Genetic characteristics of the Brucella melitensis strain with a rough phenotype
YANG Xiao-wen, HAN Xiu-rui, PIAO Dong-ri, ZHAO Hong-yan, TIAN Guo-zhong, JIANG Hai
State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Bejing 102206, China
Abstract：The aim of this study was to analyze the genetic characteristics of the B. melitensis strain with a rough phenotype. In this study, rough phenotype detection methods, whole-genome sequencing and comparative genome analysis methods were used. The results showed that this isolated strain was agglutinated with R monospecific sera and trisavin, and could be stained with crystal violet. The genome sequence of the isolation was similar to B. melitensis bv.2 str. ATCC23457 at the genomic level. The genome of the isolation consisted of two chromosomes, with a size of 3 311 700 bp and a GC content of 57.2%, including 3 379 CDS, 9 rRNA, 55 tRNA and 4 ncRNA. There are 282 variations, including 243 SNPs and 39 InDel. Among them, eight of the known LPS-related genes had missense variations. In conclusion, because these variations at the gene level hindered LPS synthesis, the phenotype of the isolation changed from smooth to rough. This study provided the basic data for rough B. melitensis, and new insights for the screening of Brucella vaccine strains.
[1] Keriel A, Botella E, Estrach S, et al.Brucella intracellular life relies on the transmembrane protein CD98 heavy chain[J]. J Infect Dis, 2015. 211(11): 1769-1778. DOI:10.1093/infdis/jiu673 [2] Boschiroli ML, Foulongne V, O’Callaghan D. Brucellosis: a worldwide zoonosis[J]. Curr Opin Microbiol, 2001,4(1): 58-64. DOI:10.1016/s1369-5274(00)00165-x [3] Bukhari EE.Pediatric brucellosis. An update review for the new millennium[J]. Saudi Med J, 2018, 39(4): 336-341. DOI: 10.15537/smj.2018.4.21896 [4] Hasanjani Roushan MR,Ebrahimpour S.Human brucellosis: An overview[J]. Caspian J Intern Med, 2015,6(1): 46-53. [5] Zia SH,Wang FL.Brucellosis in north China; a clinical, etiological and epidemiological study[J]. Am J Trop Med Hyg, 1949. 29(6): 925-936. DOI:10.4269/ajtmh.1949.s1-29.925 [6] Li ZJ, Cui BY, Chen H, et al.Molecular typing of Brucella suis collected from 1960s to 2010s in China by MLVA and PFGE[J]. Biomed Environ Sci, 2013, 26(6): 504-512. DOI: 10.3967/0895-3988.2013.06.013 [7] Piao DR, Liu X, Di DD, et al.Genetic polymorphisms identify in species/biovars of Brucella isolated in China between 1953 and 2013 by MLST[J]. BMC Microbiol, 2018,18(1): 7. DOI: 10.1186/s12866-018-1149-0 [8] Li YJ, Li XL, Liang S, et al.Epidemiological features and risk factors associated with the spatial and temporal distribution of human brucellosis in China[J]. BMC Infect Dis, 2013,13: 547. DOI: 10.1186/1471-2334-13-547 [9] Mancilla M.Smooth to Rough Dissociation in Brucella: The Missing Link to Virulence[J]. Front Cell Infect Microbiol, 2015, 5: 98. DOI: 10.3389/fcimb.2015.00098 [10] Godfroid F, Taminiau B, Danese I, et al.Identification of the perosamine synthetase gene of Brucella melitensis 16M and involvement of lipopolysaccharide O side chain in Brucella survival in mice and in macrophages[J]. Infect Immun, 1998,66(11): 5485-5493. [11] Ugalde JE, Czibener C, Feldman MF, et al.Identification and characterization of the Brucella abortus phosphoglucomutase