Transcriptomic analyses have not been as aggressively pursued in kinetoplastid organisms as they have been in other protozoan parasites, such as Plasmodium
. This is likely due to the view that the predominantly post-transcriptional nature of gene expression regulation in kinetoplastids makes microarray studies in these organisms generally less informative than microarray studies in organisms for which transcription initiation plays a larger role in gene expression regulation. In support of this, the ranges of observed mRNA ratios between life-cycle stages in kinetoplastid organisms have been relatively narrow compared to the ranges observed in organisms with canonical RNA pol II promoters. For example, 1,000-fold induction in relative transcript abundance for CD70 was observed in a microarray study comparing Human T-lymphotropic virus type 1 (HTLV-1) carrying T-cell lines versus HTLV-1-negative T-cell lines [41
], whereas in kinetoplastid microarray studies, fold inductions above ~8-fold are rarely observed [3
Further contributing to the debate over the utility of microarray studies in kinetoplastids has been the general observation that relatively few genes have exhibited significant stage regulation of mRNA relative abundances in previous microarray studies. For instance, Diehl et al., using arrays of 21,024 PCR-amplified genome shot-gun library inserts from T. brucei
to compare relative transcript abundances between in vitro
cultured human, long slender forms and procyclic parasites, observed that, although 75% of the array elements detected transcripts, only 2% displayed significant differences between the two life-cycle stages [39
]. The observation that only 2% of the detected transcripts differed significantly in relative abundance between two very different life-cycle stages supported the concept that transcript abundances in T. brucei
are largely constitutive, especially considering the two life-cycle stages were compared directly on the microarrays. This picture of predominantly constitutive genome expression at the level of transcript abundance in kinetoplastids was reinforced manifold by subsequent studies in T. brucei
] and Leishmania spp
. (reviewed in [41
In light of the findings from T. brucei
and Leishmania spp
. microarray studies as well as findings from our previous, limited microarray study in T. cruzi
], we sought to determine if transcript levels are globally variable between life-cycle stages of T. cruzi
using whole-genome oligonucleotide microarrays. When cDNAs from each life-cycle stage were co-hybridized with a reference cDNA sample comprised of all four life-cycle stages on oligonucleotide, whole genome microarrays, we observed that the relative transcript abundances for over 50% of the genes detected on the arrays were significantly regulated between the life-cycle stages.
The microarray data for selected genes were validated by qRT-PCR. There was generally good agreement between the two platforms in terms of direction of regulation, however there were some quantitative differences, especially in terms of the extent of up or downregulation estimated by these two techniques. These quantitative differences likely were the result of sequence-specific effects and differences in dynamic range for the two platforms [49
]. In addition, the RNA samples used for qRT-PCR were not the identical samples used for the microarrays.
Recent improvements in microarray design, such as the use of oligonucleotide probes designed for more uniform hybridization kinetics and with lower likelihood of cross-hybridization than amplicon-derived probes and the use of microarrays with greater genome coverage could in part account for why our study identified a higher percentage of significantly stage-regulated genes than previous microarray studies in other kinetoplastids [1
]. However, even a recent study by Rochette et al. that used Leishmania
whole genome oligonucleotide microarrays to directly compare amastigotes and procyclics reported that only 7% of the genes in Leishmania infantum
and 9.3% of the genes in L. major
were developmentally regulated [43
]. It is also possible that the use of different statistical methods for analysis of microarray data my yield widely different estimates of the frequency of regulated genes. However, using less rigorous statistical criteria for determining significance, such as fold-change, results in higher false discovery rates [29
] and thus would be predicted to overestimate the number of significantly regulated genes. Our reanalysis of the Papadopoulou data from L. infantum
using our statistical methods found 715 genes (7% of the genes fulfilling our criteria for detection) to be significantly regulated between amastigotes and promastigotes with an FDR of 1.55% (an FDR nearly 4 times greater than what we report for T. cruzi
). Thus, different statistical analysis methods cannot account for the differential estimates of the frequency of regulated genes in Leishmania
and T. cruzi
By incorporating all four life cycle stages of T. cruzi
in our analysis, in contrast to the 2 stage comparisons done with other kinetoplastids (e.g. procyclic to amastigote [43
] or procyclic to bloodstream forms [39
]) we have increased the possibility of identifying a greater number of genes regulated in expression in at least one of the 4 life cycle stages. However, the contribution of increased number of stages studied to the overall number of significantly regulated genes was greatly mitigated by the fact that we used a reference design in which each life-cycle stage was compared to a mixture of all four life-cycle stages. This design minimized the number of microarrays required to compare the four life-cycle stages (each stage vs. reference = 24 hybridizations in the present study, each stage vs. each other stage = 36 hybridizations for equivalent replication), and also simplified the analysis, but had the disadvantage of buffering the ratios observed. By contrast, previous microarray studies in the kinetoplastids involved directly comparing one life-cycle stage with another on the microarrays, an experimental design which should maximize the number of significant ratios. Thus our estimate that >50% of genes in T. cruzi
have significantly stage-regulated transcript levels is much more likely to be an underestimate than an overestimate.
