Malaria Biotechnology


Developing Drug Targeting Platforms and Decipher Drug Resistance Mechanisms

Background

Malaria still remains the most frequent global infectious disease. It is endemic to most areas in Africa, South East Asia and Latin America with up to half a million clinical cases and 1-3 millions of deaths annually. Malaria falciparum which is caused by Plasmodium falciparum represents the most severe and dangerous form, as resistances against the most common anti-malarial drugs (e.g. chloroquin, mefloquin) have widely spread. The quest for new, highly effective drugs requires more and more ‚rationale drug design‘ and in vitro based high-throughput screening assays. Such approaches are based on fundamental knowledge about the parasite’s metabolic pathways as well as the pathophysiological mechanisms in the dynamic host-parasite system. Supportive drug discovery becomes even more important since there will probably no vaccination be available in the immediate future. As the main invasion targets for the parasites are red blood cells, our malaria group within the institute will have a major focus in studying signaling mechanisms and drug responses in P.falciparum-infected erythrocytes.

Malaria Forschung

Abbildung: A, Fluo-4 ‚drug resistance patterns‘ in P. falciparum strains are characterized by a more diffuse Fluo-4 staining in drugsensitive strains (HB3) over a strcitly localized to the acid food vacuole localization in drug-resistant strains (Dd2). However, this distribution pattern of the Fluo-4 fluorescence does not reflect differences in the apparent Ca2+ concentrations between strains for this fluorochrome, as Fluo-4 is actively pumped into the vacuole by Pgh-1. Moreover, ratiometric Fura-Red Ca2+ fluorescence imaging, after careful ‚in situ‘ calibration, shows no significant differences in [Ca2+] for the different compartments and strains [from Rohrbach et al. 2006]. B, Vit. B6 de novo synthesis of Plasmodium is performed by the dodecameric PLP-synthase complex consisiting of Pdx1 (cyanblue) and Pdx2 (yellow) (YaaD/YaaE, Strohmeier et al. 2006).  We could demonstrate that the C-terminal part of PfPdx1, lying between two subunits, influences enzymatic activity and oligomerisation of the complex. Deletion mutants of the C-terminus results in lose of enzymatic function, substrate binding and dodekamer formation [Derrer et al. 2010].

Ion homeostasis and solute transport in malaria-infected erythrocytes

Once inside the erythrocyte, the parasite transforms the hosts cytosolic environment according to its needs using various accumulation and transport regimes. For instance, P.falciparum actively builds up a large Ca2+ concentration in one of its compartments (parasitophorous vacuole). This way, gradients can be used for coupled transport. In its acidic food vacuole, especially drug-resistant P.falciparum strains (e.g. Dd2) express ATP-driven multi-drug resistance transporters (e.g. Pgh-1; gene product of pfmdr1) not present in drug-sensitive strains (e.g. HB3). Such transporters actively extrude anti-malarial drugs from the food vacuole. Interestingly, some fluorochromes that are being used for Ca2+ fluorescence imaging (i.e. fluo-4) are also a substrate for these transporters and pump fluo-4 into the food vacuole. Studying fluo-4 Ca2+ fluorescence, therefore, represents an indirect tool for drug-resistance screening. Previous studies in collaboration with Prof. Rohrbach (now McGill University, Montreal) defined characteristic fluorescence patterns, i.e. a diffuse staining pattern in chloroquine-sensitive vs a localized staining confined to the acidic food vacuole in –resistant strains. The vast local accumulation of fluorescence was a result of vacuolar dye uptake and did not reflect a proportionately increased vacuolar Ca2+ concentration, as proven by ratiometric Fura-Red Ca2+ imaging and careful in situ calibration (s. figure). This result questioned a previous dogma that the food vacuole was a parasitic Ca2+ pool.

Vitamin B6-Pathway in parasites

Plasmodia have several metabolic pathways at their disposal which are not present in humans and therefore of a certain interest as a drug target for new anti-malarials. One of these pathways is the vitamin B6 de novo biosynthesis. Vitamin B6 in its phosphorylated form PLP (pyridoxal 5-phosphate) functions as co-factor in numerous enzymatic reactions and thus is essential for all living cells. Humans and animals have to take up vitamin B6 by their diet and use the salvage pathway to produce PLP. Plasmodia are, besides the salvage pathway, able to produce PLP by the PLP synthase complex. This complex consists of 12 molecules Pdx1, forming to hexamers, and 12 Pdx2 units, which are surrounding the Pdx1 (fig.XB). The Pdx1 functions as the synthase subunit which uses ribose 5-phosphate (R5P), glyceraldehyde 3-phosphate and ammonia to produce PLP. The ammonia is provided by the Pdx2 which hydrolyzes L-glutamine.

Previous data on procaryotic PLP-synthases showed that the C-terminal part of one Pdx1 stretches over the neighbouring Pdx1-subunit and interacts with an area of the C-terminus of the second Pdx1.

This interaction influences the enzymatic activity of the single Pdx1 molecules.

C-terminal deletion variants of PfPdx1, where up to 30 amino acids were truncated, revealed the function of the C-terminus for enzymatic activity and dodecamer formation. First of all, PLP-synthase activity decreased with increasing deletion, second the intermediate formation (incorporation of NH3 into the bound R5P) decreased and finally the initial step, binding of R5P, was abolished (table fig. XB). Also, the shortest variant, PfPdx1Δ270-301 could not form a dodecamer anymore.

Planned future projects

  • development of a quantitative simulation platform to predict parasitic metabolism, solute transport and ion homeostasis for drug screening assays
  • development and implementation of an automated high-throughput diagnostic in-vitro-based cell assay for fast compound screening using fluorescence microscopy workstations
  • Rational drug design based on results from live cell imaging
  • long-term microscopy of the complete parasitic stage cycle using cell culture fluorescence microscopy
  • Identification and characterization of metabolic pathways of the parasite as potential drug targets (vitamin B6 synthesis/shikimate pathway)
  • Identification of growth regulating substances of host and parasite, e.g by high-throughput screening in vitro

Literature

  • Rohrbach P, Friedrich O, Hentschel J, Plattner H, Fink RH, Lanzer M (2006). Quantitative calcium measurements in subcellular compartments of Plasmodium falciparum-infected erythrocytes. J Biol Chem 280(30): 27960-27969.
  • Rohrbach P, Sanchez CP, Hayton K, Friedrich O, Patel J, Sidhu AB, Ferdig MT, Fidock DA, Lanzer M (2006). Genetic linkage of pfmdr1 with food vacuolar solute import in Plasmodium falciparum. EMBO J 25(13): 3000-3011.
  • Derrer B, Windeisen V, Guédez Rodríguez G, Seidler J, Gengenbacher M, Lehmann WD, Rippe K, Sinning I, Tews I, Kappes B (2010). Defining the structural requirements for ribose 5-phosphate-binding and intersubunit cross-talk of the malarial pyridoxal 5-phosphate synthase. FEBS Lett. 584(19): 4169-74. doi:10.1016/j.febslet.2010.09.013