An image-based drug susceptibility assay targeting the placental sequestration of Plasmodium falciparum-infected erythrocytes.

Placental malaria is a significant cause of all malaria-related deaths globally for which no drugs have been developed to specifically disrupt its pathogenesis. To facilitate the discovery of antimalarial drugs targeting the cytoadherence process of Plasmodium-infected erythrocytes in the placenta microvasculature, we have developed an automated image-based assay for high-throughput screening for potent cytoadherence inhibitors in vitro. Parasitized erythrocytes were drug-treated for 24 h and then allowed to adhere on a monolayer of placental BeWo cells prior to red blood cell staining with glycophorin A antibodies. Upon image-acquisition, drug effects were quantified as the proportion of treated parasitized erythrocytes to BeWo cells compared to the binding of untreated iRBCs. We confirmed the reliability of this new assay by comparing the binding ratios of CSA- and CD36-panned parasites on the placental BeWo cells, and by quantifying the effects of chondroitin sulfate A, brefeldin A, and artemisinin on the binding. By simultaneously examining the drug effects on parasite viability, we could discriminate between cytoadherence-specific inhibitors and other schizonticidal compounds. Taken together, our data establish that the developed assay is highly suitable for drug studies targeting placental malaria, and will facilitate the discovery and rapid development of new therapies against malaria.

Quantum dots: a new tool for anti-malarial drug assays.

BACKGROUND: Malaria infects over 300 million people every year and one of the major obstacles for the eradication of the disease is parasite's resistance to current chemotherapy, thus new drugs are urgently needed. Quantum dot (QD) is a fluorescent nanocrystal that has been in the spotlight as a robust tool for visualization of live cell processes in real time. Here, a simple and efficient method using QD to directly label Plasmodium falciparum-infected erythrocytes (iRBCs) was searched in order to use the QD as a probe in an anti-malarial drug-screening assay. METHODS: A range of QDs with different chemical coatings were tested for their ability to specifically bind iRBCs by immunofluorescence assay (IFA). One QD was selected and used to detect parasite growth and drug sensitivity by flow cytometry. RESULTS: PEGylated-cationic QD (PCQD) was found to specifically label infected erythrocytes preferentially with late stage parasites. The detection of QD-labelled infected erythrocytes by flow cytometry was sensitive enough to monitor chloroquine anti-malarial toxicity with a drug incubation period as short as 24 h (EC50 = 113nM). A comparison of our assay with another widely used anti-malarial drug screening assay, the pLDH assay, showed that PCQD-based assay had 50% improved sensitivity in detecting drug efficacy within a parasite life cycle. An excellent Z-factor of 0.8 shows that the QD assay is suitable for high-throughput screening. CONCLUSIONS: This new assay can offer a rapid and robust platform to screen novel classes of anti-malarial drugs.

Structure of 3-deoxy-manno-octulosonate cytidylyltransferase from Haemophilus influenzae complexed with the substrate 3-deoxy-manno-octulosonate in the beta-configuration.

The enzyme 3-deoxy-manno-octulosonate cytidylyltransferase (CMP-KDO synthetase; CKS) catalyzes the activation of 3-deoxy-D-manno-octulosonate (or 2-keto-3-deoxy-manno-octonic acid; KDO) by forming CMP-KDO. CKS is unique to Gram-negative bacteria and is an attractive target for the development of antibacterial agents. The crystal structure of CKS from Haemophilus influenzae in complex with the substrate KDO has been determined at 2.30 A resolution by combining single-wavelength anomalous diffraction and molecular-replacement methods. The two monomers in the asymmetric unit differ in the conformation of their C-terminal alpha-helix (Ala230-Asn254). The KDO bound to the active site exists as the beta-pyranose form in the (5)C(2) chair conformation. The structure of CKS from H. influenzae in complex with KDO will be useful in structure-based inhibitor design.

Overexpression, crystallization, and preliminary X-ray crystallographic analysis of the alanine racemase from Enterococcus faecalis v583.

