The Cryptosporidium parvum Kinome

<p>Abstract</p> <p>Background</p> <p>Hundreds of millions of people are infected with cryptosporidiosis annually, with immunocompromised individuals suffering debilitating symptoms and children in socioeconomically challenged regions at risk of repeated infections. There is currently no effective drug available. In order to facilitate the pursuit of anti-cryptosporidiosis targets and compounds, our study spans the classification of the <it>Cryptosporidium parvum </it>kinome and the structural and biochemical characterization of representatives from the CDPK family and a MAP kinase.</p> <p>Results</p> <p>The <it>C</it>. <it>parvum </it>kinome comprises over 70 members, some of which may be promising drug targets. These <it>C. parvum </it>protein kinases include members in the AGC, Atypical, CaMK, CK1, CMGC, and TKL groups; however, almost 35% could only be classified as OPK (other protein kinases). In addition, about 25% of the kinases identified did not have any known orthologues outside of <it>Cryptosporidium spp</it>. Comparison of specific kinases with their <it>Plasmodium falciparum </it>and <it>Toxoplasma gondii </it>orthologues revealed some distinct characteristics within the <it>C. parvum </it>kinome, including potential targets and opportunities for drug design. Structural and biochemical analysis of 4 representatives of the CaMK group and a MAP kinase confirms features that may be exploited in inhibitor design. Indeed, screening <it>Cp</it>CDPK1 against a library of kinase inhibitors yielded a set of the pyrazolopyrimidine derivatives (PP1-derivatives) with IC₅₀values of < 10 nM. The binding of a PP1-derivative is further described by an inhibitor-bound crystal structure of <it>Cp</it>CDPK1. In addition, structural analysis of <it>Cp</it>CDPK4 identified an unprecedented Zn-finger within the CDPK kinase domain that may have implications for its regulation.</p> <p>Conclusions</p> <p>Identification and comparison of the <it>C. parvum </it>protein kinases against other parasitic kinases shows how orthologue- and family-based research can be used to facilitate characterization of promising drug targets and the search for new drugs.</p

The Mitochondrion-Related Organelles of Cryptosporidium Species

Amongst all apicomplexans, the mitochondrion-related organelle of Cryptosporidium species is the least studied. To date, most of our knowledge on this tiny organelle stems from observations on the remnant mitochondrion, mitosome, of Cryptosporidium parvum. In C. parvum the mitosome is structurally distinguished from the hydrogenosomes and mitosomes of other anaerobic protists by its (1) close association with the crystalloid body, an organelle unique to this apicomplexan and the function of which is currently unknown; (2) close association with the outer nuclear membrane, and possibly nuclear pores; (3) envelopment by rough endoplasmic reticulum, and in some cases an apparent direct tethering to ribosomes; and (4) atypical internal membranous compartments that lack well-defined crista junctions with the mitochondrial inner membrane, a characteristic that defines most aerobic mitochondria. Like most hydrogenosome- and other mitosome-bearing anaerobic protists, however, C. parvum lacks a mitochondrial genome, i.e. proteins are encoded by the nucleus and targeted back to the mitosome. As a consequence of this reductive evolution, there are no genes for electron transport or oxidative phosphorylation, and the only function so far ascribed to this tiny organelle is one common to all eukaryotic mitochondria, the assembly and maturation of iron-sulphur clusters. The ultrastructure and tomography of the C. parvum mitosome and crystalloid body, as well as the probable functions of these organelles, are the primary topics herein. An overview of iron-sulphur cluster biosynthesis, likely mechanisms for import into and export from the mitosome, as well as core carbohydrate and energy metabolism are also discussed. Similarities and differences in the structure and function of both organelles in the genus Cryptosporidium, with anaerobic protists in general, and with other apicomplexans specifically, are described