Molecular Markers of Radiation Induced Attenuation in Intrahepatic Plasmodium falciparum Parasites

Abstract

Experimental immunization with radiation attenuated sporozoites (RAS) and genetically attenuated sporozoites has proved to be a promising approach for malaria vaccine development. However, parasite biomarkers of growth attenuation and enhanced immune protection in response to radiation remain poorly understood. Here, we report on the effect of an attenuating dose of γ-irradiation (15 krad) on the Plasmodium falciparum sporozoite (PfSPZ) ultrastructure by electron microscopy, growth rate of liver stage P. falciparum in liver cell cultures, and genome-wide transcriptional profile of liver stage parasites by microarray. We find that γ-irradiation treated PfSPZ retained a normal cellular structure except that they were vacuous with a partially disrupted plasma membrane and inner membrane complex. A similar infection rate was observed by γ-irradiation-treated and untreated PfSPZ in human HCO-4 liver cells (0.47% versus 0.49%, respectively) on day 3 post-infection. In the microarray studies, cumulatively, 180 liver stage parasite genes were significantly transcriptionally altered on day 3 and/or 6 post-infection. Among the transcriptionally altered biomarkers, we identified a signature of seven candidate parasite genes that associated with functionally diverse pathways that may regulate radiation induced cell cycle arrest of the parasite within the hepatocyte. A repertoire of 14 genes associated with protein translation is transcriptionally overexpressed within the parasite by radiation. Additionally, 37 genes encode proteins expressed on the cell surface or exported into the host cell, 4 encode membrane associated transporters, and 10 encode proteins related to misfolding and stress-related protein processing. These results have significantly increased the repertoire of novel targets for 1) biomarkers of safety to define proper attenuation, 2) generating genetically attenuated parasite vaccine candidates, and 3) subunit candidate vaccines against liver stage malaria.

TCRβ-expressing macrophages induced by a pathogenic murine malaria correlate with parasite burden and enhanced phagocytic activity

“…To measure the effect of TCRβ expression on the macrophage transcriptome, microarray was performed in quadruplicate on RNA isolated from TCRβ + versus TCRβ − CD3ε − Ly6G − CD11b high F4/80 + CD3ε − CD4 − CD8 − macrophages purified by fluorescence-activated cell sorting of splenocytes pooled from day 3 Pb−A infected C57BL/6 mice using a FACSAria™ Fusion cell sorter (BD Biosciences, San Jose, CA). RNA isolation, amplification, and incorporation of cy3 or cy5 dyes was performed as previously described [ 33 ]. Briefly, for preparation of RNA, samples snap frozen in TRIzol ® were thawed and RNA was extracted with chloroform, precipitated with isopropanol, washed with 75% ethanol, and resuspended in water.…”

Ubiquitin-Like Modifiers: Emerging Regulators of Protozoan Parasites

“…It was demonstrated that Pf SENP1 removes SUMO moieties from modified RanGAP1 and processes SUMO precursors in vitro [ 55 ]. Upregulation of this Ulp was observed during the parasite’s intrahepatic stage following disruption by γ-irradiation of P. falciparum sporozoites, further supporting a role of SUMOylation in stress response [ 68 ]. Highlighting this enzyme’s potential as a drug target, the substrate sequence specificity of Pf SENP1 was shown to differ significantly enough from that of human SENPs, that a class of aza-epoxide small molecule inhibitors were able to specifically block replication of P. falciparum in erythrocytes, with parasite clearance and potency of Pf SENP1 inhibition showing a direct correlation [ 55 ].…”

Transcriptome analysis based detection of Plasmodium falciparum development in Anopheles stephensi mosquitoes

“…Microarray studies were performed in quadruplicate comparing RNA isolated from uninfected (day 0) versus P. falciparum infected (days 2, 4, 6, and 8) mosquito midguts to identify candidate genes that could be used to detect malaria transmission by mosquitoes. Amplification of RNA was performed to generate a sufficient quantity for microarray as previously described 22 . Briefly, the Low Input QuickAmp Labeling Kit (Agilent Technologies, Santa Clara, CA) was used for RNA amplification and incorporation of label.…”