Association Between Genotypes At Polymorphisms In The Trypanotolerance Candidate Gene, Arhgap15 And The Pathophysiology Of Trypanosomiasis Associated Anaemia

ABSTRACT

The immunobiology of African trypanosomes in the context of both parasite and host survival are tightly interconnected and ultimately determine the complicated traits of host tolerance versus susceptibility. Multigene control of variation in susceptibility to the pathological effects of trypanosomiasis, the most prominent being anaemia, is known to occur in domestic livestock populations. The aetiology of trypanosomiasis-associated anaemia in cattle is multifactorial with enhanced erythrophagocytosis and noncompensatory erythropoiesis being the key drivers. In particular, compelling evidence has accumulated implicating macrophage hyperactivation in the extravascular destruction of red blood cells due to massive erythrophagocytosis in the spleen and liver of infected cattle. Ras-related C3 botulinum toxin substrate 1 (RAC1), a member of the Rho family of small GTPases is the key intracellular signaling component regulating phagocytosis in macrophages by controlling different steps of membrane and actin dynamics. On the basis of the critical role of RAC1 in erythrophagocytosis, the RAC1 GTPase activating protein family member 15, (ARHGAP15) has been identified as the most plausible candidate gene in the quantitative trait loci contributing to trypanosomosis-associated anemia on bovine chromosome two. Sequencing of the ARHGAP15 gene in trypanotolerant N'Dama and trypanosusceptible Boran cattle breeds revealed a histidine to proline amino acid substitution. Borans carry the ancestral histidine allele, which is conserved in humans, pigs, chickens and salmon. In this study, the association between genotypes at the non-synonymous single-nucleotide polymorphism (SNP) in the ARHGAP15 gene and the pathophysiology of trypanosomiasis-associated anaemia was investigated. The two alleles were expressed in vitro and the effect of allelic substitution in the ARHGAP15 gene on the kinetic properties of the GTPase reaction of RAC1 determined by monitoring the continuous release of inorganic phosphate from RAC1-GTP in real time. Both alleles significantly increased the intrinsic rate of GTP hydrolysis by RAC1, however hydrolysis in the presence of the ARHGAP15P (N‘Dama) allele was significantly faster than with the ARHGAP15H (Boran) allele over three replicate protein extractions and activity assays. Considering the critical role of RAC1 in macrophage activation, the more efficient negative regulation of RAC1 by ARHGAP15P observed in trypanotolerant N‘Dama cattle may underlie their reduced susceptibility to trypanosomiasis associated anaemia via reduced erythrophagocytosis. Determining the effect of this SNP in more natural situations in bovine trypanosomiasis will provide considerable insights into the physiological and genetic basis of trypanotolerance. Eventually, it will be possible to integrate disease resistance with productivity traits important to farmers in tsetse infested areas.