| BackgroundTuberculosis(TB)caused by Mycobacterium tuberculosis(M.tuberculosis),which is a most common chronic infectious disease.Tuberculosis is the most deadly infectious disease in the world.The only vaccine currently available for TB is the Bacillus Calmette-Guèrin(BCG),which offers effective protection against disseminated TB in children.However,the protective efficiency against TB is variable in adults.The expression of M.tuberculosis immunodominant antigens in BCG represents an important aspect in TB vaccine research.Though a lot of effort,currently no new vaccine is available to replace traditional BCG for TB control.Therefore,it is supposed to explore new strategies for constructing new vaccine superior to current BCG in protective efficiency and safety against TB.Cyclic di-adenosine monophosphate(c-di-AMP)is one of the second messengers in bacteria.Many researches have proved that c-di-AMP not only plays important roles in various physiological processes in bacteria,but also induces innate immune responses,mucosal immunity and specific adaptive immunity.Lots of researches,include ours,reported that recombinant M.tuberculosis with elevated c-di-AMP activates type I interferon response and induces autophagy,which showed significant attenuated virulence in mice.C-di-AMP is considered as a new pathogen associated molecular pattern(PAMP)now,and treated as a new target for bacteria vaccine.ObjectThe aim of this study is to construct recombinant BCG(rBCG)with elevated c-di-AMP level and study the biological characteristics of the rBCG and investigate the autophagy incduced by the rBCG in macrophage.Further the immunological characteristic of the rBCG is evaluated in mice model,as well as the protective efficiency against M.tuberculosis.Methods and results1.Construction and biological characteristics of rBCG-DisARv3586(disA)gene(1 100bp)was successfully amplified by PCR from M.tuberculosis H37Rv genome.The fragment of disA was subcloned into E.coli-mycobacterium shuttle vector PW54,and the recombinant plasmid named PW56.PW56 was electrotransformed into BCG competent cells and the recombinant strain was identified by PCR and Western blot.The results showed that 43kDa DisA was successfully expressed in rBCG,named rBCG-DisA.Nucleotides were extracted from BCG and rBCG-DisA strains,and the HPLC analysis showed that c-di-AMP was significantly elevated in rBCG-DisA(P<0.05).The growth rate of rBCG-DisA was as same as BCG in 7H9 medium.Ziehl-Neelsen acid-fast staining assay showed that the elevated c-di-AMP had no effect on dyeing and arrangements in rBCG-DisA.Transmission electron microscopy observation showed that the bacterial length of rBCG-DisA was reduced by 20.4%relative to that of wild type BCG.2.Autophagy of macrophage incudced by rBCG-DisARAW264.7 macrophages were stimulated with c-di-AMP(10μg/mL)or infected with BCG and rBCG-DisA(MOI=10)for 12h.Total RNA was extracted to detect the transcriptional levels of autophagy-associated genes by qRT-PCR.The results showed that c-di-AMP induced significant up-regulated mRNA levels of LC3,Beclin1,Atg5 and Atg7(P<0.01,P<0.05,P<0.05,P<0.01),among which Beclin1 and Atg7 were increased significantly in rBCG-DisA infection group than BCG group(P<0.01,P<0.05).Western blot results showed that bacteria infection didn’t change the expression of LC3 and p62 in RAW264.7 cells.Bacteria infections have no effects on LC3 expression in peritoneal macrophages of mice,except for substantially increased p62 expression in both BCG and rBCG-DisA infection groups(P<0.01,P<0.01).3.Immunological characteristics of rBCG-DisAFemale BALB/c mice were randomly divided into 4 groups:Na?ve,UN,BCG and rBCG-DisA.Each mice of BCG and rBCG-DisA group was inoculated with 1×10~6 CFU respectively bacteria in 0.1mL PBS subcutaneously,and the control group with 0.1mL PBS.There were no significant differences between groups in body weights of mice(P>0.05).After immunization,antibody levels in two immunized groups were significantly increased than control group(P<0.05).The proliferation of lymphocytes were significantly increased in two immunized groups(P<0.05,P<0.05),and that of rBCG-DisA group was increased more significantly than other groups with DisA protein stimulation(P<0.001).The percentages and the ratio of CD4~+and CD8~+T cells had no differences among groups(P>0.05).The transcriptional levels of IFN-γ,IL-2 and IL-10 in the spleen and lung of two immunized groups increased significantly(P<0.05),especially in rBCG-DisA group,of which IFN-γlevels were significantly higher than BCG group(P<0.05).The productions of IFN-γ,IL-2 and IL-10 in splenic lymphocytes increased in two immunized groups,and the IFN-γproduction increased more remarkably in rBCG-DisA group(P<0.01).The proportion of TNF-α~+from CD4~+T cells was significantly increased in rBCG-DisA group(P<0.05),IFN-γ~+IL-2~+from CD4~+T cells was on the rise,though not significantly(P>0.05).After 4 weeks of immunization,mice were injected with M.tuberculosis H37Ra5×10~4CFU in 0.1mL PBS for each by tail vein.After challenged with Mtb,the antibody levels of two immunized group continued to increase,and rBCG-DisA group was considerably higher than that of non-immunized group(UN group)(P<0.05).The lymphocyte proliferation of mice in two immunization groups were significant higher than those of control group(P<0.01).The transcriptional levels of IFN-γ,IL-2 and IL-10 in two immunization groups increased significantly,and the levels of IFN-γ,IL-2 and IL-10in rBCG-DisA group were remarkably higher than those in BCG group(P<0.01,P<0.05,P<0.05).The secretions of IFN-γ,IL-2 and IL-10 in rBCG-DisA group were more significantly than those in the BCG group(P<0.01,P<0.05,P<0.05).The pathological changes of lung tissue after Mtb infection in rBCG-DisA group were similar to those in BCG immunized group.Compared with the non-immunized group(UN group),the bacterial burdens in the lung and spleen of mice in two immunized group were significantly decreased(P<0.001,P<0.001),and there was no significant difference between two immunized groups(P>0.05).ConclusionsIn this study,rBCG-DisA with elevated c-di-AMP was successfully constructed.Intracellular c-di-AMP level affects the length of the bacteria.rBCG-DisA could significantly induce autophagy associated genes expression and reduce autophagy degradation.rBCG-DisA immunization could induce humoral immune response,and Th1cell immune response,which was characterized by an increase in IFN-γexpression.After Mtb infection,both Th1 and Th2 cell immune response levels further increased,among which the rBCG-DisA group was significantly higher than the BCG group.The protective effect of rBCG-DisA immune mice on Mtb infection was comparable to BCG immunization. |
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