Adhesive Functions or Pseudogenization of Type Va Autotransporters in Brucella Species

Abstract

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.

Keywords: Brucella; adhesin; adhesion; extracellular matrix (ECM); outer membrane protein (OMP); polar localization; pseudogene; type Va autotransporter.

Figure 1

Domain organization of Bma proteins. Schematic representation of BmaA, BmaB, and BmaC, showing functional and structural domains predicted by bioinformatics (SignalP 5.0, Pfam, BLAST). Numbers indicate amino acid positions of the predicted signal peptide, β-barrel domain, and total length of the protein. Modified from Bialer et al., 2020.

Figure 2

Adherence of Brucella strains to hosts cells. (A) Synoviocyte cell line (SW982) and primary culture, (B) Osteoblast cell line (hFOB) and primary culture, (C) colorectal epithelial cell lines (HT-29 and Caco-2) and (D) trophoblast cell line (Swan71) were infected with B. suis wild type, bmaA::stops (bmaA), bmaB::Km (bmaB), ΔbmaC (bmaC) and the respective complemented strains (bmaA::st + pBBRbmaA, bmaB::Km + pBBRbmaB, and bmaC knock-in –KI-) using a MOI of 1:100. The percentage of adhered bacteria was determined based on the total of bacteria inoculated per well. The percentage of attached bacteria was expressed relative to the control strain, to which the value of 100% was assigned. The results shown correspond to the mean ± standard deviation (SD) of the average of three tests performed in triplicate. The data were analyzed with ANOVA followed by the Tukey´s post hoc test. *, significantly different from control p < 0.05, **p < 0.005, ***p < 0.0001.

Figure 3

Adherence of B. suis strains to ECM components. Binding to immobilized ligands of B. suis wild type, B. suis bmaA::stops (bmaA), B. suis bmaB::Km (bmaB), and the respective complemented strains (bmaA::st + pBBRbmaA, bmaB::Km + pBBRbmaB and bmaC KI) was assayed. Values correspond to the percentage of binding to ECM components. A value of 100% was assigned to control strains. Data represent the means and 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 one-way ANOVA followed by a Tukey´s post hoc test. *, significantly different from control p < 0.05, **p < 0.005, ***p < 0.0001.

Figure 4

Evaluation of the role of BmaA and BmaB in the adherence to ECM components by heterologous expression. Binding to immobilized ligands of E. coli CC118 with an empty pBBR1-MCS1 (EV), E. coli CC118 pBBRbmaA, and E. coli CC118 pBBRbmaB was assayed. Values correspond to the percentage of binding to ECM components. A value of 100% was assigned to control strain. Data represent the means and 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 one-way ANOVA followed by a Tukey´s post hoc test. *, significantly different from control p < 0.05, ***p < 0.0001.

Figure 5

Polar localization of BmaB. (A) Schematic representation of the 3xFLAG tag in the BmaB protein. The FLAG (in red) was cloned immediately after the predicted cleavage site of the signal peptide. (B) Immunofluorescence assays with anti-FLAG antibodies were performed in B. suis-3xFLAG-BmaB expressing the pole markers PdhS-eGFP (upper panel) and AidB-YFP (lower panel). Bacterial shapes were observed by Differential Interference Contrast (DIC) images and cells were observed by confocal microscopy using a ZEISS LSM 880 Confocal Laser Scanning Microscope. In the first panel (from left to right) the label corresponding to BmaB is observed, in the second panel the label corresponding to the pole marker (PdhS or AidB) and in the third panel the merge of the previous ones. Gray squares indicate which bacteria are represented in the cell cartoons on the right that indicate the localization of BmaB and pole markers; red dots represent 3xFLAG-BmaB green and yellow intracellular dots represent PdhS and AidB localization (old and new pole respectively). The cartoons are based on the observations of this experiment and previous data on the localization of the pole markers AidB-YFP and PdhS-eGFP (Dotreppe et al., 2011; Van der Henst et al., 2012) and BmaB subcellular fractioning analysis (Bialer et al., 2019).

Figure 6

Protein analysis of type Va autotransporters in Brucella spp. Sequence analysis of BmaA, BmaB, and BmaC orthologues in B. suis, B. abortus, and B. melitensis strains. Red cross indicates pseudogene and green tick indicate that both the signal peptide and the β-barrel domain, which are required for proper translocation of the autotransporter through the inner and outer membranes are present.