Investigation Into B. Melitensishfq Deletion Mutant Onvirulence And Immune Protective Effect

Brucellosis is one of the most common zoonotic epidemics worldwide. It is an enormous threat to people and worldwide economic construction. Brucella, the etiological pathogen of brucellosis, has unique virulence characteristics, including that no virulence components plasmid and exotoxin, the ability to survive within the host cell. Brucella is facultative intracellular pathogenic α-proteobacteria which causes undulant fever, arthritis, endocarditis and osteomyelitis in humans and abortion in domestic animals.RNA binding protein Hfq (host factor for RNA phage QP replicase) encoded by approximately half of all eubacterial species, is an important post transcriptional regulator of gene expression in bacteria. A great number of studies have shown that Hfq protein is a very important post transcriptional regulator, which can regulate the expression of target proteins. Combinng these studies about Hfq of intracellular pathogens, we find that chaperone Hfq protein may play an important regulatory role to adapt to the harsh environment of the intracellular survival, by affecting the expression level of a large number of target genes. In order to further clear Hfq and intracellular survival ability of Brucella, the relationship between the virulence and Hfq immune protective effect of B.melitensis 16M, we carried out the following experiments:Macrophage infection,mouse infection and expression of genes related to virulence during infection.Murine macrophage-like RAW264.7 cells were used to assess the survival capability of 16M,16M△hfq and 16M△hfq-C. RAW264.7 cells were infected with 16M,16M△hfq and 16M△hfq-C at a multiplicity of infection. At different time point, the supernatant was discarded, the cells were serially diluted and plated on TSA, and the CFUs were counted after 3～5 days.Female 6-8-week-old BALB/c mice were infected intraperitoneally with 200μL of bacterial suspension.At different time point post-infection mice were sacrificed by cervical dislocation, and the spleens were removed aseptically and homogenized with PBS,and the CFUs were counted after 3-5 days.Here, we analyzed the influence of hfq on genes related to virulence during mouse infection. Several important genes related to virulence,omp25, omp31, vjbR, htrA, gntR, dnaK,and their expression in 16M,16M△ hfq,16M△hfq-C during infection at different time points were analyzed.The results suggest that intracellular survival capabilities of the three strains were further confirmed. The survival of the 16M△hfqwas reduced in both macrophage and mouse model, being consistent with our previous results.Comparing to the expression level under in vitro conditions in TSB, the expression ofhfqin RAW264.7 cells indicated thathfq is mainly induced during the early stage of infection.To further analyze the expression of hfq during infection, BALB/c mice were infected with 16M, and the transcription levels of hfq were determined at different time points.The expression of hfqinBALB/c miceindicated that hfq is induced in the late stage of infection.Female 6-8-week-old BALB/c mice were immunized intraperitoneally with a bacterialsuspension containing approximately 2×107 CFU of 16MAhfq or PBS (negative control).45d post the immunization, the mice were challenged with 1×103 CFU of 16M. At 14 dand 28d post-challenge, the infected mice were sacrificed by cervical dislocation, and the spleens were removed aseptically and homogenized with 1mL of PBS containing 0.1%(v/v)TritonTM X-100.The homogenates were serially diluted and plated on TSA, and the CFUs were counted after 3～5 days.Serum samples were obtained from immunized miceat different time point, before challenge,14d and28 d post-challenge.Secretions of IL-2, IFN-y, IL-4 and IL-10 in the sera were determined by qPCR and cytokine ELISA kit essentially as recommended by manufacturer.The results suggest thatat 14 d, approximately 5.591og10CFU per mouse were isolated in the PBS group, while only 3.01log10CFU were isolated from hfq-immunized mouse. At 28d,5.191og10CFU were isolated from the PBS (control) group, but no bacteria were isolated for the 16M△hfq group.This indicated that immunization with 16M△hfqinduced protective immune response in the mice.The expression levels of cytokine genes were analyzed by qPCRand ELISA.Cytokine secretion analysis also showed levels of secreted cytokines also differed between PBS and 16M△hfq group.The secretion trends of the four cytokines were consistent with those of transcription levels between the two groups, but the extent differed.Infection with 16M△hfq induced differentimmune responses and conferred protection against futurechallenge by the virulent strain, indicating that 16M△ hfq might represent a good live attenuated vaccine candidate for brucellosis.