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Frontiers of Agricultural Science and Engineering

ISSN 2095-7505

ISSN 2095-977X(Online)

CN 10-1204/S

Postal Subscription Code 80-906

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Front. Agr. Sci. Eng.    2014, Vol. 1 Issue (4) : 331-340    https://doi.org/10.15302/J-FASE-2014032
RESEARCH ARTICLE
Attenuation mechanism of Brucella melitensis M5-10, implications for vaccine development and differential diagnosis
Yuanqiang ZHENG1,4,Yanchun SHI2,Chang AN4,Ruisheng LI3,Dongjun LIU1,Yuehua KE4,Kairong MAO5,Mingjuan YANG4,Zeliang CHEN2,4,6,*(),Shorgan BOU1,*()
1. National Research Center for Animal Transgenic Biotechnology, Inner Mongolia University, Hohhot 010021, China
2. Inner Mongolia Key Laboratory of Molecular Biology, Inner Mongolia Medical University, Hohhot 010058, China
3. Animal Laboratory Center, 302 Hospital of PLA, Beijing 100039, China
4. Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing 100071, China
5. China Institute of Veterinary Drug Control, Center for Veterinary Drug Evaluation, Ministry of Agriculture, Beijing 100081, China
6. College of Medicine, Shihezi University, Shihezi 832003, China
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Abstract

