Transcripts of developmentally regulated Plasmodium falciparum genes quantified by real-time RT-PCR

Abstract

Plasmodium falciparum intraerythrocytic development is a complex process. Development proceeds rapidly from the trophozoite phase of nutrient acquisition and growth through to the synthetic and reproductive schizont phase, which ends with production of new invasive merozoites. During this process, the malaria parasite must express a series of different gene products, depending on its metabolic and synthetic needs. We are particularly interested in the development of the merozoite's organelles in the apical complex, which form during the later schizont stages. We have used quantitative real-time RT-PCR fluorogenic 5' nuclease assays (TaqMan) for the first time on malaria parasites for analysis of erythrocytic stage-specific gene expression. We analyzed transcripts of the P.falciparum eba-175 and other erythrocyte binding-like (ebl) family genes in temperature-synchronized parasites and found ebl genes have tightly controlled, stage-specific transcription. As expected, eba-175 transcripts were abundant only at the end of schizont development in a pattern most common among ebl, including baebl, pebl and jesebl. The maebl transcript pattern was unique, peaking at mid-late trophozoite stage, but absent in late-stage schizonts. ebl-1 demonstrated another pattern of expression, which peaked during mid-schizont stage and then significantly diminished in late-stage schizonts. Our analysis demonstrates that using real-time RT-PCR fluorogenic 5' nuclease assays is a sensitive, quantitative method for analysis of Plasmodium transcripts.

Figure 1

Schematic representations defining the multi-exon gene structure for P.falciparum ebl. Boxed regions denote exons and horizontal lines signify flanking untranslated regions and introns. Regions of similarity are designated and/or shaded similarly: putative signal sequence (S), dbp and eba-175 DBL ligand domains (fading gray), maebl M1/M2 domains and ama-1 subdomains 1 and 2 (black), c-cys domain (light gray), 3′ exons encoding putative transmembrane domain and two cytoplasmic tail domains, respectively (dark gray).

Figure 2

Southern blot hybridization confirms the ebl are single-copy genes. Plasmodium falciparum gDNA (2 µg) was digested with either EcoRI (E) or NsiI (N) restriction enzymes, separated by standard electrophoresis and blotted. Each blot was probed with an ebl gene-specific probe (see Materials and Methods). Left, P.falciparum gDNA derived from clones Dd2 and 3D7 on independent blots probed for baebl, jesebl, pebl and ebl-1. Right, P.falciparum HB3 and Dd2 gDNA on a single blot probed first for maebl, stripped, and probed for eba-175. Similar hybridization results were found for maebl and eba-175 using 3D7 gDNA. DNA standard markers are listed in the margins adjacent to each panel.

Figure 3

Preliminary TaqMan® 5′ nuclease assays determine optimal ebl gene-specific primer concentrations. ebl forward and reverse TaqMan® primers were tested in real-time 5′ exonuclease reactions at three concentrations (50, 300 and 900 nM) in all combinations with a constant probe concentration (100 nM). The plots relate PCR cycle number to change of detected fluorescence with background removed (ΔR_n) on a linear (A) and logarithmic scale (B). Three isolated groups based on total ΔR_n resulted: (1) reactions containing 300 or 900 nM of each primer (green), (2) reactions containing 300 or 900 nM of one primer and 50 nM of the other (blue), and (3) reactions containing 50 nM of each primer (red). Therefore, 300 nM of each primer was the optimal concentration achieving highest fluroscence (ΔR_n) and lowest threshold cycle (C_t) while being the lowest concentration in group 1. Amplification plots show the results for varying eba-175 primer set concentrations only. The remaining ebl genes demonstrated similar results. Images were taken from data analysis provided by the Sequence Detection Software (version 1.6.3 or 1.7) for the ABI Prism 7700. The optimization strategy was followed from manufacturer’s recommendations (PE Biosystems).

Figure 4

Validation of TaqMan® real-time RT–PCR for quantitative detection of P.falciparum ebl. (A) Amplification plot of P.falciparum 3D7 gDNA with eba-175 and maebl TaqMan® primer/probe sets. maebl (yellow) and eba-175 (blue)-specific primer probes sets were used in the 5′ nuclease TaqMan® assay with varying gDNA concentrations (10-fold dilutions from 6 µg to 600 pg/reaction). The darkened horizontal bar represents the chosen threshold cycle (C_t) during the linear phase of the PCR. PCRs with higher initial gDNA concentrations cross the C_t at an earlier cycle number with subsequent dilutions crossing the C_t in later cycles. Due to the stringent and similar design for TaqMan® primer and probe sets, maebl and eba-175-specific products produced near exact amplification plots. These results verified an optimized PCR, achieving the theoretical gap of ~3.33 cycles per 10-fold dilution of gDNA. The primer/probe sets for baebl, ebl-1, jesebl and pebl showed similar and reproducible results. These and similar plots validated the use of each ebl primer and probe set as well as the use of gDNA to construct a standard curve. (B) A representative standard curve constructed from the eba-175 profile given in (A) with its replicate. The logarithmic plot relates starting DNA concentration (ng) versus C_t for the gDNA dilutions. Replicate values almost perfectly overlay each other (circles). A similar curve was created for each of the ebls for every TaqMan® experiment. (C) The lsa-1 (violet) and replicate (orange) show similar amplification profiles using four dilutions of gDNA. (D) As in (A), five dilutions (10-fold dilutions from 6 µg to 600 pg/reaction) of genomic DNA validate real-time quantitative RT–PCR using TaqMan® chemistry for the 18S rRNA control primer and probe set using the VIC reporter dye. The replicates shown (red and blue) almost perfectly overlay with one another indicating reproducibility. These images were taken from data analysis provided by the Sequence Detection Software (version 1.6.3 or 1.7) for the ABI Prism 7700.

Figure 5

The timings for temperature-cycling synchronization of clone 3D7 P.falciparum malaria parasites. The approximate times are indicated for ring-form parasites (R), trophozoites (T) and schizonts (S). Invasion occurs during the overlap between S and R. During the time at 17°C, the ring form parasites have minimal development (they are in suspended animation), and during the time at 39.8°C, the trophozoites are prevented from maturing into schizonts. Before sampling for transcription analysis, the cultures were moved to a constant temperature 37°C incubator for uninterrupted development, during which the total cycle time was ∼40 h. Sampling at seven time points at 6 h intervals covered development from early rings to late schizonts at 37°C. Synchronization of parasites was ∼90% pure as determined from examining Giemsa-stained blood smears. A detailed description of the cycling conditions is given in the Materials and Methods and Haynes and Moch (10).

Figure 6

Real-time quantitative RT–PCR using TaqMan® chemistry identified ebl stage-specific transcripts from synchronized blood-stage P.falciparum. Periodic (∼6 h) time points taken from synchronized 3D7 cultures spanning the erythrocytic cycle are represented on the abscissa with merozoite invasion occurring just before the first time point. Ordinate values of the transcript levels are given in normalized genome equivalents (see Materials and Methods). Actual normalized genome equivalent values are given in the data table beneath each graph. Standard deviation of the C_ts for each ebl on a particular plate never reached >0.40. (A) eba-175, jesebl, baebl and pebl are expressed at high levels in late-stage schizonts (time point 7). (B) maebl transcript levels peaked during the late trophozoites (time point 3) and ebl-1 were most abundant in mid-stage schizonts (time point 6). The abundance of lsa-1 transcripts was negligible and indicates the level of background detection. Digital images taken from Giemsa-stained thin blood smears show a representative parasite stage for each time point.