Abstract
TIRAP is an adaptor protein necessary for TLR4/2 inflammatory signaling in response to bacterial infection. The Brucella effector protein TcpB induces TIRAP degradation, down-regulating the immune response and enhancing Brucella intracellular replication. Two single nucleotide polymorphisms (SNPs, S180L y D96N) in TIRAP exon 5 (TIRAP-E5) were associated with an enhanced resistance to M. tuberculosis, P. falciparum and other infections in humans. The aim of this work was to evaluate if SNPs within caprine TIRAP-E5 influencethe susceptibility to B. melitensis infection in goats. A candidate-gene association study was conducted using 100 DNA samples from Brucella-seropositive (cases) and seronegative (controls) female creole goats. Animals were unvaccinated against Brucella spp. and belonged to ten flocks with prevalence of brucellosis (10 -72%). Genotyping of TIRAP-E5 was carried out by PCR and DNA sequencing. Genetic associations were evaluated by the Fisher’s exact test. Monocyte derived macrophages (MDMs) cultures were obtained from peripheral blood of seronegative goats, and infected with B. melitensis 16 M (MOI 1:10) for 30 min. At different times post-infection, intracellular Brucella counts and cytokine mRNA expression were determined by a gentamicine protection assay and qRT-PCR after RNA isolation, respectively. Sequence alignment of caprine and human TIRAP-E5 showed an identity of 76%. We detected three polymorphic SNPs (rs914(T/C), rs459(C/G) and S189N(G/A)) at TIRAP-E5 in the studied goat population. The rs914-C allele was significantly more frequent in controls than cases (p=0.002). Moreover, the genotypes rs914-CC+CT and the haplotype C-C-G (rs914- rs459-S189N) were associated with absence of Brucella-specific antibodies (p=0.001), while rs459-CG and T-C-A were associated with presence of Brucella-specific antibodies (p=0.043; p=0.045). Bioinformatic analyzes suggested that the associated polymorphisms could affect RNA folding and/or protein function. Besides, MDMs with the rs914-CT genotype controlled more efficiently Brucella intracellular growth at 24 h post-infection (p.i.) (p=0.02), and expressed higher levels of pro-inflammatory cytokines IL-12 e IL-18 at 4 h p.i. than rs914-TT MDMs (p < 0.05). Consistent with previous reports in humans, the results presented here suggest that genetic variability in TIRAP exon 5 would influence the outcome oBf.melitensis exposure in goats.
