Proteomics Study And Screening Of Protective Antigens For Brucella Vaccine Strain 104M

Brucella is a Gram-negative,facultative intracellular bacterium that causes zoonotic brucellosis in humans and various animals.Brucellosis is one of the most common zoonotic diseases that has become serious in recent years.Brucella is able to survive in the environment and has been identified as agents amenable for use in bio-terrorism.In this way,Brucella poses a serious threat to human health and biosecurity,and it merits study.At present,vaccination is the most effective approach to prevent and control brucellosis.Veterinary Brucella vaccines have been widely used and play an important role in the control of brucellosis epidemics.However,no human Brucella vaccine has been approved abroad.The 104 M strain is the only attenuated live vaccine that has been widely used in China for the control and prevention of human brucellosis.The main reasons for the difficulty in the development of human Brucella vaccines may be the multiple species and complicated protective antigens of Brucella.The availability of sequence data for entire bacterial genomes led to the advent of reverse vaccinology(RV).RV is a computational approach that can identify putative vaccine candidates in the protein coding genomes of pathogens.The establishment of a RV strategy allows researchers to predict,screen and validate all possible candidate protein antigens of the pathogen using histological data of pathogenic microorganisms.There has been some research about using RV to screen antigens from the protein coding genome of Brucella,but the relevant bacterial genome annotation has yet to be perfected.Recent developments in proteomics have enabled absolute protein levels to be measured on a system-wide level in microbes,making it possible to re-annotate genomes using tandem mass spectrometry data.Proteomics analysis can also be an effective approach to exploring the physiological metabolism and pathogenesis of Brucella.In this study,we utilized a proteogenomic approach that combined subcellular fractionation and peptide fractionation to perform a whole-proteome analysis and genome reannotation of 104 M using high-resolution mass spectrometry.Then,we used a quantitative proteome approach that described protein profiles of 104 M underlying hostile environments to better understand and characterize the molecular mechanism of intracellular survival of Brucella.In addition,we established a new protective antigen screening strategy based on the omics and bioinformatics.Potential vaccine candidates against Brucella were predicted and identified using this approach,which can be used for the development of novel vaccines against human brucellosis.In the study of proteogenomics,we utilized an approach that combined subcellular fractionation and peptide fractionation to perform a whole-proteome analysis and genome reannotation of 104 M using high-resolution mass spectrometry.In total,1,729 proteins(56.3% of 3,072)were identified.The distribution of identified proteins with respect to isoelectric point,molecular weight,hydrophobicity,transmembrane region of 104 M was consistent with the annotated proteins.Almost all of these COG categories(20/22)contain key functional proteins important for cell growth and reproduction were identified.Here,14 pivotal virulence factors and 17 known protective antigens were confirmed at the protein level following induction by the 104 M vaccine;which may help us elucidate the mechanisms that underpin residual toxicity and immune protection pertaining to the 104 M vaccine.Here,218 hypothetical proteins of 104 M that were functionally uncharacterized in preliminary studies were also confirmed at the protein level during this study;27 were predicted to be localized to the outer Membrane or extracellular;and 57 were predicted to be “probable antigens” or “virulent proteins” capable of influencing the bacterial virulence and vaccine protection.In this study,six novel ORFs and three modified existing ORFs were identified as part of this study following validation by reverse transcription-PCR,which improved the quality of the 104 M genome annotation.In addition,differences in protein abundance between the two chromosomes in 104 M were observed at the proteomic level,suggesting that changes in protein expression in large chromosomes may be more pronounced than in small chromosomes.We also explored the mechanism leading to this difference.In the study of quantitative proteomics,we used a label free quantitative proteome approach that described protein profiles of 104 M underlying hostile environments to better understand and characterize the molecular mechanism underlying intracellular survival of Brucella.In this study,1 normal laboratory culture control group(TSB);7 