Bovine And Horse SP110 Gene Identification And Their Association With Tuberculosis Susceptibility

Bovine Tuberculosis(BTB)is a disease caused by chronic bacterial infection,and causes major economic burden and raises public health concern worldwide.A recent investigation of prevalence of bovine tuberculosis from china showed the positive rate of TB-infected cow was up to 6.4% with a total of 2201 blood samples collected from 14 provinces.Although the project of TB eradication from cattle herds has made great achievement in developed countries,the worldwide prevalence of bovine TB still remains an estimated annual loss of more than 3 billion US dollars.The process to reach TB-free bovine herd status was coupled with the relatively high cost of compulsory skin testing and slaughter program,which indicates a need to investigate more sustainable control strategies.As a comparatively hardy kind of animal,horses were considered to be naturally highly resistant to mycobacterium tuberculosis(MTB)infections,although rare tuberculosis infection cases in horses have been reported.For a better understanding of the horse immune system during infection,the availability of species specific reagents for analyzing the major host immune regulatory proteins are essential.The nuclear body protein,SP110,has previously shown to be a genetic determinant of host resistance to the intracellular pathogen infection in mouse and human.However,its relevant biological information in large non-primate animals still remains unknown.Comparing the potential defferent functions of Bovine and Horse SP110 on macrophage resistance to MTB,might contribute to reveal the genetic reason to different host tuberculosis susceptibility and find a new way to produce anti-tuberculosis bovine breed.1.Here we report the novel discovery of three bovine SP110 transcript variants.The variant SP110? with the longest open reading frame translates into the complete form of SP110 containing four functional domains.SP110? variant has partial deletion of the C terminus.And SP110? transcription halts before the SAND domain,leaving the whole C terminus untranscribed.2.Here we report the novel discovery and characterization of three transcript variants of horse SP110.The transcript variant 1(Tv1)of horse SP110 with the longest open reading frame has four domains(Sp100,SAND,PHD and Bromo domain).Tv2 and Tv3 share the same N-terminal sequence as Tv1,which contains Sp100 and SAND.We show that Tv2 is generated from alternative splicing and deletion of Exon17-Exon18 segment,while Tv3 is generated by pre-mature transcriptional termination at Exon16.3.Bovine and Horse SP110 promoters were cloned,and luciferase activity analysis demonstrated that bovine and horse SP110 promoters,just like human and mouse SP110 promoters,have cell specific activities.LPS could reduce the transcription ability of all four SP110 promoters,and MTB could increase the transcription ability of all four SP110 promoters.There was no significant different transcription ability between Bovine and Horse SP110 promoters under certain conditions.4.Accordingly,ectopic expression of bovine SP110 ? and ?,but not ?,significantly inhibited the survival of MTB in macrophages.The anti-MTB activities of SP110 ? and ? are associated with increased phagolysosomes fusion and enhanced inducible nitricoxide synthase(iNOS)activity.Besides,we demonstrate that cathepsin B,D,L,or Caspase-1 are necessary for SP110 ? and ? mediated increase in MTB-phagocytosing and growth-inhibiting capacity of macrophages.5.Furthermore,we demonstrate that the heterologous expression of horse SP110 variants stimulate macrophages into a tuberculostatic activation phenotype.The macrophages underwent a shift in enhancing the secretion of cytokines(interleukin-1(IL-1)and TNF-?)and accelerating inducible nitric oxide synthase(iNOS)activity,and eventually went into apoptotic cell death to eradicate mycobacterial tuberculosis.Intriguingly,horse SP110 Tv1 showed more capability to trigger the immune activities compared to Tv2 and Tv3.6.Comparison tests showed different variants of bovine and horse SP110 all have activation effects on macrophages.There were no significant differenct activation effects between Bovine SP110? and Horse SP110-Tv1,or between Bovine SP110? and Horse SP110-Tv2.However,there were differenct immune function in the three transcripts,Bovine SP110?,? and ?.7.SP110 gene expression model analysis showed that both horse SP110 isofroms have basic expression level in horse monocytes and could be increased when MTB infection,while the bovine SP110? is the main expressioin isoform wether the MTB infection or not.A-rich embedded motif,just after 3' UTR cleavage site of SP110 ? and ?,could significantly contribute to gene transcription termination,which might lead to premature of bovine SP110.Although there are different genetic elements between bovine and horse,we found that there were no functional difference between bovine and horse SP110,including the promoter activity and gene immune effect.Interestingly,there were different expression models between bovine and horse SP110.In cattle monocytes,the defective SP110? transcript variant is the mainform of SP110 gene expression,which may be the cause for cattle's deficiency of the protective functions of SP110 towards TB infection.