Cloning and sequencing of yajC and secD homologs of Brucella abortus and demonstration of immune responses to YajC in mice vaccinated with B. abortus RB51

Abstract

To identify Brucella antigens that are potentially involved in stimulating a protective cell-mediated immune response, a gene library of Brucella abortus 2308 was screened for the expression of antigens reacting with immunoglobulin G2a antibodies from BALB/c mice vaccinated with B. abortus RB51. One selected positive clone (clone MCB68) contained an insert of 2.6 kb; nucleotide sequence analysis of this insert revealed two open reading frames (ORFs). The deduced amino acid sequences of the first and second ORFs had significant similarities with the YajC and SecD proteins, respectively, of several bacterial species. Both the YajC and SecD proteins were expressed in Escherichia coli as fusion proteins with maltose binding protein (MBP). In Western blots, sera from mice vaccinated with B. abortus RB51 recognized YajC but not SecD. Further Western blot analysis with purified recombinant YajC protein indicated that mice inoculated with B. abortus 19 or 2308 or B. melitensis RM1 also produced antibodies to YajC. In response to in vitro stimulation with recombinant MBP-YajC fusion protein, splenocytes from mice vaccinated with B. abortus RB51 were able to proliferate and produce gamma interferon but not interleukin-4. This study demonstrates, for the first time, the involvement of YajC protein in an immune response to an infectious agent.

FIG. 1

(A) Schematic diagram of clone MBP68, which contains the yajC and secD genes of B. abortus. The locations of various primers used for PCR amplification of the genes are shown as arrows. The restriction enzyme sites engineered into the primers are indicated. (B) Nucleotide sequences of the primers used for the amplification of the yajC and secD genes.

FIG. 2

Nucleotide and deduced amino acid sequences of the yajC and secD genes of B. abortus, present in clone MBP68. The putative promoter region (−10 and −35), transcription start site (+1), and ribosomal binding sites (RBS) are indicated. Inverted repeat sequences (IR) with the potential to form a hairpin loop are also indicated.

FIG. 3

Multiple alignment of deduced amino acid sequences of the YajC (A) and SecD (B) proteins. Alignment was performed with the Clustal method of the MegAlign program of LaserGene software. Aa, Aquifex aeolicus; Ba, B. abortus; Bs, Bacillus subtilis; Ec, E. coli; Ef, Enterococcus faecalis; Hi, Haemophilus influenzae; Hp, Helicobacter pylori; Rc, Rhodobacter capsulatus. Gaps are indicated by dashes. Amino acids identical for the two sequences are boxed. Based on this multiple alignment, the similarities between the YajC protein of B. abortus and those of the other bacteria are as follows: Aa, 26.5%; Bs, 30.3%; Ec, 27.9%; Ef, 31.7%; Hi, 22.7%; and Hp, 28.1%. The similarities between the SecD protein of B. abortus and those of the other bacteria are as follows: Aa, 23.4%; Ec, 24.7%; Hi, 24.9%; Hp, 26.2%; and Rc, 29.2%.

FIG. 4

Hydrophobic profiles of SecD proteins of E. coli (I) and B. abortus (II). Analysis was performed with the Kyte-Doolittle option of the Protean program of LaserGene software. The numbers at the bottom are the amino acid positions. The profiles are divided into segments for easy explanation in the text.

FIG. 5

Western blot reactivities of recombinant YajC and SecD proteins. (A and B) E. coli extracts expressing MBP fusions of LacZα peptide (lane 1), YajC (lane 2), and SecD (lane 3) were reacted with rabbit antisera to MBP (A) and antisera to B. abortus RB51 (B). (C) Reaction of affinity column-purified MBP-YajC fusion protein with mouse antisera to B. abortus 2308 (lane 1) and 19 (lane 2) and B. melitensis RM1 (lane 3). Horseradish-peroxidase-conjugated secondary antibodies specific to mouse IgG whole molecules and the IgG2a subisotype were used to develop the blots in panels B and C, respectively. Numbers at the left in panel A are approximate protein molecular masses, in kilodaltons. The appearance of a double band in the MBP-YajC lanes is due to some fusion protein with uncleaved signal sequence of the MBP.

FIG. 6

In vitro proliferation of splenocytes from vaccinated and naive mice. The assays were set up in triplicate, and the cells were either left unstimulated (media alone) or stimulated with antigens. The radioactivity of incorporated [³H]thymidine was measured after 3 or 5 days of culturing. Results were expressed as mean counts per minute ± standard deviation (n = 3). Groups with an asterisk are significantly different from other groups of cultures measured for the same number of days but not from each other.

FIG. 7

Western blot detection of YajC protein in B. abortus RB51. The antigens present in the lanes are as follows: lane 1, MBP-YajC fusion protein partially cleaved by factor Xa; lane 2, extracts of B. abortus RB51/pBByajC; lane 3, extracts of strain RB51 with plasmid alone; lane 4, extracts of strain RB51. The blot was reacted with mice antisera to B. abortus RB51 and horseradish-peroxidase-conjugated goat antibodies specific to mouse IgG whole molecules. The arrow at the left shows the YajC band. The numbers at the right are approximate protein molecular masses, in kilodaltons.