single-stress-condition scenarios: Serum stress,Nutrient starvation stress,Physical/chemical stress,Peroxide/nitric oxide stress,Oxygen deficiency stress,Iron-limited stress,Antimycobacterial stress;and 1 multi-stress condition scenario were used to simulate the hostile environments.The results showed that the survival rates of 104 M to differ under different stress conditions.Nutrient starvation,physical/chemical,peroxide/nitric oxide,and anaerobic stress showed greater impact than other stresses.The proteins of 104 M strain grown under each stress condition were identified using label-free mass spectrometry.A total of 2,289 proteins were identified in the 9 culture conditions.Among them,1,221 proteins were significantly different from the overall group under stress conditions.The differentially expressed proteins identified in this study were mainly enriched in oxidative phosphorylation,ABC transporters,two-component systems,biosynthesis of secondary metabolites,Porphyrin and chlorophyll metabolism,glycerol phospholipid metabolism,citrate cycle(TCA cycle),thiamine metabolism,nitrogen metabolism or associated with Carbon metabolism.This could provide further insight into the global physiological changes associated with distinct environmental conditions stress.In the study of screening protective antigens,we have summarized a number of factors associated with the prediction of protective antigens,and ultimately selected 6 related factors,subcellular localization,protectome,antigen,mature epitope density,virulence,and adhesion.These 6 factor indicators were scored,the resulting dates were standardized and normalized.Finally,all the proteins were sorted according to the synthesized scoring situation.The new protective antigen screening strategy “Multi-factor Prediction of Protective Antigens”(MPPA)was established.The results showed that it could effectively distinguish between protective antigens and non-protective antigens from the known antigens database.The predictive effect of MPPA is better than any other screening methods.A total of 26 high probability protective antigens in 104 M were screened by MPPA,which contained a number of known Brucella protective antigens,including Omp19,Omp25,Omp31,SodC and Invasion protein B.This further confirmed the feasibility of MPPA screening methods.At the same time,a number of potential new protective antigens,including outer membrane lipoprotein LipA,type I secretion system protein HlyD,type IV secretion system protein VirB8,efflux protein TolC,receptor protein TonB were reported.This provides basic data for the development of new Brucella vaccines.In the study of candidate antigens,four representative protective antigens Omp19,VirB8,HlyD,and Lip A were selected for the evaluation.Omp19,Vir B8,HlyD,and LipA recombinant proteins were obtained by recombinant expression and purification of Escherichia coli expression systems,respectively.The immunogenicity and protective efficacy were evaluated in a mouse model.The results of serological tests showed that Omp19,VirB8,and HlyD were able to stimulate mice to produce strong humoral immune response.The total antibody IgG titer was 8.2 × 105,3.0 × 105,and 3.3 × 106,respectively.The antibody subclass of Omp19 and VirB8 is mainly IgG1(Th2);while the antibody subclass of HlyD is IgG2a(Th1).Different levels of cytokines TNF-a,IFN-?,IL-4 and IL-10 were detected by immunizing mouse spleen cells with Omp19,VirB8 and Hlyd stimulation.The results showed that Omp19,VirB8,and HlyD can induce the body to produce TNF-?/IFN-? characterized Th1 type immune response,and IL-6/IL-10 characterized Th2 type immune response.Brucella A19 challenge test results showed that Omp19,VirB8,HlyD immunized mice can significantly reduce the bacteria CFU in spleen and liver,with partially protective ability.In conclusion,this study used a proteogenomic approach for whole-proteome analysis and genome reannotation in 104 M which helped to produce high-coverage database of Brucella proteomics and improve genome annotation quality.The results of our label free quantitative proteome analysis of 104 M under stress conditions to improve understanding of the proteins involved in the metabolic pathway response to multiple environmental stresses,which may improve understanding and characterize the molecular mechanism of intracellular survival of Brucella.The new protective antigen screening strategy “Multi-factor Prediction of Protective Antigens”(MPPA)was established,and several potential new protective antigens were obtained from 104 M.Three candidate antigens Omp19,Vir B8,and HlyD were able to stimulate the mice to produce strong specific humoral immune response and cellular immune response and showed partial protective ability.This may provide valuable clues towards development of an effective Brucella vaccine.