The number of significantly stage-regulated transcripts reported here for T. cruzi
is also likely to be underestimated because >2,000 of the oligonucleotide probes detected transcripts in two or fewer stages and were removed from further analysis for statistical reasons (significance determination for such genes would have been dubious, because SAM analysis of four groups (life-cycle stages) was unreliable for genes present in less than three groups). Also, almost certainly some of the genes that we reported as non-significantly regulated are significantly regulated in transitional stages of the life-cycle. For instance, Saxena et al., using microarrays of ~8300 PCR-amplified genome survey sequencing (GSS) clone inserts, identified 344 protein coding genes significantly regulated during axenic promastigote-to-amastigote differentiation in Leishmania donovani
]. Of these, 136 genes, nearly 40% of the total number of differentially regulated genes identified in the study, displayed transient up or downregulation that would have been missed by only comparing fully differentiated parasites. Thus our analysis of only fully differentiated stages likely underestimated the percentage of genes we found significantly regulated at the transcript level during the T. cruzi
life cycle, suggesting that time course studies looking at transitional stages in T. cruzi
would be fruitful.
The disparity in the degree of transcript abundance regulation between T. cruzi
and the other sequenced kinetoplastids suggests there may be differences in the number and kind of RNA binding proteins in their genomes, because almost all regulation of mRNA abundance in these organisms is at the level of mRNA stability. However, the genomes of T. cruzi
, T. brucei
, and L. major
all have remarkably similar complements of RNA binding proteins (reviewed in [44
]). Of the 77 proteins with RNA Recognition Motifs (RRMs) in T. cruzi
, T. brucei
, and L. major
, only two were unique to T. cruzi
, RBP4 (Tc00.1047053508901.20) and DRBD8 (Tc00.1047053503709.10, Tc00.1047053509581.50), both of which were upregulated in metacyclic trypomastigotes.
Present knowledge of the kinetoplastids does not elicit an obvious biological explanation for why gene expression regulation in T. cruz
i might be so different from its closest relatives. Perhaps the most striking difference between T. cruzi
and T. brucei
and L. major
is that only T. cruzi
has both intracellular and extracellular life-cycle stages in the mammalian host. Amastigotes must replicate intracellularly and, although sequestered from the actions of antibodies and complement, are subjected to attack by CD8+ cytotoxic T lymphocytes (CTL) [45
] and to the effects of host cytokines [46
] and antimicrobials, including reactive oxygen species and nitric oxide [47
]. Trypomastigotes are extracellular and thus must evade host defenses including complement, antibodies, and phagocytes. An enhanced capacity for regulating transcript abundances may have provided T. cruzi
with the agility necessary to rapidly shift between such different environments.
The finding that T. cruzi
divergently regulates mRNA relative abundances for members of paralog clusters was unexpected, given the post-transcriptional mode of gene expression regulation in this parasite. Divergent expression of duplicate genes is believed to occur by the accumulation of mutations in non-coding cis
regulatory sequences, such as promoters [48
]. Since kinetoplastids appear to be largely devoid of canonical promoters, it will be interesting to study the 3' untranslated regions of paralogs with divergent expression patterns to identify the sequences responsible for the differential mRNA abundances.
The differentially regulated T. cruzi duplicate genes identified here will be useful to study the retention and neofunctionalization of duplicate genes in an ancient eukaryote lacking transcriptional control of gene expression. In such an environment, the contributions of mRNA stability control and sub-cellular localization to the evolution of new genes can be studied in isolation from the effects of transcriptional control. Gene knockout studies are currently underway to determine if the array-identified, divergently-expressed paralogs, such as the trypomastigote upregulated 60S ribosomal L18 protein, have novel functions.