BtaE, an adhesin that belongs to the trimeric autotransporter family, is required for full virulence and defines a specific adhesive pole of Brucella suis

Abstract

Brucella is responsible for brucellosis, one of the most common zoonoses worldwide that causes important economic losses in several countries. Increasing evidence indicates that adhesion of Brucella spp. to host cells is an important step to establish infection. We have previously shown that the BmaC unipolar monomeric autotransporter mediates the binding of Brucella suis to host cells through cell-associated fibronectin. Our genome analysis shows that the B. suis genome encodes several additional potential adhesins. In this work, we characterized a predicted trimeric autotransporter that we named BtaE. By expressing btaE in a nonadherent Escherichia coli strain and by phenotypic characterization of a B. suis ΔbtaE mutant, we showed that BtaE is involved in the binding of B. suis to hyaluronic acid. The B. suis ΔbtaE mutant exhibited a reduction in the adhesion to HeLa and A549 epithelial cells compared with the wild-type strain, and it was outcompeted by the wild-type strain in the binding to HeLa cells. The knockout btaE mutant showed an attenuated phenotype in the mouse model, indicating that BtaE is required for full virulence. BtaE was immunodetected on the bacterial surface at one cell pole. Using old and new pole markers, we observed that both the BmaC and BtaE adhesins are consistently associated with the new cell pole, suggesting that, in Brucella, the new pole is functionally differentiated for adhesion. This is consistent with the inherent polarization of this bacterium, and its role in the invasion process.

Fig 1

BtaE domain organization. A schematic representation of BtaE showing the N-terminal signal, the conserved domains, and motifs predicted by bioinformatics (SignalP, Pfam, and BLAST) is presented. Numbers indicate amino acid positions within BtaE.

Fig 2

Binding of BtaE-expressing bacteria to ECM components. Plastic wells were coated with collagen type I, hyaluronic acid, fetuin, and fibronectin and subsequently incubated with the different strains. After being washed, bound bacteria were harvested with trypsin-EDTA; then, serial dilutions were done and the bacteria were plated on appropriate media. (A) E. coli pBBRbtaE and E. coli pBBR1MCS (control strain) were assayed. (B) The wild-type (wt) strain of B. suis and the B. suis ΔbtaE and ΔbtaE pBBRbtaE isogenic strains were analyzed. Values correspond to the percentages of total bacteria recovered from the wells after incubation with reference to the control strain (E. coli pBBR1MCS in panel A and B. suis wt in panel B), to which a value of 100% was assigned. Data represent the means ± standard deviations (SD) of the results of a representative experiment done in triplicate. Three independent experiments were performed with similar results. Data were analyzed by Student's t test and by one-way ANOVA followed by a Tukey a posteriori test. *, significantly different from control (P < 0.05), with 95% confidence.

Fig 3

Adhesion to and invasion of HeLa cells. Approximately 5 × 10⁶ bacteria were used to challenge 5 × 10⁴ HeLa cells (MOI, 100:1). Total numbers of adherent bacteria (A) and intracellular or invasive bacteria (B) were determined, and the invasive cell/adherent cell ratio was calculated (C). Total numbers of adherent and intracellular bacteria are expressed relative to wild-type B. suis 1330, defined as 100%. (D) A competitive assay was carried out in which HeLa cells were coinoculated with wild-type and B. suis ΔbtaE bacteria. The number of adherent bacteria was expressed as CFU/ml. (E) Inhibition of bacterial binding to HeLa cells by preincubation with hyaluronic acid (HA); data are expressed as total CFU/ml. Values represent the means ± SD of the results of an experiment representative of three independent assays done in triplicate or quadruplicate. Data were analyzed by one-way ANOVA followed by a Tukey a posteriori test. *, significantly different from the wild type (P < 0.05).

Fig 4

Adhesion to and invasion of A549 lung epithelial cells. Approximately 5 × 10⁶ bacteria were used to challenge 5 × 10⁴ A549 cells (MOI, 100:1). Total numbers of adherent bacteria (A) and intracellular bacteria (B) were determined, and the invasive cell/adherent cell ratio was calculated (C). Total numbers of adherent and intracellular bacteria are expressed relative to the adhesion and invasion values of the wild type, defined as 100%. Values represent the means ± SD of the results of an experiment representative of three independent assays done in triplicate. Data were analyzed by one-way ANOVA and a Tukey a posteriori test. *, significantly different from the wild type (P < 0.05).

Fig 5

Role of BtaE in the virulence of B. suis in BALB/c mice. BALB/c mice (10 mice per group) were inoculated by intragastric delivery of the B. suis wild type, B. suis ΔbtaE, or the complemented strains. Five mice from each group were sacrificed at 7 (A) and 30 (B) days postinfection, and then spleens were removed. Dilutions of spleen homogenates were plated in duplicate, and CFU were counted and expressed as the log₁₀ value per spleen. The CFU data were normalized by log transformation and evaluated by one-way ANOVA followed by Dunnett's a posteriori test. The experiment was repeated twice with similar results. *, significantly different from the wild type (P < 0.05).

Fig 6

BtaE localization. (A and C) Western blot analysis of whole-membrane fractions from B. suis (A) and E. coli pBBRbtaE (C) was carried out. Membrane samples were submitted to a strong denaturing process. Samples were electrophoresed by SDS-PAGE and transferred to a PDVF membrane. Blots were incubated with anti-BtaE antisera and with anti-mouse HRP-conjugated secondary antibody and finally revealed using ECL Plus. On the right, a Coomassie blue pattern is shown as a loading control (ctrl). (B and D) Detection of BtaE (in red) on the B. suis surface (B) and on the E. coli pBBRbtaE surface (D) by immunofluorescence of GFP-tagged bacteria. Cultures of GFP-labeled strains were fixed, incubated with anti-BtaE antibodies, and then probed with a CY3-conjugated donkey anti-mouse antibody preparation. Samples were observed with a Plan-Aprochromat 100×/1.4 oil DIC objective on a Zeiss LSM 5 Pascal confocal microscope.

Fig 7

BtaE and BmaC are localized to the new bacterial pole. (A and B) Immunofluorescence microscopy using anti-BmaC antibodies was carried out with fixed cultures of B. abortus strains expressing PdhS-eGFP (A) and AidB-YFP (B) as old and new pole markers, respectively. DAPI staining and phase contrast (PC) were used to visualize the bacterial DNA content and shape, respectively. Samples were observed with a confocal LSM 510 Meta microscope using a Plan-Apochromat 60×/1.4 oil DIC objective. Representative images are shown. BmaC, PdhS-eGFP, and AidB-YFP are indicated with red, green, and yellow arrows, respectively. A schematic representation is also displayed. An intensity profile of the channels (constructed through the cyan dotted arrow) is presented, expressed in arbitrary units. (C and D) The same analysis was carried out for BtaE in B. suis with both PdhS-eGFP (C) and AidB-YFP (D).