Temperature Shift and Host Cell Contact Up-Regulate Sporozoite Expression of Plasmodium falciparum Genes Involved in Hepatocyte Infection

Abstract

Plasmodium sporozoites are deposited in the skin by Anopheles mosquitoes. They then find their way to the liver, where they specifically invade hepatocytes in which they develop to yield merozoites infective to red blood cells. Relatively little is known of the molecular interactions during these initial obligatory phases of the infection. Recent data suggested that many of the inoculated sporozoites invade hepatocytes an hour or more after the infective bite. We hypothesised that this pre-invasive period in the mammalian host prepares sporozoites for successful hepatocyte infection. Therefore, the genes whose expression becomes modified prior to hepatocyte invasion would be those likely to code for proteins implicated in the subsequent events of invasion and development. We have used P. falciparum sporozoites and their natural host cells, primary human hepatocytes, in in vitro co-culture system as a model for the pre-invasive period. We first established that under co-culture conditions, sporozoites maintain infectivity for an hour or more, in contrast to a drastic loss in infectivity when hepatocytes were not included. Thus, a differential transcriptome of salivary gland sporozoites versus sporozoites co-cultured with hepatocytes was established using a pan-genomic P. falciparum microarray. The expression of 532 genes was found to have been up-regulated following co-culture. A fifth of these genes had no orthologues in the genomes of Plasmodium species used in rodent models of malaria. Quantitative RT-PCR analysis of a selection of 21 genes confirmed the reliability of the microarray data. Time-course analysis further indicated two patterns of up-regulation following sporozoite co-culture, one transient and the other sustained, suggesting roles in hepatocyte invasion and liver stage development, respectively. This was supported by functional studies of four hitherto uncharacterized proteins of which two were shown to be sporozoite surface proteins involved in hepatocyte invasion, while the other two were predominantly expressed during hepatic parasite development. The genome-wide up-regulation of expression observed supports the hypothesis that the shift from the mosquito to the mammalian host contributes to activate quiescent salivary gland sporozoites into a state of readiness for the hepatic stages. Functional studies on four of the up-regulated genes validated our approach as one means to determine the repertoire of proteins implicated during the early events of the Plasmodium infection, and in this case that of P. falciparum, the species responsible for the severest forms of malaria.

Translational Control in Plasmodium and Toxoplasma Parasites

“…For example, liver stage-associated proteins 1 and 2 (LSAP-1/PFL0065w and LSAP-2/PFB0105c, respectively) are among the most abundant transcripts in the salivary gland transcriptome (33) but have not been detected in proteomic surveys of sporozoites (38,39). Rather, these genes are translated only in liver stages (40). In contrast, the translation of the abundant proteins CSP and TRAP from the salivary gland sporozoites starts in oocyst sporozoites (41).…”

Disruption of Plasmodium Sporozoite Transmission by Depletion of Sporozoite Invasion-Associated Protein 1

“…Subsequently, the Plasmodium yoelii ortholog was isolated in a suppression subtractive hybridization screen for genes that are specifically upregulated in sporozoites compared to blood stages (6). More recently, the P. falciparum ortholog was identified as a gene that is transiently upregulated in activated P. falciparum sporozoites (14).In this study, we fluorescently tagged the Plasmodium berghei SIAP-1 gene (PbSIAP-1) and show enrichment of the protein in the apical pole of the sporozoite. Targeted gene deletion resulted in viable blood stage parasites that displayed a specific and complete block in sporozoite transmission to the vertebrate host.…”

Epigenetics in Plasmodium: What Do We Really Know?

“…Transcriptional profiling has demonstrated distinct profiles in all of these stages: the gametocyte (103), ookinete (90), oocyst sporozoite (59), salivary gland sporozoite (73), hepatocyte stage (95,105), and erythrocyte stage (62). Additional shifts in gene expression probably occur as the sporozoite migrates through the skin and invades a hepatocyte (102), after which replication occurs many hundredfold, giving rise to unique metabolic requirements. Likewise, many genes must be up-or downregulated at the abrupt transition to the insect gut with its distinct temperature and metabolic profile.…”