Brucellosis is a worldwide zoonosis. Vaccination is the most efficient means to prevent and control brucellosis. The current licensed attenuated vaccines for animal use were developed by sequential passage in non-natural hosts that decreased virulence in its original hosts. The attenuation mechanism of these strains remains largely unknown. In the present study, we sequenced the genome of Brucella melitensis vaccine strain M5-10. Sequence analysis showed that a large number of genetic changes occurred in the vaccine strains. A total of 2854 genetic polymorphic sites, including 2548 SNP, 241 INDEL and 65 MNV were identified. Of the 2074 SNPs in coding regions, 1310 (63.2%) were non-synonymous SNPs. Gene number, percent and N/S ratios were disproportionally distributed among the cog categories. Genetic polymorphic sites were identified in genes of the virB operon, flagella synthesis, and virulence regulating systems. These data indicate that changes in some cog categories and virulence genes might result in the attenuation. These attenuation mechanisms also have implications for screening and development of new vaccine strains. The genetic changes in the genome represent candidate sites for differential diagnosis between these vaccine strains and other virulence ones. Transcription analysis of virulence genes showed that expression of dnaK, vjbR were reduced in M5-10 strain when compared with that in 16M. A duplex PCR targeting virB6 and dnaK was successfully used to differentiate between M5-10 and the virulent 16M strain. The genome re-sequencing technique represents a strong strategy not only for evaluation of vaccines, but also for development of new vaccines.
Keywords Brucella      live attenuated vaccine      attenuation      differential diagnosis     
Corresponding Author(s): Zeliang CHEN,Shorgan BOU   
Online First Date: 16 January 2015    Issue Date: 10 March 2015
 Cite this article:   
Yuanqiang ZHENG,Yanchun SHI,Chang AN, et al. Attenuation mechanism of Brucella melitensis M5-10, implications for vaccine development and differential diagnosis[J]. Front. Agr. Sci. Eng. , 2014, 1(4): 331-340.
 URL:  
https://academic.hep.com.cn/fase/EN/10.15302/J-FASE-2014032
https://academic.hep.com.cn/fase/EN/Y2014/V1/I4/331
Fig.1  Length and coverage distribution of the assembled contigs. (a) Length distribution of the assembled contigs; (b) average reads coverage of the contigs.
Variant type Number Coding region Non-coding region Non-synonymous Synonymous
SNP 2548 2074 474 1310 764
MNV 65 50 15 – –
INDEL 241 151 90 – –
Tab.1  SNP, MNV and INDEL sites identified in the genome sequence
Reference Base no. Variant Base Variant no. Syn no. Non-syn no. Frequency/% Transition/% Transversion/%
A 513 C 92 24 68 17.9 – 17.9
G 381 152 229 74.3 – 74.3
T 40 10 30 7.80 7.80 –
C 558 A 99 28 71 17.7 17.7
G 99 20 79 17.7 17.7 –
T 360 153 207 64.5 – 64.5
G 506 A 341 141 200 67.4 – 67.4
C 81 26 55 16.0 16.0 –
T 84 18 66 16.6 – 16.6
T 497 A 36 7 29 7.24 7.24 –
C 373 161 212 75.1 – 75.1
G 88 24 64 17.7 – 17.7
Tab.2  Base change frequency of SNPs
Indel type Number CDS Intergenic
Deletion 1 126 79 47
Deletion 2 12 4 8
Deletion 3 5 1 4
Deletion 4 2 1 1
Insertion 1 91 3 88
Insertion 2 4 2 2
Insertion 3 1 0 1
Tab.3  Distribution of small deletions and insertions
Fig.2  Distribution of SNPs in genes of different functional categories. (a) Gene number of different COG categories; (b) percent of genes with genetic changes in each of the cog categories; (c) N/S ratio for each of the cog categories. N/S: the ratio of non-synonymous to synonymous SNPs; Syn: synonymous; Non-syn: non-synonymous.
Fig.3  Reduced expressions of virulence genes and differential diagnosis by PCR. (a) Relative expression of selected virulence genes in M5-10 compared with 16M; (b) duplex amplification of M5-10 and 16M. Lane 1, 3: amplification of virB6; lanes 2, 4: amplification of dnaK; lanes 5, 6: duplex amplification of virB6 and dnaK. Lanes 1, 2 and 5: M5-10; lane 3,4 and 6: 16M.
Synonym Gene Product Non-syn Syn Total Length SNPs/kb nSNPs/kb sSNPs/kb cog single
BMEII0026 virB2 VirB2 – 2 2 ?318 6.29 0.00 6.29 –
BMEII0033 virB9 Channel protein VirB9 homolog 1 1 2 ?744 2.69 1.34 1.34 U
BMEII0030 virB6 Channel protein VirB6 homolog 2 2 1044 1.92 1.92 0.00 U
BMEI0948 vceC Hypothetical protein 2 2 1086 1.84 1.84 0.00 –
BMEII0028 virB4 Atpase VirB4 homolog 1 1 2496 0.40 0.40 0.00 U
BMEI1830 omp25 25 kDa outer-membrane immunogenic protein precursor 1 1 2 ?693 2.89 1.44 1.44 M
BMEI1249 omp25 25 kDa outer-membrane immunogenic protein precursor – 1 1 ?642 1.56 0.00 1.56 M
BMEI0748 L7/L12 50S ribosomal protein L7/L12 – 1 1 ?375 2.67 0.00 2.67 J
BMEII1048 GroEL Chaperonin GroEL 1 1 2 1641 1.22 0.61 0.61 O
BMEI2002 dnaK Molecular chaperone dnaK 1 – 1 1926 0.52 0.52 0.00 O
BMEI0754 EF-2 Elongation factor EF-2 2 – 2 2085 0.96 0.96 0.00 J
BMEII1086 flgG Flagellar basal-body rod protein flgG 3 – 3 ?789 3.80 3.80 0.00 N
BMEII0154 – Flagellar motor protein 3 – 3 1101 2.72 2.72 0.00 N
BMEII1085 – Flagellar basal body P-ring biosynthesis protein 1 – 1 ?414 2.42 2.42 0.00 N
BMEII1110 flim Flagellar motor switch protein flim 1 1 2 ?951 2.10 1.05 1.05 N
BMEII1107 flgF Flagellar basal-body rod protein flgF 1 – 1 ?522 1.92 1.92 0.00 N
BMEI1692 flgJ Flagellar protein flgJ 3 – 3 1917 1.56 1.56 0.00 N
BMEII0151 fliF Flagellar m-ring protein fliF – 1 1 ?729 1.37 0.00 1.37 N
BMEII1080 – Flagellar biosynthesis protein 1 – 1 ?741 1.35 1.35 0.00 N
BMEII1109 – Flagellar motor protein 1 – 1 ?972 1.03 1.03 0.00 N
BMEII0152 – Flagellar m-ring protein fliF 1 – 1 1002 1.00 1.00 0.00 N
BMEI0324 – Flagellar motor protein – 1 1 1032 0.97 0.00 0.97 N
BMEII0159 – Flagellar hook protein 1 – 1 1191 0.84 0.84 0.00 N
BMEII0167 – Flagellar biosynthesis protein – 1 1 1770 0.56 0.00 0.56 N
BMEI0881 gntR5 Transcriptional regulator, gntR family – 2 2 762 2.62 0.00 2.62 K
BMEI1913 lysR13 Transcriptional regulator, lysR family 2 – 2 900 2.22 2.22 0.00 K
BMEI0305 gntR2 Transcriptional regulator, gntR family 1 – 1 633 1.58 1.58 0.00 K
BMEI0320 gntR17 Transcriptional regulator, gntR family – 1 1 693 1.44 0.00 1.44 K
BMEI2036 – Transcriptional regulatory protein chvi – 1 1 720 1.39 0.00 1.39 T
BMEII0390 lysR12 Transcriptional regulator, lysR family 1 – 1 933 1.07 1.07 0.00 K
BMEI1709 – Oxidoreductaseucpa 3 1 4 735 5.44 4.08 1.36 I
BMEI1305 – Porin 2 3 5 1128? 4.43 1.77 2.66 -
BMEII0322 – Cytochrome c oxidase polypeptide iv 1 – 1 240 4.17 4.17 0.00 -
BMEI1980 – DNA protection during starvation conditions 1 – 1 498 2.01 2.01 0.00 P
BMEI1483 – 50S ribosomal protein L9 – 1 1 570 1.75 0.00 1.75 J
BMEI0319 – Bioy protein – 1 1 576 1.74 0.00 1.74 R
BMEII1053 gluP Glucose/galactose transporter 2 – 2 1239? 1.61 1.61 0.00 G
BMEI1985 – Phosphate transport system protein phou – 1 1 723 1.38 0.00 1.38 P
BMEI0330 – Opgc 1 – 1 1203? 0.83 0.83 0.00 S
BMEI2035 – Sensor protein chvg – 1 1 1806? 0.55 0.00 0.55 T
Tab.4  Selected virulence genes and virulence related genes with SNPs
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