Adhesive Functions or Pseudogenization of Type Va Autotransporters in Brucella Species

Adhesion to host cells is a key step for successful infection of many bacterial pathogens and may define tropism to different host tissues. To do so, bacteria display adhesins on their surfaces. Brucella is an intracellular pathogen capable of proliferating in a wide variety of cell types. It has been described that BmaC, a large protein that belongs to the classical (type Va) autotransporter family, is required for efficient adhesion of Brucella suis strain 1330 to epithelial cells and fibronectin. Here we show that B. suis 1330 harbors two other type Va autotransporters (BmaA and BmaB), which, although much smaller, share significant sequence similarities with BmaC and contain the essential domains to mediate proper protein translocation to the bacterial surface. Gain and loss of function studies indicated that BmaA, BmaB, and BmaC contribute, to a greater or lesser degree, to adhesion of B. suis 1330 to different cells such as synovial fibroblasts, osteoblasts, trophoblasts, and polarized epithelial cells as well as to extracellular matrix components. It was previously shown that BmaC localizes to a single bacterial pole. Interestingly, we observed here that, similar to BmaC, the BmaB adhesin is localized mostly at a single cell pole, reinforcing the hypothesis that Brucella displays an adhesive pole. Although Brucella species have strikingly similar genomes, they clearly differ in their host preferences. Mainly, the differences identified between species appear to be at loci encoding surface proteins. A careful in silico analysis of the putative type Va autotransporter orthologues from several Brucella strains showed that the bmaB locus from Brucella abortus and both, the bmaA and bmaC loci from Brucella melitensis are pseudogenes in all strains analyzed. Results reported here evidence that all three autotransporters play a role in the adhesion properties of B. suis 1330. However, Brucella spp. exhibit extensive variations in the repertoire of functional adhesins of the classical autotransporter family that can be displayed on the bacterial surface, making them an interesting target for future studies on host preference and tropism.Fil: Bialer, Magali Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Ferrero, Mariana Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Delpino, María Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Inmunología, Genética y Metabolismo. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Inmunología, Genética y Metabolismo; ArgentinaFil: Ruiz Ranwez, Verónica. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Posadas, Diana Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Baldi, Pablo Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Zorreguieta, Ángeles. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentin

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

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

BtaE and BtaF domain organization.

<p>Schematic representation of BtaE and BtaF showing functional and structural domains predicted by bioinformatics (SignalP, Pfam, BLAST and daTAA), and the fragment of the protein used for antibody production (underlined region). Numbers indicate amino acid positions within the adhesin.</p

BtaF localization.

<p>Detection of BtaF (in red) on the <i>B. suis</i> wild type surface (A) and on the <i>E. coli</i> pBBR<i>btaF</i> surface (B) by immunofluorescence of GFP-tagged bacteria. Cultures of GFP-labeled strains were fixed without permeabilization, incubated with anti-BtaF antibodies, and then probed with a CY3-conjugated donkey anti-mouse antibody. Samples were observed with a Plan-Aprochromat 100×/1.4 oil DIC objective on a Zeiss LSM 5 Pascal confocal microscope. Immunofluorescence microscopy using anti-BtaF antibodies of fixed cultures of <i>B. suis</i> strains expressing Pdhs-eGFP (C) and AidB-YFP (D) as old and new pole markers, respectively. Samples were observed with a confocal LSM 510 Meta microscope, using a Plan-Aprochromat 60×/1.4 oil DIC objective. Representative images are shown. BtaF is indicated with red arrows, and pole markers with green arrows. A schematic representation is presented. Lines indicate how the intensity profile (expressed in arbitrary units) was constructed.</p

Comparison of BtaF orthologues from different species and strains of <i>Brucella</i>.

a<p>Domains as determined by daTAA (<a href="http://toolkit.tuebingen.mpg.de/data" target="_blank">http://toolkit.tuebingen.mpg.de/data</a>). Numbers of domains predicted to be part of the head, stalk, connector or anchor are indicated.</p>b<p>Percentage of Identity/Similarity shared with BtaF from <i>B. suis</i> 1330.</p>c<p>Locus not annotated; a gene encoding a predicted full length protein homologue was identified between positions 1786495 and 1785911 (585 nucleotides) of chromosome I.</p

Both, BtaE and BtaF, are required for full virulence of <i>B. suis</i> in mice.

<p>BALB/c mice were inoculated with the <i>B. suis</i> strains by intragastric delivery, sacrificed at 7 and 30 days p.i., and spleens were removed. Dilutions of spleen homogenates were plated 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 Tukey's <i>a posteriori</i> test. The experiment was repeated twice with similar results. *, significantly different from the wild type (P<0.05); ** Significantly different from wild type and simple mutants (P<0.05).</p

<i>In vivo</i> expression of BtaE and BtaF in pigs.

<p>Extracts from <i>E. coli</i> pBBR<i>btaE</i>, <i>E. coli</i> pBBR<i>btaF</i> and the control <i>E. coli</i> pBBR1MCS were used to perform indirect ELISA using sera from healthy and sick pigs infected with <i>Brucella</i>. The values correspond to the means ±SD of the results of a representative experiment of three independent assays done in duplicate. Data were analyzed by Student's t test. Values between healthy and sick pigs were significantly different (P<0.001 for BtaE and P<0.05 for BtaF)</p

BtaF is involved in resistance to porcine serum.

<p>Early logarithmic phase cultures were used to evaluate resistance to porcine serum of <i>B. suis</i> (A) and <i>E. coli</i> (B) strains. Survival after one hour of incubation in serum with complement activity was evaluated and then expressed as survival percentage (%) relative to wild type <i>B. suis</i> 1330 or <i>E. coli</i> pBBR1MCS-1, defined as 100%. Total CFU/ml are also indicated in the right Y-axis for <i>Brucella</i> survival (A). <i>E. coli</i> survival was also assessed in buffer and heated serum (B). Values represent the means ±SD of the results of an experiment representative of three independent assays done in triplicate. Data were analyzed by Student's t test and one-way ANOVA followed by a Tukey <i>a posteriori</i> test. *, significantly different from control (P<0.05).</p

Adhesion and invasion to HeLa and A549 cells.

<p>Total numbers of adherent and intracellular (invasive) bacteria were determined, and the invasive/adherent cell ratio was calculated. HeLa cells were infected with <i>E. coli</i> pBBR<i>btaF</i> and <i>E. coli</i> pBBR1MCS (A). HeLa (B) and A549 (C) cells were infected with <i>B. suis</i> wild type, <i>B. suis</i> Δ<i>btaF</i> and <i>B. suis</i> Δ<i>btaF</i> pBBR<i>btaF</i>. Values are expressed relative to the control strains (<i>E. coli</i> pBBR1MCS or the wild type strain of <i>B. suis</i>), defined as 100%. Values represent the means ±SD of the results of a representative experiment of three independent assays done in triplicate. Data were analyzed by Student's t test and by one-way ANOVA followed by a Tukey <i>a posteriori</i> test. *, significantly different from control (P<0.05).</p