gene: role of lipopolysaccharide in virulence and intracellular multiplication[J]. Infect Immun, 2000,68(10): 5716-5723. DOI: 0.1128/iai.68.10.5716-5723.2000 [12] Alton GG,Jones LM.Laboratory techniques in brucellosis[J]. Monogr Ser World Health Organ, 1967,55: 1-92. [13] Li H,Durbin R.Fast and accurate short read alignment with Burrows-Wheeler transform[J]. Bioinformatics, 2009,25(14): 1754-1760. DOI: 10.1093/bioinformatics/btp324 [14] Li H, Handsaker B, Wysoker A, et al.The sequence alignment/map format and SAMtools[J]. Bioinformatics, 2009,25(16): 2078-2087. DOI: 10.1093/bioinformatics/btp352 [15] Zerbino DR, Birney E.Velvet: algorithms for de novo short read assembly using de Bruijn graphs[J]. Genome Res, 2008,18(5): 821-830. DOI: 10.1101/gr.074492.107 [16] Tatusova T, DiCuccio M, Badretdin A, et al. NCBI prokaryotic genome annotation pipeline[J]. Nucleic Acids Res, 2016. 44(14): 6614-6624. DOI: 10.1093/nar/gkw569 [17] McKenna A, Hanna M, Banks E, et al. The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data[J]. Genome Res, 2010,20(9): 1297-1303. DOI: 10.1101/gr.107524.110 [18] Faison WJ, Rostovtsev A, Castro-Nallar E, et al.Whole genome single-nucleotide variation profile-based phylogenetic tree building methods for analysis of viral, bacterial and human genomes[J]. Genomics, 2014, 104(1): 1-7. DOI:10.1016/j.ygeno.2014.06.001 [19] Darling AC, Mau B, Blattner FR, et al.Mauve: multiple alignment of conserved genomic sequence with rearrangements[J]. Genome Res, 2004. 14(7): 1394-403. DOI: https://doi.org/10.1101/gr.2289704 [20] Godfroid F, Cloeckaert A, Taminiau B, et al.Genetic organisation of the lipopolysaccharide O-antigen biosynthesis region of brucella melitensis 16M (wbk)[J]. Res Microbiol, 2000,151(8): 655-668. DOI: 10.1016/s0923-2508(00)90130-x [21] Gonzalez D, Grillo MJ, De Miguel MJ, et al.Brucellosis vaccines: assessment of Brucella melitensis lipopolysaccharide rough mutants defective in core and O-polysaccharide synthesis and export[J]. PLoS One, 2008,3(7): e2760. DOI:10.1371/journal.pone.0002760 [22] Conde-Alvarez R, Arce-Gorvel V, Iriarte M, et al.The lipopolysaccharide core of Brucella abortus acts as a shield against innate immunity recognition[J]. PLoS Pathog, 2012,8(5): e1002675. DOI: 10.1371/journal.ppat.1002675 [23] Gil-Ramirez Y, Conde-Alvarez R, Palacios-Chaves L, et al.The identification of wadB, a new glycosyltransferase gene, confirms the branched structure and the role in virulence of the lipopolysaccharide core of Brucella abortus[J]. Microb Pathog, 2014,73: 53-62. DOI: 10.1016/j.micpath.2014.06.002 [24] Cardoso PG, Macedo GC, Azevedo V, et al.Brucella spp noncanonical LPS: structure, biosynthesis, and interaction with host immune system[J]. Microb Cell Fact, 2006,5: 13. DOI: 10.1186/1475-2859-5-13 [25] Feng Y, Peng X, Jiang H, et al.Rough brucella strain RM57 is attenuated and confers protection against Brucella melitensis[J]. Microb Pathog, 2017,107: 270-275. DOI: 10.1016/j.micpath.2017.03.045 [26] Rautemaa R, Meri S.Complement-resistance mechanisms of bacteria[J]. Microbes Infect, 1999, 1(10): 785-794. DOI: 10.1016/s1286-4579(99)80081-1 [27] Moreno E.Retrospective and prospective perspectives on zoonotic brucellosis[J]. Front Microbiol, 2014, 5: 213. DOI: 10.3389/fmicb.2014.00213
