Release of periplasmic proteins of Brucella suis upon acidic shock involves the outer membrane protein Omp25

Abstract

The survival and replication of Brucella in macrophages is initially triggered by a low intraphagosomal pH. In order to identify proteins released by Brucella during this early acidification step, we analyzed Brucella suis conditioned medium at various pH levels. No significant proteins were released at pH 4.0 in minimal medium or citrate buffer, whereas in acetate buffer, B. suis released a substantial amount of soluble proteins. Comparison of 13 N-terminal amino acid sequences determined by Edman degradation with their corresponding genomic sequences revealed that all of these proteins possessed a signal peptide indicative of their periplasmic location. Ten proteins are putative substrate binding proteins, including a homologue of the nopaline binding protein of Agrobacterium tumefaciens. The absence of this homologue in Brucella melitensis was due to the deletion of a 7.7-kb DNA fragment in its genome. We also characterized for the first time a hypothetical 9.8-kDa basic protein composed of five amino acid repeats. In B. suis, this protein contained 9 repeats, while 12 were present in the B. melitensis orthologue. B. suis in acetate buffer depended on neither the virB type IV secretory system nor the omp31 gene product. However, the integrity of the omp25 gene was required for release at acidic pH, while the absence of omp25b or omp25c displayed smaller effects. Together, these results suggest that Omp25 is involved in the membrane permeability of Brucella in acidic medium.

FIG. 1.

Release of proteins into bacterial supernatant increases in acidic acetate buffer (pH 4). Wild-type B. suis 1330 cells were incubated for 150 min in various media before the bacteria were centrifuged and the different supernatants were concentrated 1,000-fold. Brucella proteins from the lysate or the concentrated supernatants were separated by SDS-PAGE and stained with Coomassie blue (A) or blotted and immunostained with an anti-Omp25 antibody (B). Lanes: 1, bacterial lysate; 2, supernatants composed of ammonium acetate containing 120 mM NaCl, pH 4; 3, PBS, pH 7; 4, RPMI, pH 7.5; Markers, molecular mass markers.

FIG. 2.

Two groups of proteins (GF-A and GF-B) were separated by gel filtration of wild-type B. suis acidic supernatant. Proteins from wild-type Brucella incubated in ammonium acetate containing 120 mM NaCl, pH 4 (supernatant 1 [SN1]) were concentrated (SN3) and subjected to ultracentrifugation at 126,000 × g. Proteins from the various fractions were loaded into a gel filtration column, and separation was monitored at 280 nm and quantified as arbitrary units × 10⁻³ (mAu) (A) Initial supernatant (SN3) (trace a), ultracentrifuged supernatant (SN4) (trace b), and the resuspended pellet resulting from the ultracentrifugation (trace c). Proteins from the GF-A fraction had an apparent molecular mass of >2,000 kDa, while proteins of the GF-B group exhibited an apparent molecular mass of <100 kDa. (B and C) Proteins from the various fractions were further separated by SDS-PAGE and stained with Coomassie blue (B) or subjected to immunoblotting with monoclonal antibodies directed against Omp31 and Omp25 (C). Lanes: M, molecular mass markers; 1, initial bacterial supernatant (SN3); 2, GF-A fraction; 3, GF-B fraction; 4, resuspended pellet.

FIG. 3.

Electron micrographs reveal the production of Brucella blebs in various media and within phagosomes. (A) Vesicles present in the pellet obtained after ultracentrifugation of the GF-A fraction. (B) Spontaneous blebbing of wild-type B. suis in RPMI medium used for macrophage infections.

FIG. 4.

Comparison of the constitutive production of Rib- and Nop-like periplasmic proteins in various Brucella species. After overnight culture, the different Brucella species were centrifuged and the resulting bacterial pellets were lysed in Laemmli electrophoretic sample buffer. Brucella proteins were separated by SDS-PAGE and blotted, and the Nop (A) and Rib (B) proteins were detected using our rabbit polyclonal antibodies.

FIG. 5.

Comparison of schematic diagram of ORF region encoding B. suis Nop protein with that of corresponding B. melitensis DNA sequence. (A) In B. melitensis, deletion of the 7,737-bp fragment present in B. suis resulted in the loss of five full-length putative genes (BRA0631 to BRA0635) and truncated two genes (BRA0630 and BRA0636) located at the extremities of the deleted DNA fragment. (B) Close-up view of nucleotide sequences within which deletion occurred. Note that the unique B. melitensis sequence could be derived from the B. suis sequence by deletion of a 7,737-bp DNA fragment, which could occur at several different positions provided that the deleted part included the extra G nucleotide (-ccttG-).

FIG. 6.

Sequence comparison of bacterial orthologues of B. suis protein 13. Alignment of the full-length mature orthologues of B. suis protein 13 clearly indicates the repetitive conserved stretches of five amino acids. Shaded columns indicate identical (*), strongly similar (:), or weakly similar (.) amino acids.

FIG. 7.

Wild-type B. suis periplasmic proteins were not released in acidic media containing assimilable citrate buffer. Wild-type B. suis cells were incubated at pH 4 in different media for 150 min at 37°C. Proteins were separated by SDS-PAGE and analyzed by Western blotting with an anti-Omp25, anti-Rib, or anti-Nop antibody. Lanes: 1, ammonium acetate and NaCl; 2, sodium acetate and NaCl; 3, sodium citrate and NaCl; 4, minimum medium containing citrate (minimal medium A [MMA]) (see Materials and Methods).

FIG. 8.

Knockout of omp25 prevents the release of periplasmic proteins Rib and Nop. Wild-type (WT) B. suis organisms or B. suis mutants (2.5 × 10¹⁰/ml) were incubated for 150 min at 37°C in 25 mM ammonium acetate buffer containing 120 mM NaCl, pH 4. Proteins from lysates obtained after centrifugation (A) and from concentrated supernatants (B) were separated by SDS-PAGE and analyzed by Western blotting with an anti-Rib (α-Rib) or anti-Nop antibody. Lanes: 1, WT B. suis; 2, Δomp25 B. suis; 3, Δomp25/omp25⁺ B. suis; 4, omp31 mutant B. suis. Arrows indicate Rib (left panels) or Nop (right panels) immunoreactivities.

FIG. 9.

Effects of Omp25/Omp31 protein impairment on release of the periplasmic protein Rib. Wild-type (WT) B. suis (lane 1) and the B. suis strains with omp25b (lane 2), omp25c (lane 3), omp25d (lane 4), and omp31 (lane 5) knocked out were incubated in ammonium acetate as described in Materials and Methods. The amounts of periplasmic proteins released by each mutant were assessed in the supernatant using anti-Rib antibody.