An improved single-step lysis protocol to measure luciferase bioluminescence in Plasmodium falciparum
© Hasenkamp et al; licensee BioMed Central Ltd. 2012
Received: 16 December 2011
Accepted: 10 February 2012
Published: 10 February 2012
This report describes the optimization and evaluation of a simple single-step lysis protocol to measure luciferase bioluminescence from genetically modified Plasmodium falciparum. This protocol utilizes a modified commercial buffer to improve speed of assay and consistency in the bioluminescence signal measured by reducing the manipulation steps required to release the cytoplasmic fraction. The utility of this improved assay protocol is demonstrated in typical assays that explore absolute and temporal gene expression activity.
The use of reporter assay systems in the human malarial parasite Plasmodium falciparum have proven an invaluable tool in the functional characterization of cis-acting sequences that govern the promoter and terminator activities of gene flanking sequences [1–3]. The two most commonly used reporter systems utilize either a bacterial chloramphenicol acetyltransferase (cat) gene or a Photinus pyralis luciferase (luc) gene [4, 5]. Both assay systems offer attractive properties in terms of sensitivity and large linear range for quantification of activity. The non-isotopic bioluminescence assay provided by luc, however, offers additional attributes such as speed of assay (no need for lengthy incubations for the end-point of the cat assay), easier handling and waste disposal characteristics, and ability to exploit recent advances in instrumentation for the detection of bioluminescence. One final advantage of the luc assay, its flexibility, has recently been evidenced in the malarial field by the development of a high throughput anti-malarial drug screening assay using transgenic parasites expressing luc [6–9].
There are, however, inherent limitations in the utilization of the luc reporter assay system. Whilst valuable in demonstrating the effect of modified gene flanking sequences in determining the absolute level of gene expression, the concatamerization of episomal plasmids, unequal segregation of plasmids during mitotic division, and frequent use of mis-matched 5' and 3' gene flanking sequences, limit their use in the exploration of molecular mechanisms that govern temporal patterns of gene expression [1, 10]. These limitations, however, can be mitigated by the use of matched 5' and 3' flanking sequences in combination with the bxb1 integrase system to ensure production of homogenous populations of parasites that each contain a single reporter gene cassette [11, 12]. However, the repeated lysis and washing steps required to produce a cleared parasite cytoplasmic fraction from the intraerythrocytic stages of parasite development often generate a variation in the bioluminescence signal that needs to be carefully controlled (typically through cell count and protein concentration quantification). This variation is particularly confounding when attempting a multi-variant analysis, resulting in data that may demonstrate a trend but falls short of demonstrating significance.
Here the optimization and validation of a modified luciferase reporter gene assay protocol for the asexual intraerythrocytic stages of P. falciparum is described. This revised protocol uses a simple single-step lysis protocol with an improved lysis buffer formulation to generate a more consistent bioluminescent signal output. The utility of this modified assay is demonstrated by showing its ease of use in generating a comprehensive portrait of temporal gene expression and how consistency in data can provide significant information from a multi-variant promoter deletion study where previously only trends had been described.
Plasmodium falciparum clones and culture
The genetically modified P. falciparum clone expressing luciferase (Pfluc) used here has previously been described . This clone was originally derived from the AHE1 clone (Dd2attB) using the mycobacteriophage bxb 1 integration system [11, 12]. In Pfluc, the luc reporter gene is flanked by 1417 bp and 647 bp of 5' and 3' flanking sequences, respectively of Pfpcna (PF13_0328). Clones Δ2 and Δ3 contain 1160 bp and 990 bp of 5' Pfpcna flanking sequence, respectively . All clones were cultured under standard conditions (complete growth medium, 2% haematocrit, atmosphere of 1% O2, 3% CO2, and 96% N2) and maintained using 5 nM WR99210 and 2.5 μg/ml BSD (blasticidin S) drug selection . Staging and parasitaemia was determined by Giemsa-stained thin blood smears and light microscopy. Synchronization of cultures was attained using sequential sorbitol treatment .
Standard reporter lysis assay
Plasmodium falciparum-infected erythrocytes (IE) were collected by centrifugation (5 min, 3000xg, RT) and 20 μl samples of packed IE lysed using 10 volumes of 1× phosphate buffered saline (PBS)/0.1% saponin at room temperature for 5 min. Following centrifugation, the parasite pellet was washed three times in 1× PBS and re-suspended in 100 μl of 1× Reporter Lysis Buffer (Promega), and subjected to three cycles of freeze (liquid nitrogen) and thaw (37°C) with a final centrifugation step of 13000 rpm for 2 min to clear the parasite supernatant. Bioluminescence readings were measured using the single-tube luminometer (Glomax 20/20, Promega) from 20 μl of cleared parasite supernatant and 100 μl of Luciferase Substrate Reagent (Promega).
