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

Abstract

Brucella organisms are facultative intracellular bacteria that may infect many species of animals as well as humans. The smooth lipopolysaccharide (S-LPS) has been reported to be an important virulence factor of these organisms, but the genetic basis of expression of the S-LPS O antigen has not yet been described. Likewise, the role of the O side chain of S-LPS in the survival of Brucella has not been clearly defined. A mini-Tn5 transposon mutant library of Brucella melitensis 16M was screened by enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies (MAbs) directed against the O side chain of Brucella. One mutant, designated B3B2, failed to express any O side chain as confirmed by ELISA, Western blot analysis, and colony coloration with crystal violet. Nucleotide sequence analysis demonstrated that the transposon disrupted an open reading frame with significant homology to the putative perosamine synthetase genes of Vibrio cholerae O1 and Escherichia coli O157:H7. The low G+C content of this DNA region suggests that this gene may have originated from a species other than a Brucella sp. The survival of B. melitensis mutant strain B3B2 in the mouse model and in bovine macrophages was examined. The results suggested that S-LPS or, more precisely, its O side chain is essential for survival in mice but not in macrophages.

FIG. 1

Immunoblot analysis of Brucella whole-cell lysates probed with MAbs 12G12 and 2E11 (directed against Brucella S-LPS) (a) and MAb A68/3F03/D05 (directed against R-LPS of Brucella) (b). Lanes: A, B. melitensis 16M (parental strain); B, B. melitensis B115; C, B. melitensis H38 rough mutant; D, B. melitensis B3B2 mutant.

FIG. 2

(a) pKSTn5R and primers used in this study. (b) Gene replacement strategy used to create B. melitensis 16M DR identical to the B3B2 insertion mutant. The construction of pKSTn5R and pSKoritTn5R is described in the text. Open regions, B. melitensis chromosomal DNA; light-grey regions, SalI chromosomal DNA containing the mini-Tn5 in the B3B2 mutant; solid regions, kanamycin resistance gene and cat reporter gene of the mini-Tn5.

FIG. 3

The nucleotide sequence and the deduced amino acid sequence of the B. melitensis 16M perosamine synthetase gene. The putative ribosome binding site (RBS) is underlined. The asterisk denotes the termination codon. The arrow indicates the site of the mini-Tn5 insertion in strain B3B2.

FIG. 4

Simultaneous multiple alignment of perosamine synthetase amino acid sequences from B. melitensis 16M (1), V. cholerae O1 (2), and E. coli O157:H7 (3). The matching regions for the three sequences are outlined by boxes. Amino acids are numbered above the sequence. Shaded boxes indicate identities. Lowercase letters indicate unaligned residues.

FIG. 5

Growth of B. melitensis 16M (parental strain) and the rough insertion mutant B3B2 in bovine macrophages. The data presented are means ± standard deviations of quintuplicate plate counts and are representative of two experiments. p.i., postinfection.