Transcription in P. falciparum and P. vivax parasites appears to be tightly regulated, resulting in a ‘continuous cascade’ of gene expression during blood-stage development
[4, 5, 45]. There is some evidence that this transcriptional programme continues unabated even in the presence of external stresses. In one study, anti-folate treatment of cultures of drug-sensitive P. falciparum blood-stage parasites did not lead to increased expression of the defined drug target, dihydrofolate reductase-thymidylate synthatase. Additionally, there were no genes differentially regulated greater than two-fold between control and drug treated cultures
. Similarly, Gunasekera et al. found little variation in transcription patterns between untreated and chloroquine-treated P . falciparum parasites growing asynchronously in culture. These results were unexpected, as genome-wide studies in organisms such as Saccharomyces cerevisiae, Mycobacterium tuberculosis and Candida albicans revealed differential expression of hundreds of genes in response to drug treatment, and these changes often confirmed or revealed associated drug targets and affected pathways
[48–51]. However, other studies in Plasmodium examining the effects of drug treatment or manipulation of culture conditions have found more substantial transcriptional changes (~300-400 differentially expressed genes), especially in genes predicted to encode proteins exported to the host cell cytoplasm and the RBC membrane
[52, 53]. The work shown here extends these efforts significantly by focusing on malaria parasites replicating in vivo, in a more complex setting. With P. yoelii 17X blood-stage parasites, global gene expression profiles were remarkably consistent even in the presence of a changing host environment.
Initially, three genetically identical, age-matched BALB/cByJ mice were infected with the same inoculum of P. yoelii 17X iRBCs obtained from a donor mouse. P. yoelii RNA was isolated from each animal after a 10–11 day period of replication in vivo. As the in vivo environment is expected to be comparable in this setting, only a small set of differentially expressed genes was anticipated. Parasite gene expression patterns in each of the three animals were virtually identical with only five to six differentially expressed genes. Between the initiation of infection and harvest of iRBCs for RNA isolation, P. yoelii parasites in this study completed 10–12 replication cycles in vivo. Over this time, the number of differentially expressed genes was expected to increase somewhat relative to the population of P. yoelii iRBCs used to initiate the infection. This was the case, but the increase was relatively small involving only 40–60 genes out of the set represented by ~ 6,700 oligonucleotides on the arrays. A small cluster of genes expressed in late-stage parasites and potentially involved in merozoite invasion of host cells were differentially expressed between the donor mouse and recipients. This may not be surprising as iRBCs were harvested early during ascending parasitaemia when P. yoelii 17X parasites can be found in varying degrees in both normocytes and reticulocytes. This change in gene expression may reflect utilization of multiple invasion pathways. Finally, a number of potentially influencing variables were further increased by comparing gene expression profiles of P. yoelii 17X blood-stage parasites harvested from four independent infections initiated with iRBCs obtained from separate donor mice. Remarkably, the number of differentially expressed genes in pair-wise comparisons remained low (10–50 genes). These represent genes that are involved in diverse or unknown functions and at present, the associated changes do not appear to reflect a biologically significant response to the host environment. This, combined with a false discovery rate of 1% associated with this analysis suggest that detection of a low number of differentially expressed but largely unrelated genes could simply represent ‘noise’ inherent to large microarray analysis.
Non-lethal P. yoelii 17X parasites replicate preferentially in reticulocytes but do invade and develop within mature RBCs when reticulocytes are limiting. Parasites harvested on day 10 of P. yoelii 17X infection will be present in both reticulocytes and normocytes. As a result of an influx of new reticulocytes into circulation, P. yoelii 17X parasites will be found almost exclusively in reticulocytes by day 14 post-infection. As such, substantial differences in patterns of P. yoelii 17X gene expression in parasites harvested on day 10 versus day 14 were expected. In addition to the shift in host cell tropism, the ongoing infection increases parasite burden, host stress and immune pressure that could also influence P. yoelii 17X expression profiles. While the number of differentially expressed genes in this comparison increased to 95, these were not clustered based on related function or biological process and the majority were of unknown function. In an early study, utilizing lethal P. yoelii 17XL parasites, a similar number of genes altered in association with a shift in host cell preference from normocytes to reticulocytes were identified
. The function of these genes will be revisited as the annotation of both P. falciparum and P. yoelii genomes progresses. To further examine the role of host cell availability and immune pressure, gene expression in parasites isolated on day 10 and day 18 of infection was compared. Parasitaemia at these two time points is comparable. However on day 18, 70-75% of RBCs in circulation are reticulocytes and P. yoelii iRBCs are rapidly being cleared from circulation by immune-mediated mechanisms. Nearly 200 genes were differentially expressed in P. yoelii iRBCs on day 18 relative to day 10 of infection; the largest set in this study. On day 18, several genes encoding components of the cytoplasmic translation machinery were down-regulated while a second set associated with merozoite invasion were up-regulated. In P. falciparum, transcription of genes encoding the cytoplasmic translation machinery generally peaks in ring-stage parasites at ~12 hours post-invasion, followed by a marked down-regulation in trophozoite- and schizont-stage parasites. In contrast, transcription of genes involved in schizont rupture and merozoite invasion is relatively low through most of the asexual cycle, peaking at ~42 hours post-invasion
[4, 5]. Differences noted in gene expression in P. yoelii parasites on day 18 versus day 10 of infection may partially be explained by a modest shift noted in distribution of parasite stages in circulation toward increased schizonts and decreased trophozoites. It is also clear that infected blood isolated on day 18 contains damaged host cells and dying parasites, as well as free extracellular parasites. These factors likely contributed to a mixed alteration in expression profiles at this late time point during infection.
