The online version of this article (doi:10.1186/1475-2875-11-265) contains supplementary material, which is available to authorized users.
The authors declare that they have no competing interests.
ACO conceived, designed and performed the experiments and wrote the paper. JMB conceived and designed experiments, analyzed the data, and wrote the paper. ABV analyzed the data. LWB analyzed the data and contributed reagents/materials. TMD contributed reagents/materials. All authors read and approved the final manuscript.
Microarray studies using in vitro cultures of synchronized, blood-stage Plasmodium falciparum malaria parasites have revealed a ‘just-in-time’ cascade of gene expression with some indication that these transcriptional patterns remain stable even in the presence of external stressors. However, direct analysis of transcription in P. falciparum blood-stage parasites obtained from the blood of infected patients suggests that parasite gene expression may be modulated by factors present in the in vivo environment of the host. The aim of this study was to examine changes in gene expression of the rodent malaria parasite, Plasmodium yoelii 17X, while varying the in vivo setting of replication.
Using P. yoelii 17X parasites replicating in vivo, differential gene expression in parasites isolated from individual mice, from independent infections, during ascending, peak and descending parasitaemia and in the presence and absence of host antibody responses was examined using P. yoelii DNA microarrays. A genome-wide analysis to identify coordinated changes in groups of genes associated with specific biological pathways was a primary focus, although an analysis of the expression patterns of two multi-gene families in P. yoelii, the yir and pyst-a families, was also completed.
Across experimental conditions, transcription was surprisingly stable with little evidence for distinct transcriptional states or for consistent changes in specific pathways. Differential gene expression was greatest when comparing differences due to parasite load and/or host cell availability. However, the number of differentially expressed genes was generally low. Of genes that were differentially expressed, many involved biologically diverse pathways. There was little to no differential expression of members of the yir and pyst-a multigene families that encode polymorphic proteins associated with the membrane of infected erythrocytes. However, a relatively large number of these genes were expressed during blood-stage infection regardless of experimental condition.
Taken together, these results indicate that 1) P. yoelii gene expression remains stable in the presence of a changing host environment, and 2) concurrent expression of a large number of the polymorphic yir and pyst-a genes, rather than differential expression in response to specific host factors, may in itself limit the effectiveness of host immune responses.
Additional file 1: yir and pyst-a oligonucleotides on Plasmodium yoelii DNA microarrays that mapped to a single gene. In assessing the number of yir and pyst-a genes expressed in a given sample, only data obtained with yir and pyst-a oligos that mapped to a single gene were considered. Mapping data available on PlasmoDB[ 3] was used to focus the analysis on 464/859 yir oligos (top portion of Table) and 74/140 pyst-a oligos (bottom portion of Table) on P. yoelii DNA microarrays. (XLSX 13 KB)
Additional file 2: Differential gene expression between Plasmodium yoelii 17X parasites isolated from individual mice. Three animals (M1, M2 and M3) were infected with P. yoelii 17X iRBCs from a single donor mouse and gene expression analysed using P. yoelii DNA microarrays. Of three possible pair-wise comparisons, significant differential gene expression was only seen between M2 and M3 (black text) and M1 versus M3 (blue text). For each differentially expressed gene, oligo ID and gene name are listed[ 3]. Log 2 Fold Changes and adjusted p -values are also included, as are the predicted number of amino acids (AA) and molecular weight in Daltons (MW). Where available, P. falciparum orthologs and associated Plasmodb.org accession numbers are included (Pf Ortholog # and Pf Name). Functional groupings (Category) are listed at the far right of the Table. For detailed information regarding data analysis and gene categorization, please see the Methods. (XLSX 12 KB)
Additional file 3: Comparative expression of Plasmodium yoelii genes encoding mitochondria associated proteins. Data on the differential expression of genes encoding mitochondria associated proteins is shown for all comparisons. The gene set included P. yoelii orthologs of putative P. falciparum mitochondrial proteins[ 7, 41] Mather and Vaidya, unpublished data] for which signal intensity across arrays was consistently above the 50 th percentile. Shown are the P. falciparum Gene ID and Name, P. yoelii Oligo ID and Name and for each comparison, the corresponding the log 2 fold change and adjusted p -value. Log 2 fold change values >1 are shaded in red, log 2 fold change values < −1 are shaded in green and adjusted p -values <0.01 are shaded in yellow. (XLSX 104 KB)
Additional file 4: Differential gene expression between parasites isolated from a single donor mouse and three individual animals. Three animals (M1, M2 and M3) were infected with P. yoelii 17X iRBCs from a single donor mouse (D) and gene expression analysed using P. yoelii DNA microarrays. Three pair-wise comparisons were possible: D versus M1 (black text), D versus M2 (blue text) and D versus M3 (red text). For differentially expressed genes, oligo ID, gene name, log2 fold change, adjusted p -value, predicted number of amino acids, predicted MW, available P. falciparum ortholog information and functional categories are provided as described for Additional file 2. (XLSX 27 KB)
Additional file 5: Differential gene expression between Plasmodium yoelii 17X parasites isolated from independent infections. Groups of animals were infected on four separate occasions (I1, I2, I3 and I4) using four separate donor animals and gene expression analysed using P. yoelii DNA microarrays. Six possible pair-wise comparisons were considered: I1 versus I2 (black text), I1 versus I3 (blue text), I1 versus I4 (red text), I2 versus I3 (green text), I2 versus I4 (purple text) and I3 versus I4 (blue text). For differentially expressed genes, oligo ID, gene name, log2 fold change, adjusted p -value, predicted number of amino acids, predicted MW, available P. falciparum ortholog information and functional categories are provided as described for Additional file 2. (XLSX 32 KB)
Additional file 6: Differential gene expression on days 10/14, 10/18 and in the absence/presence of host antibody responses. Groups of wild-type animals were infected with P. yoelii 17X iRBCs and parasite RNA was isolated on day 10 (D10), day 14 (D14) or day 18 (D18) post-infection. Immunologically intact (WT) and B-cell deficient JHD (JHD) animals were similarly infected and parasite RNA was isolated on day 10. A third set of animals was immunized three times with a preparation of P. yoelii reticulocyte membrane proteins plus Quil A as adjuvant (RMP) or with Quil A alone (QA) prior to P. yoelii 17X challenge. Parasite RNA was then isolated on day 10 (QA) or day 12 (RMP) post-infection. Gene expression was analysed using P. yoelii DNA microarrays and the following comparisons were made: D10 versus D14 (black text), D10 versus D18 (blue text), WT versus JHD (red text), and QA versus RMP (green text). For differentially expressed genes, oligo ID, name, log2 fold change, adjusted p -value, predicted number of amino acids, predicted MW, available P. falciparum ortholog information and functional categories are provided as described for Additional file 2. (XLSX 46 KB)
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- Remarkable stability in patterns of blood-stage gene expression during episodes of non-lethal Plasmodium yoelii malaria
Thomas M Daly
Akhil B Vaidya
Lawrence W Bergman
James M Burns Jr
- BioMed Central
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