Characterisation of benzimidazole binding with recombinant tubulin from Giardia duodenalis, Encephalitozoon intestinalis, and Cryptosporidium parvum.

The binding kinetics of several benzimidazole compounds were determined with recombinant tubulin from benzimidazole-sensitive and -insensitive organisms. This study utilised the naturally occurring high efficacy of the benzimidazoles for the parasitic protozoa Giardia duodenalis and Encephalitozoon intestinalis, and low efficacy with Cryptosporidium parvum. Direct kinetic analysis of the benzimidazole-beta-tubulin interaction was performed using a fluorescence-based quenching method to determine the apparent association (k(on)) and dissociation (k(off)) rate constants from which the affinity constant (K(a)) was calculated. The binding kinetics were determined with recombinant alpha- and beta-tubulin from the parasitic protozoa with several benzimidazole R(2)-carbamate analogues. The affinity constant for the binding of several benzimidazoles with beta-tubulin from benzimidazole-sensitive protozoa was found to be significantly greater than binding to beta-tubulin from benzimidazole-insensitive protozoa. Additionally, the high affinity of several benzimidazole derivatives (albendazole, fenbendazole, mebendazole) for monomeric beta-tubulin and heterodimeric alphabeta-tubulin from benzimidazole-sensitive protozoa was also clearly demonstrated. The affinity constants determined with beta-tubulin from G. duodenalis and E. intestinalis also supported the observed in vitro efficacy of these compounds. The binding characteristics of the benzimidazoles with the highest in vitro efficacy (albendazole, fenbendazole, mebendazole) was reflected in their high association and slow dissociation rates with the beta-tubulin monomer or dimer from benzimidazole-sensitive protozoa compared with insensitive ones. Benzimidazole-bound alphabeta-tubulin heterodimers also had a significantly lower rate of microtubule assembly compared with benzimidazole-free alphabeta-heterodimers. The incorporation of benzimidazole-bound alphabeta-heterodimers into assembling microtubules was shown to arrest polymerisation in vitro although the addition of benzimidazole compounds to assembled microtubules did not result in depolymerisation. These findings indicate that a benzimidazole-beta-tubulin cap may be formed at the growing end of the microtubule and this cap prevents elongation of the microtubule.

A fluorescent based PCR assay for the detection and quantitation of Giardia duodenalis genotypes in mixed populations.

A method based on the polymerase chain reaction has been developed for differentiating between genotypically and phenotypically distinct strains of Giardia duodenalis and quantifying the amount of initial template of the different genotypes in mixed populations. The assay relies on a sequence-specific probe, labelled with two fluorescent dyes, designed to bind within the small subunit ribosomal (SSU) RNA gene. This target region is amplified by primers specific for either Group 1 or Group 2-type isolates of G. duodenalis and the probe binds within the primer-targeted region. This quantitative method takes advantage of the 5' nuclease activity of Taq DNA polymerase, which, on encountering a probe bound within the target DNA sequence cleaves it, causing it to become dissociated from the template. When the two fluorescent dyes bound to the probe are in close proximity (when the probe is intact), the interaction of the two dyes prevents the reporter dye from fluorescing. However, during the extension phase of amplification, the activity of the DNA polymerase causes the dyes to become separated and hence the reporter dye increases its fluorescent intensity. This release of fluorescence is directly related to the amplified amount of target template. This assay was developed with the aim of providing a unique method with which to investigate interactions within mixed populations of genetically distinct strains of G. duodenalis.

Characterization of factors favoring the expression of soluble protozoan tubulin proteins in Escherichia coli.

The alpha- and beta-tubulin genes of the parasitic protozoa Giardia duodenalis, Cryptosporidium parvum, and Encephalitozoon intestinalis have been overexpressed in soluble form using Escherichia coli-based expression systems. Several expression systems were compared in terms of the amount of soluble protein produced with different fusion partners, strains of E. coli BL21, and expression temperatures. The cleavability of the fusion partner was also assessed in terms of post-expression applications of the recombinant protein. The maltose-binding protein (MBP) and glutathione S-transferase (GST) fusion partners produced the highest expression levels for all six proteins without the formation of inclusion bodies. The expression system also provided a means of purifying the soluble protein using affinity and anion-exchange chromatography while minimizing protein losses. The yield and purity were therefore very high for both the MBP and GST systems. The tubulin monomers were demonstrated to be assembly-competent using a standard dimerization assay and also retained full antigenicity with monoclonal antibodies. This study presents several methods which are suitable for producing soluble tubulin monomers and, thus, circumventing the formation of inclusion bodies which necessitates re-folding of the tubulin.

A review of chemotherapeutic approaches to the treatment of cryptosporidiosis.

This review focuses on chemotherapies used against the parasite, Cryptosporidium parvum, the causative agent of cryptosporidiosis. Populations at risk from severe morbidity or mortality from cryptosporidiosis are discussed with particular reference to those infected with HIV. The review then examines chemotherapies used in the clinical setting, as well as a number of in vitro and in vivo experimental studies. It begins with a discussion of the targets within Cryptosporidium that have been the foci of past treatments and then examines novel target sites that may present an exploitable alternative. Some of the novel target sites discussed include the recently discovered apicomplexan plastid and its associated pathways. Lastly, the review examines tubulin as a potential anticryptosporidial target in view of the fact that it has been exploited successfully for almost 50 years for the treatment of helminthiasis. The review concludes with a five-year outlook on the future of anticryptosporidial drug design.