Molecular Mechanism Of Apoptosis Induced By The Outer Membrane Proteins Of Brucella Melitensis

Brucellae, gram-negative pathogens, are facultative intracellular bacteria, which mainly caused a kind of widespread zoonotic infectious disease--Brucellosis. It was identified as class B by World Organisation for Animal Health (OIE) and the Law on Prevention and Control of Infectious Diseases of the People’s Republic of China. Brucella priority replicates within phagocytic cells of the reticuloen dothelial system, and in the pregnant animal, inside placental trophoblasts. In reservoir animals, brucellosis could be mainly manifested included abortion, infertility and epididymitis. In humans, displaying a series of the clinical symptoms, such as fever, orchitis, hepatosplenomegaly, endocarditis, arthritis and meningitis et al, chronic infections may be lead to great damage on human important organs such as liver, spleen, kidney, joint, nervous system and hunman immune system. Some cases can lead to abortion, even though vertical transmitted from mother to infant. Under natural conditions, human infected Brucella is usually throuth direct contact with infected animals and consumption of unpasteurized dairy products. In China, Brucella mainly mixed infection, B. melitensis accounted for 80% of epidemic strains, B. abortus accounted for 10%, B. suis is less than 1%. According to CDC China, there was 57222 cases in 2014 and more than 59.7% increase compared with the number of 2009. According to the newest data, this number has increased to 56989 in 2015, which means the incidence rate raised to 4.1828/100000.Brucellae can grow within vacuoles of macrophages, dendritic cells (DCs) and placental trophoblasts. The process of bacteria infected host divided into five steps: adhesion, biochemical, invasion, survival and reproduction. The intracellular lifestyle of Brucella limits exposure to thehost innate and adaptive immune responses, sequesters the organism from the effects of some antibiotics, and drives the unique features of pathology in infected hosts:invades and disseminates in host tissue, and the chronic phase that can eventually result insevere organ damage and death of the host organism. Toll-like receptors (TLRs) is mainly expressed in the immune system including dendritic cells, macrophages and neutrophils and lymphocytes surface. Initiated host natural immune response of inflammation by identifying the expression in the broad spectrum of bacteria, viruses and fungi constituents PAMPs. Natural infection, macrophages are brucella infected target cells, because it express high abundance ratios of TLRs.Inside mononuclear phagocytic cells, Brucella reside in aspecial vacuole (Brucella-containing vacuole, BCV). The BCV will respectively interact with endosome, lysosome and endoplasmic reticulum (ER) exit sites toreach an ER-like compartment where bacteria will actively proliferate. In anin vitro brucellosis infection model, expression of a type IV secretion system (T4SS) early after infection is essential for intracellular survival and multiplication inside mammalian cells. Trophoblast cell damage such as inflammation, affect the implantation cause miscarriage. Brucella neither contain the typical pathogenic factors such as toxins, external enzyme protein, enzyme and other extracellular enzyme, nor express pathogenic factors such as fimbriae, flagella, antigenic variation, plasmid or lysogenic phages. So after immune antibody fully recognize Brucella antigen, the bacteria activates the host immune response caused tissue damage. TLR2 is activated by lapidated outer membrane proteins (L-OMP16 and L-OMP19); TLR4 is activated by B. abortus LPS and unlipidated outer membrane protein-16 (U-OMP16); and TLR9 is activated by B. abortus DNA. TLR activation leads to intracellular signaling via MyD88 and IRAK-4 resulting in the activation