Screening And Identification Of Murine Macrophage Proteins Interacting With Omp25and VjbR Of Brucella Melitensis

Brucellosis is a worldwide zoonosis caused by brucella. At present, brucellosis isalways outbreak in developing countries. The disease is increasing from2000, whichcauses tremendous harm to animal and human. The obstacles for prevention andcontrolling of brucellosis are mainly unclear on the mechanism of pathogenesis andintracellular parasitic. Brucella could infect allied and non-allied macrophage, andthen survive and replicate. Macrophage is the first object of infection. The interactionof bacterial protein and host cell proteins might provide the condition for brucellaintracellular survival, but the mechanism is not uncovered.Omp25is one of Omp25/Omp31family proteins, which belong to the thirdgroup outer membrane protein of brucella. Brucella melitensis Omp25mutant couldnot cause portion, and could survive and replicate in vivo. The virulence and humoralimmunity of Omp25mutant compared parent strain was weakened. All thesesuggested that Omp25was not only virulent factors, but also immuno-protein. VjbR isa transcription factor of Qurum sensing system (QS) belonging to LuxR family, whichaffects IV secretion system (T4SS) and flagellum expression and play important rolesin virulence and intracellular survival.In this paper, we constructed cDNA library of Brucella melitensis16M infectingmurine macrophage, and screened and identified interaction proteins using Omp25and VjbR as bait protein. All these could lay the foundation for researching thepathogenesis and intracellular survival mechanism.First, we cloned Omp25and VjbR, and ligased to pSos vector and constructedbait vecter after detecting bait vector toxicity, activation and membrane localization.Second, we constructed the model of Brucella melitensis infecting murinemacrophage, and extracted total RNA and prepared mRNA for cDNA libraryconstruction, and then cDNA library was convered to pMyr vector.Finally, Co-transformed bait vectors and pMyr-cDNA into cdc25HSaccharomyces cerevisiae competents for screening interaction proteins, andverificated by co-immunoprecipitation. Results:1. Omp25and VjbR gene were cloned and connected with pSos vector, and thebait vectors were non-toxicity, non-self reactivation, and located at right membraneposition suitable for yeast hybrid test.2. By indirect immunofluorescence test and transmission electron microscopetechnology, the model of Brucella melitensis16M infecting murine macrophage wasestablished at the bacteria and cells ratio was500:1.3. cDNA library was constructed by Stratangen mRNA kits, and convered cDNAlibrary to pMyr bait vectors, and constructed pMyr-cDNA library. The plasmidcapacity was1.6×109cfu/mL, and recombination ratio was very high.The bait plasmid and pMyr-cDNA library were co-transformed into cdc25Hyeast competent for yeast hybrid. Two positive clones were screened, and thesequence showed that they were Mus musculus NADH dehydrogenase (ubiquinone)1alpha subcomplex, assembly factor2(NM₀01127346.1),Ndufaf2, and (Musmusculus ribosomal protein S5(NM₀09095.2),RPSs5. Ndufaf2could interact withOmp25, and RPSs5could interact with VjbR by co-immunoprecipitation. Datashowed:(1) Omp25interacted with Ndufaf2could enhance NADH concentration, andincreased NAHD activity, reducing the oxidative derivatives for brucella intracellularsurvival.(2) VjbR connected RPS5could inhibite RPS5activity, and inhibit apoptosisof murine macrophage providing brucella infection environment.In this study, the foundation was established for paint the network of proteinsinteraction of brucella and murine macrophage, which provide foundations for futurestudy on pathogenesis and intracellular parasitic mechanisms.