Functional Characterization of a Novel Thioredoxin Domain-Containing Protein of the Malaria Parasite Plasmodium

A novel thioredoxin domain-containing protein of the malaria parasite Plasmodium was identified and found to be conserved among eukaryotes. This protein belongs to the phosducin-like family of proteins (PhLPs), and was therefore assigned the name PhLP1, since it is the first phosducin-like protein to be identified in Plasmodium. PhLPs have been found to have various roles in G-protein signaling, cell cycle progression, and protein folding. However, the biochemical mechanism by which PhLPs perform their function is unknown. Here is described the cloning and biochemical characterization of both PhLP1 and its human homolog TXNDC9. Both purified PhLP1 and TXNDC9 showed enzymatic activity in the insulin reduction assay and were also active in the thioredoxin-couple reduction assay. Sequence alignment and homologous modeling of PhLP1 and TXNDC9 indicated a conserved, putative atypical active site in place of the typical catalytic CXXC motif found in classical redox active thioredoxins. Site-directed mutagenesis of the putative redox active cysteine (C106) in PhLP1 abolished the redox functions of the protein, confirming the role of C106 in the catalytic mechanisms of the protein. These results show for the first time that PhLPs are redox active enzymes that can be efficiently reduced by the thioredoxin system. These findings shed new light on the biochemical mechanism and biological function of these highly conserved proteins

Expression of Cytosolic Peroxiredoxins in Plasmodium Berghei Ookinetes is Regulated by Environmental Factors in the Mosquito bloodmeal

The Plasmodium ookinete develops over several hours in the bloodmeal of its mosquito vector where it is exposed to exogenous stresses, including cytotoxic reactive oxygen species (ROS). How the parasite adapts to these challenging conditions is not well understood. We have systematically investigated the expression of three cytosolic antioxidant proteins, thioredoxin-1 (Trx-1), peroxiredoxin-1 (TPx-1), and 1-Cys peroxiredoxin (1-Cys Prx), in developing ookinetes of the rodent parasite Plasmodium berghei under various growth conditions. Transcriptional profiling showed that tpx-1 and 1-cys prx but not trx-1 are more strongly upregulated in ookinetes developing in the mosquito bloodmeal when compared to ookinetes growing under culture conditions. Confocal immunofluorescence imaging revealed comparable expression patterns on the corresponding proteins. 1-Cys Prx in particular exhibited strong expression in mosquito-derived ookinetes but was not detectable in cultured ookinetes. Furthermore, ookinetes growing in culture upregulated tpx-1 and 1-cys prx when challenged with exogenous ROS in a dose-dependent fashion. This suggests that environmental factors in the mosquito bloodmeal induce upregulation of cytosolic antioxidant proteins in Plasmodium ookinetes. We found that in a parasite line lacking TPx-1 (TPx-1KO), expression of 1-Cys Prx occurred significantly earlier in mosquito-derived TPx-1KO ookinetes when compared to wild type (WT) ookinetes. The protein was also readily detectable in cultured TPx-1KO ookinetes, indicating that 1-Cys Prx at least in part compensates for the loss of TPx-1 in vivo. We hypothesize that this dynamic expression of the cytosolic peroxiredoxins reflects the capacity of the developing Plasmodium ookinete to rapidly adapt to the changing conditions in the mosquito bloodmeal. This would significantly increase its chances of survival, maturation and subsequent escape. Our results also emphasize that environmental conditions must be taken into account when investigating Plasmodium-mosquito interactions

Automatic flower cluster estimation in apple orchards using aerial and ground based point clouds

Chemical and mechanical thinning processes have long been used in stone and pome fruit production. During the thinning of apple flowers, growers use chemicals to regulate the tree load. Hand thinning is applied after the June drop to prune trees with excess crop load. The process of thinning can be unpredictable especially in biennial bearing cultivars. Thus, incentives to optimise chemical usage and to reduce expensive manual labour is ever increasing. Ground based machine vision systems have grown in popularity in orchard management due to the level of detail as well as plant coverage they can inspect with. Additionally, unmanned aerial vehicles (UAV) -based remote sensing technology is becoming a popular non-invasive quality inspection solution. This work proposes a framework for combining UAV and ground based RGB image data to detect flowering intensity in a Dutch Elstar apple orchard. The framework, based on point cloud reconstruction, presents automatic point cloud handling techniques as well as automated unsupervised flowering intensity estimation methods. Two linear regression models based on unsupervised machine learning methods were trained and validated from the framework that estimate flowering intensity in the orchard with both models having R2 > 0.65, RRMSE < 20% and p-stat < 0.005 for the correlation between the image derived flower index and the flower cluster number counted in field. The proposed methods provide a novel strategy for guiding flower thinning using simple RGB images and location data only. Moreover, the proposed methods also reveal the flexibility of intra-tree inspection by checking its sub-volumes

Characterization of a protozoan Phosducin-like protein-3 (PhLP-3) reveals conserved redox activity.