of NF-kB and MAPKs producing inflammatory cytokines.The relative involvement of TLR2 and TLR4 in mediating B. abortus induced cytokine production merits discussion. HKB A induces production of TNF-a and IL-6 via TLR2. LPS is not a mediator of the proinflammatory activity of HKBA, and provided, in addition, proof of concept that B. abortus lipoproteins would be the TLR2 ligands used by the bacterium to trigger the release of pro-and anti-inflammatory mediators. L-OMP16 and L-OMP19 induced the secretion of TNF-a, IL-6, IL-10, and IL-12 in a time-and dose-dependent fashion. Neither U-OMP16 nor U-OMP19 induced cytokine secretion in THP-1 cells, demonstrating that acylation of the lipoprotein moleculeis required for the production of cytokines in cells of the monocyte or macrophage lineage. L-OMP19 induced endothelial cells secret IL-6, chemotactic factor and adherence factor. HKBA and L-OMP19 induceastrocyte proliferation and apoptosis via TNF receptor (TNFR) 1, promote the secretion of IL-6, IL-1β,TNF-a, MCP-1 and KC. Many reports have shown that OMPs is immune protective antigen, interacted with virulence genes, so many studies of bacterial pathogenic mechanism and the immune response mechanism focuses on the study of OMPs. Brucellae as facultative intracellular bacteria, trophoblast cells and macrophage cells are Brucella’s host cells, which OMPs interacted with signaling proteins in natural immune TLR signal pathway? Led to bacteria survival and replicate in trophoblast cells and macrophage cells via negative regulation mechanism of TLRs? How do OMPs play role on infected cells’two opposite fate:first, cause the host immune tolerance and resistance to apoptosis; second, cause pregnant animal abortion? The biological function and mechanism of different forms of OMP16 and OMP19 is not clear.Objective:Aim to study the relation of Brucella OMPsand apoptosis of macrophages and/or trophoblast cells and the mechanism, to find out the intracellular proteins which interacted with OMPs to discover the new virulence factor during Brucella invasion host cells, to provide reference for prevetion and control of Brucella via understanding the molecular mechanism and characteristic of Brucella intracellular survive pathway.Methods:(1) Preparation of B. melitensis lipoproteins:B. melitensis lipoprotein’s genes were amplified from M5-90 genome. The fragment was expressed using prokaryotic expression system pET-28a (+) E.coli. The expression of the fusion protein was confirmed by SDS-PAGE and Western-Blot. Proteins was purified by inclusion bodies resolution, refolding and the affinity chromatography of Ni-NTA.(2) Apoptosis induced by B. melitensis OMPs:B. melitensis OMPs incubated with Huh7.5.1, JEG-3, nonactivated THP-1 and activated THP-1 respectively, collected cells to test apoptosis rate by flow cytometry after 24,48 and 72 hours.(3) B. melitensis OMPs induced the secretion of cytokines:B. melitensis OMPs incubated with Huh7.5.1, JEG-3, nonactivated THP-1 and activated THP-1 respectively, collected cell culture supernatants to test the level of cytokines by ELISA.(4) Detection of the apoptosis related proteins:B. melitensis L-OMP19, U-OMP19, L-OMP16, U-OMP16 and U-OMP10 incubated with activated THP-1 respectively, collected cells after 72h. Cell lysis and measure protein concentration by BCA, send to RayBiotech detecting related apoptosis protein chips.Results:(1) Preparation of B. melitensis lipoproteins:Amplified B. melitensis lipoproteins gene from the M5-90 vaccine strain genome. Amplication of L-OMP10 and U-OMP10 were about 381bp and 324bp, L-OMP16 and U-OMP16 were about 507bp and 435bp, U-OMP19 was about 510bp. By analyzing the sequence we found that there are bacteria lipoprotein precursor in lipoproteins’ amina acid sequence:amino terminal signal peptide end with a four peptide sequence, accord with the form lipoprotein shearing and processing the required sequence. The fragment were expressed in pET-28a(+) E.coli. Except L-OMP10 the others expressed successfully. The molecular weight of U-OMP10 fusion protein was about 15kDa, L-OMP16 and U-OMP16 were about 18kDa and 17kDa, U-OMP19 was about 23kDa, which were identical to the theoretical size that prognosticated by Western Blot. Inclusion body were washed, dissolved and then refolded, while secretory protein were purified with Ni-NTA.