Analysis for significance in the bioluminescent signals determined for the reporter deletion series was carried out using n = 6 independent measurements for each construct at each developmental stage and analysed using one-way analysis of variants (ANOVA), with Tukey's post tests for significance, on MiniTab version 14. All other experiments were carried out using n = 5 independent measurements. Where indicated, t-tests were carried out in GraphPad Prism version 5.0 (GraphPad Software, San Diego California USA).
Evaluation of parameters for a simplified single-lysis step luciferase reporter assay
The standard luciferase reporter assay protocol for P. falciparum requires release of the parasite from the host erythrocyte, extensive washing of the parasite pellet to remove haemoglobin, and then a repeated process of freeze-thaw lysis of the parasite to release the cytoplasmic fraction [5, 12]. This protocol was devised prior to the improved performance and sensitivity of modern luminometers and it was reasoned that a revaluation of the lysis conditions should be explored in an attempt to simplify the protocol to use only a single lysis step.
To explore the possibility of improving the bioluminescent signal obtained using the single-step lysis protocol, two variables were explored. First was the use of different lysis buffers, and second the pellet-volume of lysis buffer used. A range of commercial lysis buffers optimized for bioluminescence assays are available; here the performance of the previously used RLB was compared against cell culture lysis reagent (CCLR, Promega, UK) and passive lysis buffer (PLB, Promega, UK), in each case assessing its use in the presence or absence of a 0.1% saponin supplement. Using five-pellet volumes (i.e. 100 μl) of each lysis buffer, the bioluminescent activity of the same IE culture was assayed and compared to a control using the standard lysis protocol and a five-pellet volume of RLB (Figure 1B). Saponin was observed to significantly enhance the bioluminescent signal obtained using all lysis buffers (t-test, p < 0.005). Comparison of the bioluminescent signal using the different buffers shows a clear preference for the modified PLB/0.1% saponin buffer. A revised assay was thus adopted using this modified buffer as the data would indicate that some 65% of the bioluminescent signal lost on adoption of the single-step assay can be recovered without adversely compromising the consistency of the data produced.
The final step in the validation of the revised single-step lysis protocol was a demonstration that the bioluminescent signal generated is directly proportional to the number of parasites being assayed. A range of different volumes of Pfluc IE (2-16 μl) were subject to single-step lysis using PLB/0.1% saponin and the bioluminescent signal plotted against parasite number (Figure 2B). To explore the effect of haemoglobin from uninfected and infected erythrocytes on this relationship, the volume of the IE pellet was made up to 20 μl using either phosphate-buffered saline (PBS) or packed uninfected erythrocytes (uIE). The bioluminescent signal obtained from IE supplemented with uIE was, in all cases, significantly less than that when supplemented with PBS. Plotting the bioluminescence signal against a linear scale of the number of parasites, reveals a strong correlation between IE number and signal for both conditions; with trend lines that would appear to be converging as the total number of erythrocytes (uIE and IE) becomes equivalent. This presumably reflects the known quenching effect of haemoglobin on luciferase bioluminescence . Whatever assay condition applied, however, this revised single-step lysis protocol produces a bioluminescent signal directly proportional to the number of parasites irrespective of the presence of haemoglobin and would indicate that washing away of haemoglobin is not required.
Demonstrating the utility of a single-step lysis protocol to investigate absolute and temporal changes in gene expression
Here a revised luciferase reporter gene assay protocol is described, based on the use of a single lysis-step, which offers a simpler and quicker procedure whilst delivering a more consistent bioluminescent signal. Reduction in the bioluminescent signal due to the presence of haemoglobin does not affect the specificity of the assay, and this loss in signal can be ameliorated using a saponin-supplementation of a commercial buffer. The utility of this revised assay in typical studies that examine the absolute and temporal properties of P. falciparum promoters is demonstrated. It is anticipated, however, that this protocol could be more widely applied to bioluminescence reporter gene assays in other intraerythroctytic pathogens.
This work was supported by funds from the Royal Society to SH and PH and the BBSRC through a New Investigator Award to PH. We would like to thank Alister Craig for critically reading the manuscript.
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