Identification Of Brucella Secreted Effector Specially Interacting With Small GTPase Rab1A

Brucellosis is a classical zoonotic disease caused by Brucella infection. Brucellais an intracellular bacterial pathogen that causes abortion and infertility in mammalsand leads to a debilitating febrile illness that can progress into a long lasting diseasewith severe complications in humans. Its virulence depends on survival andreplication properties in host cells. However, the underlying mechanism of Brucellaintracellular survival remains unclear. Although it has been reported that theformation of replicative Brucella-containing vacuole (rBCV) is crucial for survivaland replication of Brucella, how rBCV is formed in host cells remains unknown untilnow.Rab is the largest member of the small GTPase superfamily, which is thought toact as molecular switches that regulate member transport or formation. Moreimportantly, Rab1A, one member of Rab, plays a key role in mediating the Brucellaintracellular replication. Therefore, we propose that the effective proteins secreted byBrucella could coopt Rab1A for the formation of rBCV to regulate its intracellularsurvival.To confirm our hypothesis, the following experiments were performed. Firstly,the J774A.1macrophage of Rab1A knock-down was constructed through lentiviruspackage’s method. Briefly, the recombinant lentivirus was packaged by co-tranfecting293T cells using expression plasmid, packaging plasmid and envelope plasmid. Theexpression of protein Rab1A was detected by Western blotting. The role of Rab1A inregulating Brucella growth was measured via comparing colony formation units (CFU)between wild type and Rab1A knock-down J774A.1. Then mice Rab1A was obtainedby RT-PCR. Rab1A [S25N] and Rab1A [Q70L] site mutant genes were constructedvia ordinary molecular clone technology, and subsequently were sub-cloned intoprokaryotic expression vector pGEX-4T-1to create pGEX-4T-1-Rab1A, pGEX-4T-1-Rab1A[S25N] and pGEX-4T-1-Rab1A[Q70L], respectively. IPTG was utilized toinduce the expression of GST-Rab1A, GST-Rab1A[S25N] and GST-Rab1A[Q70L],which were purified by agrose affinity column. And then these purified GST fusionproteins were incubated with cytosolic proteins from wild type. The different bands were identified through SDS-PAGE electrophoresis and silver staining. Thesignificantly different bands were chosen for LC/MALDI-TOF through comparisonand analysis.The potential candidates were screened further by bioinformatics amongthe Result of mass spectrometry. Finally, co-immunoprecipitation (CO-IP) andimmunofluorescence (IF) were utilized to verify interaction between the effector andRab1A in vitro.Identification of restriction enzyme digestion and DNA sequencing showed thelentiviral vector was successfully constructed. The green fluorescent cells wereobserved under the fluorescence microscope48h after the recombinant lentivirusestransduced cells, which demonstrated that the recombinant lentiviruses knocking-down Rab1A gene were packaged successfully. Rab1A expression was decreasedsignificantly in Rab1A lentivirus infected J774A.1cells. The number of intracellularbacteria (CFU) was increased in the Rab1A knock-down J774A.1cells. GST Pull-down, bioinformatics, and LC/MALDI-TOF showed that effector protein is HSP70;Results from CO-IP and IF further testified the Rab1A interaction of Brucella effectorprotein HSP70. These studies potentially give an insight into how to manipulate thebacterial effector to achieve more efficient elimination of intracellular Brucella. Thisis a topic of significance in the development of drugs to prevent and treat Brucellosis.