Tandem affinity purification of exosome and replication factor C complexes from the non-human infectious kinetoplastid parasite Crithidia fasciculata.

Kinetoplastid parasites are responsible for a range of diseases with significant global impact. Trypanosoma brucei and Trypanosoma cruzi cause human African trypanosomiasis and Chagas disease, respectively, while various Leishmania species are responsible for cutaneous, mucocutaneous and visceral leishmaniasis. Understanding the biology of these organisms is key for effective diagnosis, prophylaxis and treatment. The insect parasite Crithidia fasciculata offers a safe and low-cost alternative for studies of kinetoplastid biology. C. fasciculata does not infect humans, can be cultured to high yields in inexpensive serum-free medium in a standard laboratory, and has a completely sequenced publically available genome. Taking advantage of these features, however, requires the adaptation of existing methods of analysis to C. fasciculata. Tandem affinity purification is a widely used method that allows for the rapid purification of intact protein complexes under native conditions. Here we report the application of tandem affinity purification to C. fasciculata for the first time, demonstrating the effectiveness of the technique by purifying both the intact exosome and replication factor C complexes. Adding tandem affinity purification to the C. fasciculata toolbox significantly enhances the utility of this excellent model system.

Kipandula W ，Smith TK ，MacNeill SA 《-》

Screening of the MMV and GSK open access chemical boxes using a viability assay developed against the kinetoplastid Crithidia fasciculata.

Diseases caused by the pathogenic kinetoplastids continue to incapacitate and kill hundreds of thousands of people annually throughout the tropics and sub-tropics. Unfortunately, in the countries where these neglected diseases occur, financial obstacles to drug discovery and technical limitations associated with biochemical studies impede the development of new, safe, easy to administer and effective drugs. Here we report the development and optimisation of a Crithidia fasciculata resazurin viability assay, which is subsequently used for screening and identification of anti-crithidial compounds in the MMV and GSK open access chemical boxes. The screening assay had an average Z' factor of 0.7 and tolerated a maximum dimethyl sulfoxide concentration of up to 0.5%. We identified from multiple chemical boxes two compound series exhibiting nanomolar potency against C. fasciculata, one centred around a 5-nitrofuran-2-yl scaffold, a well-known moiety in several existing anti-infectives, and another involving a 2-(pyridin-2-yl) pyrimidin-4-amine scaffold which seems to have pan-kinetoplastid activity. This work facilitates the future use of C. fasciculata as a non-pathogenic and inexpensive biological resource to identify mode of action/protein target(s) of potentially pan-trypanocidal potent compounds. This knowledge will aid in the development of new treatments for African sleeping sickness, Chagas disease and leishmaniasis.

Kipandula W ，Young SA ，MacNeill SA ，Smith TK ... -  《-》

Dramatic changes in gene expression in different forms of Crithidia fasciculata reveal potential mechanisms for insect-specific adhesion in kinetoplastid parasites.

Filosa JN ，Berry CT ，Ruthel G ，Beverley SM ，Warren WC ，Tomlinson C ，Myler PJ ，Dudkin EA ，Povelones ML ，Povelones M ... -  《PLoS Neglected Tropical Diseases》

Application of magnetically induced hyperthermia in the model protozoan Crithidia fasciculata as a potential therapy against parasitic infections.

Grazú V ，Silber AM ，Moros M ，Asín L ，Torres TE ，Marquina C ，Ibarra MR ，Goya GF ... -  《International Journal of Nanomedicine》

Crithidia fasciculata adenosine transporter 1 (CfAT1), a novel high-affinity equilibrative nucleoside transporter specific for adenosine.

Most eukaryotic organisms including protozoans like Crithidia, Leishmania, and Plasmodium encode a repertoire of equilibrative nucleoside transporters (ENTs). Using genomic sequencing data from Crithidia fasciculata, we discovered that this organism contains multiple ENT genes of highly similar sequence to the previously cloned and characterized adenosine transporter CfNT1: CfAT1 and CfNT3, and an allele of CfAT1, named CfAT1.2. Characterization of CfAT1 shows that it is an adenosine-only transporter, 87% identical to CfNT1 in protein sequence, with a 50-fold lower Km for adenosine. Site directed mutation of a key residue in transmembrane domain 4 (TM4) in both CfNT1 and CfAT1 shows that lysine at this position results in a high affinity phenotype, while threonine decreases adenosine affinity in both transporters. These results show that C. fasciculata has at least two adenosine transporters, and that as in other protozoan ENTs, a lysine residue in TM4 plays a key role in ligand affinity.

Arendt CS 《-》