| Brucellosis is a zoonosis, which not only seriously affects the development ofaquaculture, also poses human health a huge threat. Prevention and control of humanand animal brucellosis is difficult for many reasons, but pathogenesis (including themechanism of intracellular parasitism) and immune mechanism are unclear that is themain reason.Lipopolysaccharide (LPS) is one of the major Brucella virulence factors, and isone of the major antigens in mammals immunized. Currently Brucella LPSsynthesis pathway is not completely clear, and several critical LPS synthesis-relatedgenes have been found, wbkA, gmd, wzm, wzt, wbkB, wbkC et al., in which wzm andwzt genes are major components.ABC transporter, in bacterial cells, is a major transporter of the machine consistsof a series of homologous genes encoding an operon. The operon is substantiallycomposed of three parts, ATP-binding proteins, membrane proteins, protein subunits,and is typically formed two integral membrane proteins by six transmembranesegments. ABC transport system is one important transport corridors in outermembranes on biological macromolecules synthesized, involved in the synthesis ofextracellular polysaccharides. In addition, ABC transporters also play an importantrole in transporting nutrient and exporting some molecules. However, the effect of theABC transporter system on bacterial virulence, immunogenicity, physiologicalmetabolism needs to be proved.In this study, a plasmid was constructed for knockout wzm/wzt gene by allelicexchange methods, and LPS deletion mutants were constructed. The spleen index and CFU of infected mice spleen was evaluated. And the differences of the parent strainand mutants on antibody level, cytokine level, lymphocyte proliferation and proteindifferences by2D electrophoresis. The effect of gene mutation on virulence,immunogenicity, and gene expression was evaluated, in order to further reveal thepathogenesis and immune mechanism of Brucella, and lay the foundation for thedevelopment of the rough Brucella vaccine.The results showed:1. The lethal plasmid was constructed for wzm/wzt knockout, and amplified byPCR and digested by restriction enzymes, containing sacB gene as a reverse selectionmarker, wzmf/wztf and wzmr/wztr arms of target genes fragments. The allelicexchange plasmid was electro-transformed into vaccine strain B. abortus S19, andscreened on TSB agar solid medium (kanr) with5%sucrose, and then screened byPCR amplification. The mutant sites were confirmed correct, and511bp,446bp genewere deleted. The specific primers multiple-PCR identification results showed asBrucella. LPS crude extracts of two mutant strains were analyzed by SDS-PAGE, andthe results showed that the mutants LPS molecule distribution is missing. Acridineorange agglutination of mutants and the test of serum by Rose Bengal plateagglutination test proved O antigen deleted on bacteria as rough mutants.2. The results showed that the logCFU of infected spleen of S19, Δwzm andΔwzt strains were4.11±0.31,2.86±0.52, and2.52±0.41, and the spleen indexswere1.30±0.23,0.67±0.12, and0.62±0.52respectively. And the Δwzm and Δwztmutants were decreased significantly compared to the parental strain S19, thatindicated wzm/wzt gene deletion caused virulence decline.3. The lymphocyte proliferation and lymphocyte transformation were analyzedthat Δwzm and Δwzt mutants induced lymphocyte CD4+/CD8+ratio higher than theparental strain S19, but the number of CD3+lymphocytes and lymphocyteproliferation decreased compared with the parent strain. IgG antibody levels detectedby ELISAin serum showed immunized antibody levels of Δwzm and Δwzt mutants in mice were higher compared to the parental strain S19, and longer maintained durationof the antibody. Mice spleen lymphocyte cells of mutants cultured in vitro to produceIFN-γ, IL-2, IL-4, IL-10, TNF-, and NO showed different degrees of declinecompared with the parental strain S19. And Δwzt mutant induced lower production ofIFN-γ thanΔwzm mutant.4. Results showed that wzm/wzt gene deletion caused2,3-bisphosphoglycerate-dependent phosphogl-ycerate mutase, glyceraldehyde-3-phosphate dehydrogenase,Acetyl-coenzyme A carboxylase carboxyltransferase subunit alpha,50S ribosomalprotein L9, Phosphomethyl-pyrimidine kinase, ATP/GTP-binding protein, and ABCtransporter substrate-binding protein expression down-regulation. wzm deletioncaused the decline of Nucleoside diphosphate kinase, fructose-bisphosphate aldolase,glyceraldehyde-3-phosphate dehydrogenase, Ribosome recycling factor,Thiamine-phosphate pyrophosphorylase, Glycine oxidase ThiO proteins, while causedThiazole synthase, Molybdate ABC transporter, Two component transcriptionalregulator, porin omp2b,6,7-dimethyl-8ribityllumazine synthase protein up-regulated.wzt gene deletion caused6-phosphogluconolactonase, electron transfer flavoproteinsubunit alpha, DNA protection during starvation protein, Superoxidedismutase[Cu-Zn],50S ribosomal protein L25, Aldo-keto reductases, Protein-exportprotein secB, ATP-dependent Clp protease proteolytic subunit, Twin-argininetanslocation pathway signal sequence domain-containing protein down-regulated,while caused TRAP transporter solute receptor, FeS assembly ATPase SufC,ubiquinol-cytochrome c reductase, iron-sulfur subunit, elongation factor Tsup-regulated.These results indicated that Brucella wzm and wzt genes deletion caused roughmutants, and further described that wzm and wzt genes were involved in bacterialLPS-O side chain synthesis. The virulence of two mutant strains was decreased thanthe parental virulent strains that revealed wzm and wzt genes were related to bacterialvirulence. Two mutants could induce higher antibody titer than the parent, but decreased levels of cell-mediated immunity, which indicated wzm and wzt genesaffected the bacterial immunogenicity. Comparative proteomics study found that manyproteins expression are affected by wzm and wzt genesand mostly decreased, whichneed further study on the regulation of protein expression. |
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