Novel mechanisms of programmed cell death in the protozoan parasite blastocystis

Master'sMASTER OF SCIENC

Structural, mechanistic and functional characterization of glycoside hydrolases of family GH99

Glycosylation is a very common post-translational modification and the glycans can be attached to oxygen (O-linked), nitrogen (N-linked) or carbon (C-linked). N-linked glycosylation has implications for protein folding and is also essential in viral infectivity and cell-cell signalling. Endo-α-1,2-mannosidase from family GH99 is a unique enzyme within the N-glycosylation pathway as it is the only one which does not cleave the terminal sugar from the reducing end of the glycan, but instead releases an α-Glc-1,3-Man disaccharide, with overall retention of stereochemistry at the anomeric carbon. Previously it was proposed that GH99 endo-acting mannosidases and mannanases proceed through a neighbouring group participation mechanism with a 1,2-anhydrosugar as a reaction intermediate. This Thesis contains evidence supporting this hypothesis. Chapter 2 presents structures of the bacterial GH99 with its substrate, with mimics of the reaction intermediate and with the products of the reaction. Kinetic and structural data on various intermediate mimics show that the compound whose structure is the closest to the intermediate is turned over by the enzyme. In Chapter 3, analysis of different designs of GH99 inhibitors and their conformation on-enzyme is presented. Chapter 4 presents purification and solution of the crystal structure of the catalytic domain of the human endomannosidase (MANEA). Multiple crystal forms were obtained, which made it possible to look at the conformation of a feature present in the eukaryotic but not bacterial GH99: a loop spanning residues 191–201. This loop was disordered when no ligand was present in the –2/–1 sites, and ordered when these sites were occupied. Chapter 5 explores attempts at producing MANEAL, a paralog of MANEA which is found in bony vertebrates. The Thesis concludes with an analysis of the phylogeny of endomannosidase genes and perspectives for future research: studies of endomannosidase in mammalian model organisms are needed to understand its significance

Ultrasensitive detection of toxocara canis excretory-secretory antigens by a nanobody electrochemical magnetosensor assay.

peer reviewedHuman Toxocariasis (HT) is a zoonotic disease caused by the migration of the larval stage of the roundworm Toxocara canis in the human host. Despite of being the most cosmopolitan helminthiasis worldwide, its diagnosis is elusive. Currently, the detection of specific immunoglobulins IgG against the Toxocara Excretory-Secretory Antigens (TES), combined with clinical and epidemiological criteria is the only strategy to diagnose HT. Cross-reactivity with other parasites and the inability to distinguish between past and active infections are the main limitations of this approach. Here, we present a sensitive and specific novel strategy to detect and quantify TES, aiming to identify active cases of HT. High specificity is achieved by making use of nanobodies (Nbs), recombinant single variable domain antibodies obtained from camelids, that due to their small molecular size (15kDa) can recognize hidden epitopes not accessible to conventional antibodies. High sensitivity is attained by the design of an electrochemical magnetosensor with an amperometric readout with all components of the assay mixed in one single step. Through this strategy, 10-fold higher sensitivity than a conventional sandwich ELISA was achieved. The assay reached a limit of detection of 2 and15 pg/ml in PBST20 0.05% or serum, spiked with TES, respectively. These limits of detection are sufficient to detect clinically relevant toxocaral infections. Furthermore, our nanobodies showed no cross-reactivity with antigens from Ascaris lumbricoides or Ascaris suum. This is to our knowledge, the most sensitive method to detect and quantify TES so far, and has great potential to significantly improve diagnosis of HT. Moreover, the characteristics of our electrochemical assay are promising for the development of point of care diagnostic systems using nanobodies as a versatile and innovative alternative to antibodies. The next step will be the validation of the assay in clinical and epidemiological contexts

Automatic cell classification and population estimation in blastocystis autophagy images

10.1109/ICIP.2010.5652386Proceedings - International Conference on Image Processing, ICIP4349-435