Alanine racemase, a bacterial enzyme belonging to the fold-type III group of pyridoxal 5'-phosphate (PLP)-dependent enzymes, has been shown to catalyze the interconversion between L- and D-alanine. The alanine racemase from the pathogenic bacterium Enterococcus faecalis v583 has been overexpressed in E. coli and was shown to crystallize an enzyme at 295 K, using polyethylene glycol (PEG) 8000 as a precipitant. X-ray diffraction data to 2.5 A has been collected using synchrotron radiation. The crystal is a member of the orthorhombic space group, C222(1), with unit cell parameter of a=94.634, b=156.516, c=147.878 A, and alpha=beta;=gamma=90 degrees. Two or three monomers are likely to be present in the asymmetric unit, with a corresponding Vm of 3.38 A3 Da(-1) and 2.26 A Da(-1) and a solvent content of 63.7% and 45.5%, respectively.

Crystallization and preliminary X-ray crystallographic analysis of UDP-N-acetylglucosamine enolpyruvyl transferase from Haemophilus influenzae in complex with UDP-N-acetylglucosamine and fosfomycin.

The bacterial enzyme UDP-N-acetylglucosamine enolpyruvyl transferase catalyzes the first committed step of peptidoglycan biosynthesis, i.e., transfer of enolpyruvate from phosphoenolpyruvate to UDP-N-acetyl-glucosamine. We have overexpressed the enzyme from Haemophilus influenzae in Escherichia coli and crystallized it in the apo-form, as well as in a complex with UDP-N-acetylglucosamine and fosfomycin using ammonium sulfate as the precipitant. X-ray diffraction data from a crystal of the apo-form were collected to 2.8 A resolution at 293 K. The crystal quality was improved by co-crystallization with UDP-N-acetylglucosamine and fosfomycin. X-ray data to 2.2 A have been collected at 100 K from a flash-frozen crystal of the complex. The complex crystals belong to the orthorhombic space group I222 (or I212121) with unit-cell parameters of a = 63.7, b = 124.5, and c = 126.3 A. Assuming a monomer of the recombinant enzyme in the crystallographic asymmetric unit, the calculated Matthews parameter (VM) is 2.71 A3 Da-1 and solvent content is 54.6%.

Crystallization and preliminary X-ray crystallographic analysis of the tRNA-specific adenosine deaminase from Streptococcus pyogenes.

The tRNA-specific adenosine deaminase from the pathogenic bacteria Streptococcus pyogenes (spTAD) has been overexpressed in Escherichia coli and crystallized in the presence of Zn2+ ion at 295 K using ammonium sulfate as a precipitant. Flash-cooled crystals of spTAD diffracted to 2.0 A using 30%(v/v) glycerol as a cryoprotectant. X-ray diffraction data have been collected to 2.0 A using synchrotron radiation. The crystal belongs to the tetragonal space group P4(2)2(1)2, with unit-cell parameters a = b = 81.042, c = 81.270 A. The asymmetric unit contains one subunit of spTAD, with a corresponding crystal volume per protein weight (VM) of 3.3 A3 Da(-1) and a solvent content of 62.7%.

Crystallization and preliminary X-ray crystallographic studies of 3-deoxy-manno-octulosonate cytidylyltransferase from Haemophilus influenzae.

The enzyme 3-deoxy-manno-octulosonate cytidylyltransferase (CMP-KDO synthetase; CKS) catalyzes the activation of 3-deoxy-manno-octulosonate (KDO) by forming CMP-KDO. It is essential for the biosynthesis of lipopolysaccharides in Gram-negative bacteria and is a potential target for the discovery of antibacterial agents. L-CKS from Haemophilus influenzae was overexpressed with a C-terminal hexahistidine tag in Escherichia coli and crystallized in the presence of the substrate KDO at 297 K using PEG 4000 as a precipitant and ethylene glycol as an additive. The diffraction limit and spot shape of the native crystal could be improved significantly by dehydration/annealing. X-ray diffraction data were collected to 2.5 A resolution from a native crystal. The crystals are orthorhombic, belonging to the space group P2(1)2(1)2(1), with unit-cell parameters a = 48.6, b = 83.1, c = 117.3 A. The presence of two monomers of recombinant L-CKS in the crystallographic asymmetric unit gives a reasonable V(M) of 2.05 A(3) Da(-1), with a solvent content of 40.0%.