In 2013, the Apicomplexan Molecular Physiology Section continued studies into the molecular basis and physiological role of increased erythrocyte permeability after infection with malaria parasites. Our studies implicate an unusual ion channel at the host erythrocyte membrane after infection with malaria parasites. This channel, the plasmodial surface anion channel (PSAC), is determined by clag3 genes, which are conserved in and restricted to malaria parasites. The precise composition of PSAC remains debated because the clag3 genes do not have obvious homology to known ion channels in other organisms. We are working to address this uncertainty with biochemical, molecular and genetic studies. In one line of studies, we obtained independent evidence for clag3 genes and additionally the clag2 gene, a paralog on parasite chromosome 2. Here, we used two blasticidin S-resistant parasite lines with altered PSAC activity to examine molecular basis. Whole-genome sequencing did not reveal DNA level changes in either mutant. Expression profiling instead revealed that clag3 and clag2 underwent marked silencing these mutants (18- to 140-fold reduced expression relative to the wild-type parental lines). Whole-genome expression microarray experiments excluded global changes in parasite gene expression. Silencing of the clag genes was due to specific epigenetic marks on associated histones. Biochemical studies revealed that silencing aborted production of the channel protein. DNA transfection to prevent silencing by expression of the clag3 gene under a constitutive promoter prevented acquisition of blasticidin S resistance, revealing that gene silencing is required by this drug resistance mechanism. This study contributes to our understanding of PSAC structure and function by quantifying the effects of clag gene silencing on PSAC activity. It also reveals a novel epigenetic mechanism of antimalarial drug resistance relevant to diverse parasite targets. We also explore the precise roles served by increased erythrocyte permeability after infection. Studies from numerous groups have suggested that PSAC may 1) function in parasite nutrient acquisition, 2) serve to remodel erythrocyte cation concentrations for parasite benefit, or 3) serve no essential role for the intracellular parasite. In one study, we explore nutrient acquisition as a possible PSAC role by examining parasite killing by PSAC inhibitors. We found that PSAC inhibitors from various classes exhibited markedly improved efficacy when external nutrient concentrations are reduced to physiological levels;similar studies with antimalarials that do not block PSAC showed no change in efficacy, excluding nonspecific effects of our engineered media. Linkage analysis, DNA transfection experiments, and selections yielding a specific ectopic homologous recombination all implicated clag3 genes in parasite growth inhibition by PSAC inhibitors. These findings indicate that PSAC functions in intracellular parasite nutrient acquisition. They should stimulate drug discovery efforts targeting this parasite channel. In an independent study that examined PSACs role further, we challenged generally accepted assumptions about the parasites ionic requirements by establishing continuous culture in novel sucrose-based media. With sucrose as the primary osmoticant and K+ and Cl- as the main extracellular ions, we obtained parasite growth and propagation at rates indistinguishable from those in physiological media. These conditions abolish long-known increases in intracellular Na+ via PSAC, excluding a requirement for erythrocyte cation remodeling. We also dissected Na+, K+, and Cl- requirements and found that unexpectedly low concentrations of each ion meet the parasites demands. Surprisingly, growth was not adversely affected by up to 148 mM K+, suggesting that low extracellular K+ is not an essential trigger for erythrocyte invasion. At the same time, merozoite egress and invasion required a threshold ionic strength, suggesting critical electrostatic interactions between macromolecules at these stages. These findings provide insights into transmembrane signaling in malaria and reveal fundamental differences between host and parasite ionic requirements.

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Ito, Daisuke; Schureck, Marc A; Desai, Sanjay A (2017) An essential dual-function complex mediates erythrocyte invasion and channel-mediated nutrient uptake in malaria parasites. Elife 6:
Chalapareddy, S; Desai, S A (2017) Malaria parasite proteins involved in nutrient channels at the host erythrocyte membrane: advances and questions for future research. Int J Curr Multidiscip Stud 3:619-623
Cheng, Yang; Lu, Feng; Wang, Bo et al. (2016) Plasmodium vivax GPI-anchored micronemal antigen (PvGAMA) binds human erythrocytes independent of Duffy antigen status. Sci Rep 6:35581
Pain, Margaret; Fuller, Alexandra W; Basore, Katherine et al. (2016) Synergistic Malaria Parasite Killing by Two Types of Plasmodial Surface Anion Channel Inhibitors. PLoS One 11:e0149214
Sharma, Paresh; Rayavara, Kempaiah; Ito, Daisuke et al. (2015) A CLAG3 mutation in an amphipathic transmembrane domain alters malaria parasite nutrient channels and confers leupeptin resistance. Infect Immun 83:2566-74
Kasukurti, A; Eggleton, C D; Desai, S A et al. (2015) FACS-style detection for real-time cell viscoelastic cytometry. RSC Adv 5:105636-105642
Gupta, Ankit; Thiruvengadam, Girija; Desai, Sanjay A (2015) The conserved clag multigene family of malaria parasites: essential roles in host-pathogen interaction. Drug Resist Updat 18:47-54
Basore, Katherine; Cheng, Yang; Kushwaha, Ambuj K et al. (2015) How do antimalarial drugs reach their intracellular targets? Front Pharmacol 6:91
Zipprer, Elizabeth M; Neggers, McKinzie; Kushwaha, Ambuj et al. (2014) A kinetic fluorescence assay reveals unusual features of Ca?? uptake in Plasmodium falciparum-infected erythrocytes. Malar J 13:184
Nguitragool, Wang; Rayavara, Kempaiah; Desai, Sanjay A (2014) Proteolysis at a specific extracellular residue implicates integral membrane CLAG3 in malaria parasite nutrient channels. PLoS One 9:e93759

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