We recently identified three novel thioredoxin-like genes in the genome of the protozoan parasite Plasmodium that belong to the Phosducin-like family of proteins (PhLP). PhLPs are small cytosolic proteins hypothesized to function in G-protein signaling and protein folding. Although PhLPs are highly conserved in eukaryotes from yeast to mammals, only a few representatives have been experimentally characterized to date. In addition, while PhLPs contain a thioredoxin domain, they lack a CXXC motif, a strong indicator for redox activity, and it is unclear whether members of the PhLP family are enzymatically active. Here, we describe PbPhLP-3 as the first phosducin-like protein of a protozoan organism, Plasmodium berghei. Initial transcription analysis revealed continuous low-level expression of pbphlp-3 throughout the complex Plasmodium life cycle. Attempts to knockout pbphlp-3 in P. berghei did not yield live parasites, suggesting an essential role for the gene in Plasmodium. We cloned, expressed and purified PbPhLP-3 and determined that the recombinant protein is redox active in vitro in a thioredoxin-coupled redox assay. It also has the capacity to reduce the organic compound tert-Butyl hydroperoxide (TBHP) in vitro, albeit at low efficiency. Sequence analysis, structural modeling, and site-directed mutagenesis revealed a conserved cysteine in the thioredoxin domain to be the redox active residue. Lastly, we provide evidence that recombinant human PhLP-3 exhibits redox activity similar to that of PbPhLP-3 and suggest that redox activity may be conserved in PhLP-3 homologs of other species. Our data provide new insight into the function of PhLP-3, which is hypothesized to act as co-chaperones in the folding and regulation of cytoskeletal proteins. We discuss the potential implications of PhLP-3 as a thioredoxin-target protein and possible links between the cellular redox network and the eukaryotic protein folding machinery

Candidate genes of the cytosolic thioredoxin system of <i>P. berghei</i>.

<p>Candidate genes of the cytosolic thioredoxin system of <i>P. berghei</i>.</p

Challenge of ookinete cultures with increasing concentrations of the ROS producing agent paraquat (PQ).

<p>RT-qPCR data showing dose-response modulation of target gene expression in ookinete cultures in the presence of increasing concentrations of the superoxide-producing compound Paraquat (PQ, Viologen, Aldrich). Increasing concentrations of PQ were added to 12-hours <i>P. berghei</i> ookinete cultures as indicated. Shown are the ratios of normalized relative transcript quantities (RQ) of treated vs. non-treated samples (non-treated RQ = 1, represented by the dashed line). All data were normalized against the expression of 18 s rRNA A-type <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003136#ppat.1003136-Yano1" target="_blank">[29]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003136#ppat.1003136-Thompson1" target="_blank">[34]</a>. (*) indicates statistical significance (p<0.1). Shown are mean values of 3 independent experiments. Error bars indicate STDEV.</p

Time dependent transcription profiles of the cytosolic thioredoxin system in culture-derived and mosquito-derived parasites.

<p>RT-qPCR data showing relative target gene expression as fold increase over time. The 3-hour time point was used as a reference. All data was normalized against the expression of 18 s rRNA A-type <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003136#ppat.1003136-Yano1" target="_blank">[29]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003136#ppat.1003136-Thompson1" target="_blank">[34]</a>. The Mann-Whitney U test was conducted on each candidate gene from both mosquito-derived and from culture-derived parasites. Significance was assessed at p<0.1(*) due to the small sample sizes. Shown are mean values of 4 independent blood feeding experiments (n = 55 mosquitoes/time point/experiment) and 3 independent ookinete culture setups, respectively. Error bars show STDEV.</p

Expression of the Trx-1, TPx-1 and 1-Cys Prx in developing ookinetes from culture or from mosquito bloodmeal.

<p>Samples were isolated from ookinete cultures and blood fed mosquitoes at indicated time points and prepared as described in material and methods. Fixed samples were probed with <b>A</b>) anti-PbTrx-1, <b>B</b>) anti-Pf TPx-1 or <b>C</b>) anti-PbTPx-1. Primary antibodies were probed with secondary antibody coupled to AF 488 (Molecular Probes). Samples were counterstained with the nuclear dye TO-PRO-3 (Life technologies). The source of the ookinetes from either ookinete cultures or bloodfed mosquito midguts is indicated. Images are merged and overlaid onto the respective DIC image. White arrows indicate accumulation of Trx-1, TPx-1 or 1-Cys Prx at the apical ends of developing ookinetes. Scale bar = 5 µM. Quantitative <i>Relative Fluorescence Analysis</i>: Shown is the median boxed by the first and third quartiles with minimum and maximum values displayed as whiskers. Mann-Whitney U-Test was performed with samples of culture-derived ookinetes compared to mosquito-derived ookinetes (p = 0.4551 for Trx-1; p = 0.1061 for TPx-1; p = 0.0413 for 1-Cys Prx). Below each graph are representative images from IFA-epifluorescence experiments.</p