To focus on the role of the host immune response on parasite gene expression, two approaches to actively alter host immune pressure were taken. In the first approach, all antibody mediated immune pressure was removed by infecting B cell deficient JHD mice. It was expected that in the absence of antibodies, P. yoelii growth would be unchecked with an increase in the diversity of parasite populations and gene expression profiles. This did not occur as only seven genes were differentially expressed when P. yoelii parasites isolated from B cell deficient JHD mice were compared with those from immunocompetent control mice. In the second approach, immune pressure on P. yoelii 17X parasites was increased by immunizing mice with a preparation of parasite proteins associated with the membrane of infected reticulocytes. Here, immunization-induced antibodies were expected to select for the growth of parasites with altered gene expression patterns that included changes in members of the yir and pyst-a multigene families that encode proteins localized to the erythrocyte membrane. Unexpectedly, this did not occur as only 10 genes were differentially expressed. Even in these situations where the host immune response was experimentally manipulated, P. yoelii 17X gene expression proceeded unaltered. This is very reminiscent of the ‘hard-wired’ programme of gene expression observed with in vitro cultures of P. falciparum[4, 5, 46].
In addition to addressing global changes in gene expression, the expression of members of two multi-gene families in P. yoelii, the yir and pyst-a were of particular interest. Previous studies have shown that at a population level, many yir genes are transcribed during blood-stage infection, in a seemingly random order. Transcription at the level of individual parasites appears to be tightly controlled, with each parasite transcribing between one and three yir genes
. In the present study, significant differences in the expression of yir family members across experimental conditions were not observed, including the comparison of P. yoelii 17X day 10 and day 18 parasites. This is in agreement with the study by Cunningham et al. examining the expression of a subset of the yir repertoire on days 12 and 18 post-infection
. Of note, expression of a fairly large number of the yir genes (89–155) was consistently observed using signal intensity greater than the 50th percentile on each array as a benchmark. These expressed yir genes were distributed across five previously reported phylogenetic groups, with no obvious bias toward any one group
The expression of pyst-a genes followed a similar pattern to the yir family, with little to no differential expression between conditions. It appears that a large proportion of pyst-a family members are expressed in a mixed blood-stage infection. Whether pyst-a gene expression is more restricted in individual parasites has not been determined. No yir or pyst-a members were differentially expressed in wild-type versus JHD animals, in agreement with a study examining expression of a subset of yir members in wild-type versus Rag2 knockout mice, which lack mature B and T cells
. A more extensive analysis of yir and pyst-a expression during primary and secondary infections using a microarray approach would be of interest. It has been suggested that the polymorphic erythrocyte membrane antigens encoded by the yir and/or pyst-a multigene families may function as part of a parasite immune evasion strategy. For immune evasion, the current data suggest that changing the pattern of gene expression may be less important than showering the host immune system with a large repertoire of polymorphic antigens at any given time. In fact, prior immunization of mice with the Py RMP preparation may have further impeded the development of protective immune responses following challenge infection as these animals developed a persistent infection which was difficult to clear. In agreement with the current findings, members of the cir multi-gene family do not seem to be differentially expressed during the course of blood-stage P. chabaudi infection, but there is some indication that cir gene expression varies in parasites localized to different host tissues
. However, it is still possible that much like the P. falciparum EMP1 and the var gene family, the yir and/or pyst-a encoded proteins may possess specific functions required for parasite growth and development in vivo.
Overall, in vivo, P. yoelii 17X gene expression did not appear to be appreciably influenced by the host environment. These data in the P. yoelii 17X model differ from that reported by Daily et al. who detected distinct patterns of gene expression in P. falciparum parasites isolated from malaria-infected patients. In light of these data, changes in the expression of genes encoding a set of mitochondrial proteins (n = 148), across all experimental conditions was examined. Significant changes that would be consistent with distinct physiological states or a response to environmental stress were not observed (Additional file
3). Although parasite gene expression was assessed in vivo, the P. yoelii model is still less complex than with P. falciparum infected human subjects. Here, a single cloned line of P. yoelii 17X that does not produce gametocytes was utilized to simultaneously infect genetically identical mice housed in environmentally controlled, specific pathogen free conditions. In some regards, this lack of gametocyte-stage parasites is advantageous, as detection of differential gene expression due to varying gametocyte levels across samples can be ruled out. On the other hand, the repeated passage of this P. yoelii 17X line in the vertebrate host and/or the lack of exposure to the mosquito vector could have altered regulatory mechanisms that control expression of certain gene subsets (i.e. yir or pyst-a genes). It is also possible that the greater diversity in human hosts (genetic, environmental) may have a greater influence on parasite gene expression than we observed in the P. yoelii model. Finally, each isolate of P. falciparum may exhibit unique elements of a ‘hard-wired’ programme of gene expression that can be detected when comparing isolates obtained from individual malaria patients. Co-infection of human subjects with such distinct P. falciparum clones will increase diversity in the overall P. falciparum gene expression profile detected in a single host and may allow preferential growth of P. falciparum clones in specific in vivo environments.