(2) Apoptosis induced by B. melitensis:FCM results show that B. melitensis OMP31, BP26, OMP25, L-OMP19, U-OMP19, L-OMP16, U-OMP16 and U-OMP10 didn’t induce apoptosis of Huh7.5.1 and JEG-3. B. melitensis unlipidated U-OMP16 induced apoptosis of nonactivated THP-1, which is time-dependent, the apoptosis rate increased with time, while the other OMPs couldn’t induce apoptosis of nonactivated THP-1. L-OMP16, U-OMP16 and U-OMP10 incubated with cells at 24h, activated THP-1 began to show the phenomenon of apoptosis, the apoptosis rate increased with time, the phenomenon of apoptosis is most obvious induced by U-OMP16. BP26 and U-OMP19 can induce apoptosis of activated THP-1 at 48h, while the apoptosis rate induced by BP26 is up to 26% at 72h. L-OMP19 only induced a little apoptosis at 72h. OMP31 and OMP25 couldn’t induce apoptosis of THP-1.(3) The expression of cytokines induced by B. melitensis:U-OMP16 induced nonactivated THP-1 secret IL-6, TNF-a, CXCL, MCP-1, IL-1β and IL-8. Except IL-8 and TNF-a, the level of the others cytokines increased. IL-6 and CXCL increased with time, while MCP-1 and IL-1β declined after 48h. Activated THP-1 cells could secret MCP-1. BP26, L-OMP19 and U-OMP19 only induced activated THP-1 secret low level of TNF-a, IL-1β and IL-6. L-OMP16, U-OMP16 and U-OMP10 could induced relatively high level of TNF-a, IL-1β and IL-6. B. melitensis OMPs incubate with activated THP-1 cells, the level of all of cytokines didn’t changed obiviously with time.(4) Screen apoptosis related proteins in THP-1 cells:Protein chips results show that B. melitensis lipoproteins L-OMP19, U-OMP19, L-OMP16, U-OMP16 and U-OMP10 can induce the expression of Bcl-2 decreased, while the expression of HSP60, caspase3 and cytoC increased. Only L-OMP19 and U-OMP19 can induce the level of XIAP increased, meanwhile U-OMP19 can induce the level of SMAC increased. Except L-OMP19, the others can induce the level of HSP70 and p53 increased. Western Blot results shows that L-OMP19 can decrease the expression of XIAP, p-Bcl-2, Bcl-2 and Bax, and increase the expression of cytoC as time goes on. While U-OMP19 can increase the expression of p-Bcl-2, Bcl-2, Bax as time goes on. L/U-OMP16 can increase the expression of p-Bcl-2, Bcl-2, Bax and cytoC as time goes on.Conclusion:(1) Recombinant proteins L-OMP10, U-OMP10, L-OMP16, U-OMP16 and U-OMP19 were successfully obtained.(2) Reveal that B. melitensis OMPs OMP31, BP26, OMP25, L/U-OMP19, L/U-OMP16 and U-OMP10 didn’t induce apoptosis by incubated with Huh7.5.1, JEG-3 via FCM.(3) Except OMP31 and OMP25, the other B. melitensis OMPs, including BP26, L-OMP19, U-OMP19, L-OMP16, U-OMP16 and U-OMP10 can induce activated THP-1 apoptosis and promote the expression of TNF-a, IL-1β and IL-6. U-OMP16 had the strongest effect. It suggested U-OMP16 may be the key virulence factor in apoptosis, while OMP31 and OMP25 didn’t induce apoptosis.(4) OMP19 may has a dual regulatory role in the process of cell apoptosis, U-OMP19 whether depend on SMAC inhibit XIAP function lead to apoptosis need to further study.(5) According to the result of protein chips, we suppose that B. melitensis lipoproteins maybe induce apoptosis through the mitochondrial pathway, while apoptosis related protein and specific mechanism still need to further study. All of B. melitensis lipoproteins L/U-OMP19, L/U-OMP16 and U-OMP10 can induce the expression of HSP60 remarkably increased.