The present study has demonstrated that sufficient amounts of RNA can be extracted from in vivo samples to support a whole-genome microarray analysis of P. falciparum gene expression. An excellent correlation between expression levels of parasite transcripts in in vivo samples and in in vitro 3D7 samples was found. More importantly, this analysis was sensitive enough to detect specific differences in expression in vivo and in vitro. This approach has demonstrated the overexpression of genes that encode surface proteins and has identified a putative novel gene family that encodes surface proteins that may play a role in in vivo parasite biological processes.
The analysis of genes differentially expressed in vivo and in vitro has identified virulence genes in bacterial systems [31
]. This approach was adopted to study the pathogenesis of malaria in vivo. The total number of parasite transcripts detected in a small blood sample approaches the number of transcripts reported to be expressed in in vitro cultivated 3D7 ring-stage parasites [15
]. For this first analysis of whole-genome in vivo steady-state mRNAs, the potential host effect on parasite selection and RNA expression was minimized through selection of samples from patients with similar demographics and disease presentation. A surprisingly high correlation between the transcriptomes in all the samples obtained from children and those in the in vitro 3D7 samples with ring-stage parasites was found by use of a rank-based correlation statistic. The high correlation between the in vivo samples and in vitro 3D7 samples with parasites in stages preceding the ring stage is notable. Although there are clearly defined morphologic differences between the schizont, merozoite, and ring stages, biological processes may be shared in contiguous stages. For example, developmental clustering between stages was noted for a set of 300 open-reading frames involved in host cell invasion in these stages [15
]. These data demonstrate the reproducibility of this method and validate the results.
The high correlation between the in vivo transcriptomes and the 3D7 ring-stage transcriptome, as well as the lack of significant difference in malarial metabolic pathways and most GO functions, is striking. This observation implies that the basic molecular processes of natural isolates and the 3D7 strain are highly conserved, consistent with the observation that the 3D7 strain can infect human volunteers and Anopheles
mosquitoes in experimental settings [34
The lack of overall diversity among the in vivo transcriptomes obtained from a homogenous cohort of patients suggests that distinct transcriptomes, when identified, could be informative. The transcriptome derived from the only adult patient had a lower correlation with the other in vivo samples and with 3D7 stage-specific transcriptomes. The presence of parasites in stages other than rings in the peripheral blood may account for some of these differences; however, the overexpressed gene list contains transcripts that are at least 2-fold more abundant than those for all 3D7 life-cycle stages, including the schizont stage. Similar to the other in vivo samples, there was no difference in genes that encode malarial metabolic pathways or GO functions, aside from the plasma membrane GO function. The presence of late forms, as detected by microscopy, has been shown to correlate with a more severe outcome, and it is intriguing that this was found in the sample with a distinct transcriptome [35
]. A larger study that enrolls patients with differences in age and disease severity will be necessary to interpret the significance of unique transcriptomes.
The specific overexpression of genes that encode surface proteins, hypothesized to be involved in immune evasion, is consistent with the biological processes of the in vivo environment, which is rich in immune cells and factors, compared with the in vitro environment. Previous work has demonstrated changes in expression of surface-expressed proteins in P. falciparum
when the parasite is in prolonged culture or under biologic or immune selection [37
]. Gene family members that are uniquely expressed in vivo may simply represent diversity of gene expression or be required for in vivo survival. Var genes were not found to be overexpressed in vivo. Because more-recent data examining geographically distinct strains (C. Kidgell, J. Borevitz, J. Johnson, S. Volkman, D. Plouffe, K. Le Roch, D. Wirth, Y. Zhou, and E. Winzeler, unpublished data) has found marked sequence polymorphism in the region from which the var probes used in our microarray analysis were derived, our detection methods may have resulted in an underestimation of the number of var transcripts present. Because parasite-encoded surface proteins interact with the host immune system, comprehensive analysis of genes in this functional class isolated from patients who demonstrate immunity, compared with those isolated from patients who do not demonstrate immunity, may provide insight into this critical aspect of the host-pathogen interaction.
A major goal of this work was to identify, for further analysis, genes that encode hypothetical proteins that may play a role in in vivo biological processes. One gene that encodes a hypothetical protein (PF14_0752) was identified as being overexpressed in 4 of the 5 in vivo samples. Further analysis revealed additional P. falciparum homologues that encode hypothetical proteins; these homologues were predominantly located at the telomeres, with the majority containing a predicted transmembrane domain, a vacuolar export/host targeting signal, and sequence polymorphism. Taken together, these characteristics suggest a new family of surface proteins, and analysis of these genes is under way to test this hypothesis.
Parasites residing in vivo are challenged with unique features that are not present under in vitro conditions. The in vivo environment contains immune factors, endothelial ligands, and variation in microenvironments secondary to sequestration in different organs. The molecular analysis of in vivo biological processes by means of this new approach will identify genes that are important for survival of the parasite in the human host and provide additional candidates for vaccine development, to lessen disease severity and provide immunity.