High-throughput analysis of the transcriptional patterns of sexual genes in malaria

Parasites & Vectors

Methodology

Ron Rotkopf¹  Teresa G. Carvalho²  Sonia Oren³  Noam Soffer³  Abel Cruz Camacho³  Edo Kiper³  Nir Zaharoni³  Anna Rivkin³  Yael Noy³  Neta Regev-Rudzki³  Bar Ben David³  Netta Nir³  Dan Michael⁴ 

[1] Department of Life Sciences Core Facilities, Weizmann Institute of Science, 7610001, Rehovot, Israel;Department of Microbiology, Anatomy, Physiology and Pharmacology, La Trobe University, 3086, Melbourne, VIC, Australia;Faculty of Biochemistry, Department of Biomolecular Sciences, Weizmann Institute of Science, 7610001, Rehovot, Israel;Feinberg Graduate School, Weizmann Institute of Science, 7610001, Rehovot, Israel;

关键词: Malaria;    Gametocytogenesis;    RT-qPCR;    Gene expression;    Plasmodium falciparum;    Gametocyte;    Automatization;   

DOI  :  10.1186/s13071-022-05624-w

 received in 2022-05-09, accepted in 2022-12-17,  发布年份 2022

来源: Springer

【 摘 要 】

BackgroundPlasmodium falciparum (Pf) is the leading protozoan causing malaria, the most devastating parasitic disease. To ensure transmission, a small subset of Pf parasites differentiate into the sexual forms (gametocytes). Since the abundance of these essential parasitic forms is extremely low within the human host, little is currently known about the molecular regulation of their sexual differentiation, highlighting the need to develop tools to investigate Pf gene expression during this fundamental mechanism.MethodsWe developed a high-throughput quantitative Reverse-Transcription PCR (RT-qPCR) platform to robustly monitor Pf transcriptional patterns, in particular, systematically profiling the transcriptional pattern of a large panel of gametocyte-related genes (GRG). Initially, we evaluated the technical performance of the systematic RT-qPCR platform to ensure it complies with the accepted quality standards for: (i) RNA extraction, (ii) cDNA synthesis and (iii) evaluation of gene expression through RT-qPCR. We then used this approach to monitor alterations in gene expression of a panel of GRG upon treatment with gametocytogenesis regulators.ResultsWe thoroughly elucidated GRG expression profiles under treatment with the antimalarial drug dihydroartemisinin (DHA) or the metabolite choline over the course of a Pf blood cycle (48 h). We demonstrate that both significantly alter the expression pattern of PfAP2-G, the gametocytogenesis master regulator. However, they also markedly modify the developmental rate of the parasites and thus might bias the mRNA expression. Additionally, we screened the effect of the metabolites lactate and kynurenic acid, abundant in severe malaria, as potential regulators of gametocytogenesis.ConclusionsOur data demonstrate that the high-throughput RT-qPCR method enables studying the immediate transcriptional response initiating gametocytogenesis of the parasites from a very low volume of malaria-infected RBC samples. The obtained data expand the current knowledge of the initial alterations in mRNA profiles of GRG upon treatment with reported regulators. In addition, using this method emphasizes that asexual parasite stage composition is a crucial element that must be considered when interpreting changes in GRG expression by RT-qPCR, specifically when screening for novel compounds that could regulate Pf sexual differentiation.Graphical Abstract

【 授权许可】

CC BY   
© The Author(s) 2023

【 图 表 】

Fig. 1

41116_2022_35_Article_IEq110.gif

41116_2022_35_Article_IEq104.gif

41116_2022_35_Article_IEq102.gif

41116_2022_35_Article_IEq100.gif

Fig. 6

41116_2022_35_Article_IEq90.gif

Fig. 3

Fig. 1 

Fig. 5

40854_2022_443_Article_IEq20.gif

41116_2022_35_Article_IEq58.gif

41116_2022_35_Article_IEq53.gif

40249_2022_1049_Article_IEq1117.gif

40249_2022_1049_Article_IEq39.gif

40249_2022_1049_Article_IEq34.gif

Fig. 3

41116_2022_35_Article_IEq35.gif

41116_2022_35_Article_IEq13.gif

Fig. 1