Using a Relative Quantitative Proteomic Method to Identify Differentially Abundant Proteins in Brucella melitensis Biovar 3 and Brucella melitensis M5-90

Abstract

Brucellosis, caused by Brucella spp., is one of the most widespread bacterial zoonoses worldwide. Vaccination is still considered the best way to control brucellosis. An investigation into the differential proteome expression patterns of wild and vaccine strains may help researchers and clinicians differentiate between the strains to diagnose and better understand the mechanism(s) underlying differences in virulence. In the present study, a mass spectrometry-based, label-free relative quantitative proteomics approach was used to investigate the proteins expressed by the wild strain, B. melitensis biovar 3 and compare it with those expressed by B. melitensis M5-90. The higher level of virulence for B. melitensis biovar 3 compared to B. melitensis M5-90 was validated in vitro and in vivo. A total of 2133 proteins, encompassing 68% of the theoretical proteome, were identified and quantified by proteomic analysis, resulting in broad coverage of the B. melitensis proteome. A total of 147 proteins were identified as differentially expressed (DE) between these two strains. In addition, 9 proteins and 30 proteins were identified as unique to B. melitensis M5-90 and B. melitensis biovar 3, respectively. Pathway analysis revealed that the majority of the DE proteins were involved in iron uptake, quorum sensing, pyrimidine metabolism, glycine betaine biosynthetic and metabolic processes, thiamine-containing compound metabolism and ABC transporters. The expression of BtpA and VjbR proteins (two well-known virulence factors) in B. melitensis biovar 3 was 8-fold and 2-fold higher than in B. melitensis M5-90. In summary, our results identified many unique proteins that could be selected as candidate markers for differentiating vaccinated animals from animals with wild-type infections. BtpA and VjbR proteins might be responsible for the residual virulence of B. melitensis M5-90, while ABC transporters and thiamine metabolism associated proteins may be newly identified Brucella virulence factors. All of the identified DE proteins provide valuable information for the development of vaccines and the discovery of novel therapeutic targets.

Keywords: Brucella melitensis M5-90; Brucella melitensis biovar 3; Brucellosis; proteomics; vaccine.

Figure 1

Investigation of the phylogeny of B. melitensis biovar 3 and B. melitensis M5-90, and identification of survivability of B. melitensis biovar 3 and B. melitensis M5-90 in vitro and in vivo. (A) Phylogenetic tree of the IS711 concatenated sequence of B. melitensis biovar 3 (●) isolated from milk or aborted sheep fetuses in a previous study and reference sequences from B. melitensis retrieved from the GenBank database. The black triangle (▲) represents Brucella melitensis M5-90. The tree was constructed according to the neighbor-joining (NJ; 500 bootstrap replicates) and maximum-likelihood (ML, 1000 bootstrap replicates) analyses using MEGA7. The scale bar represents the inferred substitutions per nucleotide site; (B) RAW264.7 cell lines were infected with B. melitensis biovar 3 or B. melitensis M5-90 for 4, 12, 24, 48h. The CFU was determined on TSA at each time point; (C) The Balb/c mice were infected with B. melitensis biovar 3 or B. melitensis M5-90 and PBS (negative control) with four mice in each group. At 7, 14, 21, 28 days post-infection, the spleen was isolated and individual spleens were assessed for colonization and weight index (D). Splenic weight was calculated as organ weight (in grams) per gram of mouse body weight.

Figure 2

Identification of DE proteins of B. melitensis biovar 3 and B. melitensis M5-90. The volcano plot also shows the DE proteins of B. melitensis biovar 3 and B. melitensis M5-90; red points represent hDE proteins and blue points represent lDE proteins. The protein dataset in this study is classified according to the relative abundance of the protein entries, calculated on the inter-sample (i.e. Y3 vs M5) basis.

Figure 3

Classification of GO secondary annotation and subcellular location of DE proteins. (A) The biological function of DE proteins was characterized by biological process, cellular component, and molecular function; (B) The pie chart shows the proportion of subcellular location of DE proteins.

Figure 4

Expression of BtpA and vjbR genes in RAW264.7 cells. RAW264.7 cell lines were infected with B. melitensis biovar 3 or B. melitensis M5-90 for 4, 8, 12, 24, 48 h. BtpA (A) and vjbR (B) gene expression were analyzed using real-time quantitative RT-PCR and normalized by the expression of 16 S.