Metabolism Characteristics Of Mycoplasma Ovipneumoniae And Mycoplasma Mycoides Subsp.Capri And Optimization Of Culture Medium Based On Transcriptomic Analysis

Mycoplasmal pneumonia of sheep and goats(MPSG)is a highl y contagious infectious disease that is prevalent among she ep and goats.In China,its main pathogens include Mycoplasma ovipneumoniae(Mo)and Mycoplasma mycoides subsp.capri(Mmc).Compared with Mmc,the culture for Mo is difficult providing low yield,which limits the conventional identifications such as bacterial culture,growth inhibition test,growth precipitation test,agglutination test,and antigen preparation.After a ll,bacterial culture is considered to be the "golden standard" for detecting pathogens,and vaccine therapy is an effective approach for preventing and controlling the disease.Furthermore,few effective quarantine measures,inactivated vaccines or attenu ated vaccine for these diseases were reported up till now.In this study,RNA-seq was performed to characterize the genes linked to metabolism of Mmc and Mo at different growth stages.RNA samples were collected at the logarithmic,stationary,decline,and late decline phases from Mmc PG3 strain and Mo NM151 strain.A point-to-point comparison between the corresponding phases of PG3 and NM151 strain was performed.The key enz ymes of the genes that were differentiall y expressed in onl y one coded species were screened.Hypothesis was proposed and validated based on the key requirements for Mo to fill the metabolic maps,which is significant to Mo culture and application.1.Based on the growth curve of Mo NM151 strain and Mmc PG3 strain,logarithmic phase(log),stable phase(sta)and decline phase(dec),latedec phase(latedec)and NM151 died phase(die)were selected.A total 27 RNA samples(9×3 replicated samples)were sequenced.2.The reads and saturation of PG3 at four different growth stages were qualified.During the four growth stages,445 differentiall y-expressed genes(DEGs)were screened and 64 DEGs were enriched into 28 metabolic pathways.Six major metabolic pathways were s tudied.At the logarithmic phase,the RPKM value of DEGs linked to gl ycol ysis and nucleotide metabolism were the highest;At the stable phase,DEGs associated with phospholipid,nicotinic acid and nicotinamide metabolism expression level graduall y increased;At the decline phase and late decline phase,DEGs related to gl ycol ysis and nucl eotide metabolism were expressed higher slightl y;the number of DEGs encoding the aminoacyl t RNA ligase was the most,and the RPKM value varied in the whole growth periods.Expression levels of genes encoding glutam yl-t RNA ligase was significantl y up-regulated in the stationary and decline period which promotes the s ynthesis of glutamic acid and adaptabilit y to the increasing osmotic pressure of PG3 strain.3.The reads number and saturation at five different growth stages of NM151 were qualified.In this way,307 DEGs were screened and 45 DEGs were related to metabolism.31 DEGs existed in the group of NM151-latedec vs.NM151-die.In the NM151-log vs.NM151-sta group,DEGs were mainl y invoved in gl ycol ytic or nucleotide metabolism.No DEG was enriched in gl ycerophospholipid metabolism.4.In this study,corresponding growth stages of standard strain PG3 and NM151 isolate were compared.First,from the view of DEGs in PG3 strain and NM151 strain,the expression levels of most genes in PG3 decreased in decline stage and increased slightl y in late-decline stage,while the NM151 gene descendent expressed with the culture time;90% “ Shared DEGs ” slighl y expressed in PG3 strain,including many metabolism key enz ymes: Dank protein,PTS transporter EII BC,galactose ribose/ABC transporters,ATP s ynthase alpha and beta subunit,fructose-2-phosphate aldolase-II and gl ycerol kinase;The number of ?Specific DEGs? in PG3 was higher than that of NM151.The proteins encoded by these genes would significantl y enhancethe abilit y of PG3 to transport nutrients a ndensure the smooth progress of key metabolism.5.According to results mentioned above,four kinds of medium were optimized for Mo were evaluated by CFU method.The results are listed as followed: compared with the control medium,the four culture media impr oved viable counts of Moin logarithmic phase.Modified Hyflick 's both supplemented with Hepes or serine doubled the maximum viable counts in 36 h,the viable counts in glucose medium reached the highest viable counts at 48 h;all of four media improved the Mo growth to the stable stage within 12 h;the Hepes addition maintained the p H values of media,the media with Hepes had the highest viable counts in the whole period of decline.The modified Mo culture medium was optimized through comparative transcriptome of Mo and Mmc at different growth stages.It improved Mo growth titer from 1 billion CFU/m L to 2 billion CFU/m L at logarithmic phase,prolonged stable period and maintened p H values of medium.With the growth titers on the modified medium,1 m L of Mo inactivated vaccine for each lamb can obtain good immune effects,and the whole preparation process of inactivated vaccine could be conducted without concentration operations,which simplifies the producing procedures and reduces production costs.This paper solves the cuture problem of Mo including difficult culture,low viable counts and difficult passages continuousl ywhich lays an essential foundation for the preparation of inactivated vaccines.