Cu-ATPase in Leishmania major and established its role in parasite survival in host macrophages. This novel L. major Cu-ATPase, LmATP7, exhibits homology with its orthologs at multiple motifs. In promastigotes, LmATP7 primarily localized at the plasma membrane. We also show that LmATP7 exhibits Cu-dependent expression patterns and complements Cu transport in a Cu-ATPase-deficient yeast strain. Promastigotes overexpressing LmATP7 exhibited higher survival upon Cu stress, indicating efficacious Cu export compared with Wt and heterozygous LmATP7 knockout parasites. We further explored macrophage– Leishmania interactions with respect to Cu stress. We found that Leishmania infection triggers upregulation of major mammalian Cu exporter, ATP7A, in macrophages, and trafficking of ATP7A from the trans -Golgi network to endolysosomes in macrophages harboring amastigotes. Simultaneously, in Leishmania , we observed a multifold increase in LmATP7 transcripts as the promastigote becomes established in macrophages and morphs to the amastigote form. Finally, overexpressing LmATP7 in parasites increases amastigote survivability within macrophages, whereas knocking it down reduces survivability drastically. Mice injected in their footpads with an LmATP7-overexpressing strain showed significantly larger lesions and higher amastigote loads as compared with controls and knockouts. These data establish the role of LmATP7 in parasite infectivity and intramacrophagic survivability.

Leishmania major-induced alteration of host cellular and systemic copper homeostasis drives the fate of infection

“…When faced with excess intracellular copper, ATP7A vesicularizes out of Golgi to export copper from the cell (Figure S1A). Previously, studies from our group have shown that a fraction of macrophage ATP7A traffics out of Golgi 24 hours post-Leishmania major infection 8 . This post-Golgi vesicular movement of ATP7A to Leishmania-harboring compartment was attributed to mobilization of copper for killing the pathogen.…”

“…The pathogen's ability to infect and survive in host decreased when LmATP7 was knocked down. It was further demonstrated that at 24 hours post-infection, host CuATPase, ATP7A traffics from the Golgi, where it normally resides, and localises to the Leishmaniapositive compartments, possibly to exert Cu stress on the pathogen 8 .…”

“…Besides its biosynthetic role, the redox property of copper has been exploited by many mammalian hosts to fight-off a variety of pathogenic infection. We have previously reported that the parasitic kinetoplast, Leishmania is subjected to copper stress by its host macrophage in order to neutralize it 8 .…”

Copper trafficking inTrypanosoma cruzi: the transcriptional response of candidates to balance toxicity and recruitment

“…These Cu P- type ATPases also localize to the trans Golgi network (TGN) for delivery of Cu to exported copper proteins or to intracellular vesicles for Cu storage [9]. The Cu P-type ATPases of Trypanosoma congolense and T. brucei have been experimentally analyzed [10] and the function and expression profile of Cu P-type ATPases of Leishmania major was studied during the life cycle of this parasite [11]. In addition, bioinformatic analysis revealed potential P-type ATPases for Cu, Ca, Na, and other ions in other Leishmania spp., T. brucei , and T. cruzi but the function of these proteins has not been experimentally verified [12].…”

Copper Metabolism in Naegleria gruberi and Its Deadly Relative Naegleria fowleri

“…In Plasmodium falciparum , a copper efflux P-ATPase has been identified and partly characterized ( Rasoloson et al, 2004 ), as was a copper-binding protein with sequence features characteristic of copper transporters, including three transmembrane domains: an extracellular copper-binding methionine motif and transmembrane Gx 3 G and Mx 3 M motifs ( Choveaux et al, 2012 ). Copper transporting ATPases were also identified in trypanosomatid parasites ( Isah et al, 2020 ; Paul et al, 2021 ). Significantly more research has been performed on copper metabolism in pathogenic yeasts.…”