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

Outcomes from elective colorectal cancer surgery during the SARS-CoV-2 pandemic

This study aimed to describe the change in surgical practice and the impact of SARS-CoV-2 on mortality after surgical resection of colorectal cancer during the initial phases of the SARS-CoV-2 pandemic

Transport and adhesion in Brucella suis

Brucella es un patógeno intracelular facultativo, responsable de una infección zoonótica llamada brucelosis. Un aspecto clave en la virulencia de este género es su habilidad para invadir y proliferar dentro de células fagocíticas y no fagocíticas, en las que se establece. En esta tesis se identificaron y caracterizaron factores imprescindibles para la interacción de este patógeno con su hospedador. Uno de estos factores fue la proteína BepC, que pertenece a la familia TolC de proteínas de membrana externa. BepC resultó crucial en la detoxificación de la bacteria tanto in vivo como in vitro, probablemente formando parte de sistemas tripartitos de eflujo. Los otros factores fueron tres adhesinas que pertenecen a la familia de autotransportadores de tipo I. Estas proteínas, a las que denominamos BmaA, B y C (por Brucella monomeric autotransporter), participarían en: i) la adhesión de Brucella suis a las células del hospedador, ii) la interacción con otras superficies como la matriz extracelular y iii) las interacciones bacteria-bacteria, para ambas, BmaA y BmaB. Estos factores pueden ser base de estudios futuros más específicos y profundos que lleven al desarrollo de vacunas o terapias que mejoren el diagnóstico, la prevención y el tratamiento de la brucelosis.Brucella is an intracellular pathogen responsible of the zoonotic disease called brucellosis. A key feature of Brucella virulence is its ability to invade and proliferate inside professional and non-professional phagocytic cells, were it establishes. In this thesis, essential factors for the interaction of this pathogen with its host were identified and characterized. One such factor was the BepC protein, that belongs to the TolC family of outer membrane proteins. BepC was shown to be crucial for the detoxification of the bacteria both in vivo and in vitro, probably as part of tripartite efflux systems. The other factors were three adhesins belonging to the type I autotransporters family. These proteins that we called BmaA, B and C (Brucella monomeric autotransporter) were shown to be involved in: i) Brucella suis adhesion to host cells, ii) the interaction of B. suis with other surfaces such as the extracellular matrix and iii) for both BmaA and BmaB, in bacteria-bacteria interactions. These factors can be the basis of future studies, more specific and profound, that lead to the development of vaccines or therapies that improve the diagnosis, prevention and treatment of brucellosis.Fil:Posadas, Diana M.. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Interplay between Two RND Systems Mediating Antimicrobial Resistance in Brucella suis▿

The RND-type efflux pumps are responsible for the multidrug resistance phenotype observed in many clinically relevant species. Also, RND pumps have been implicated in physiological processes, with roles in the virulence mechanisms of several pathogenic bacteria. We have previously shown that the BepC outer membrane factor of Brucella suis is involved in the efflux of diverse drugs, probably as part of a tripartite complex with an inner membrane translocase. In the present work, we characterize two membrane fusion protein-RND translocases of B. suis encoded by the bepDE and bepFG loci. MIC assays showed that the B. suis ΔbepE mutant was more sensitive to deoxycholate (DOC), ethidium bromide, and crystal violet. Furthermore, multicopy bepDE increased resistance to DOC and crystal violet and also to other drugs, including ampicillin, norfloxacin, ciprofloxacin, tetracycline, and doxycycline. In contrast to the ΔbepE mutant, the resistance profile of B. suis remained unaltered when the other RND gene (bepG) was deleted. However, the ΔbepE ΔbepG double mutant showed a more severe phenotype than the ΔbepE mutant, indicating that BepFG also contributes to drug resistance. An open reading frame (bepR) coding for a putative regulatory protein of the TetR family was found upstream of the bepDE locus. BepR strongly repressed the activity of the bepDE promoter, but DOC released the repression mediated by BepR. A clear induction of the bepFG promoter activity was observed only in the BepDE-defective mutant, indicating a regulatory interplay between the two RND efflux pumps. Although only the BepFG-defective mutant showed a moderate attenuation in model cells, the activities of both bepDE and bepFG promoters were induced in the intracellular environment of HeLa cells. Our results show that B. suis harbors two functional RND efflux pumps that may contribute to virulence

Proteins Exported via the PrsD-PrsE Type I Secretion System and the Acidic Exopolysaccharide Are Involved in Biofilm Formation by Rhizobium leguminosarum

The type I protein secretion system of Rhizobium leguminosarum bv. viciae encoded by the prsD and prsE genes is responsible for secretion of the exopolysaccharide (EPS)-glycanases PlyA and PlyB. The formation of a ring of biofilm on the surface of the glass in shaken cultures by both the prsD and prsE secretion mutants was greatly affected. Confocal laser scanning microscopy analysis of green-fluorescent-protein-labeled bacteria showed that during growth in minimal medium, R. leguminosarum wild type developed microcolonies, which progress to a characteristic three-dimensional biofilm structure. However, the prsD and prsE secretion mutants were able to form only an immature biofilm structure. A mutant disrupted in the EPS-glycanase plyB gene showed altered timing of biofilm formation, and its structure was atypical. A mutation in an essential gene for EPS synthesis (pssA) or deletion of several other pss genes involved in EPS synthesis completely abolished the ability of R. leguminosarum to develop a biofilm. Extracellular complementation studies of mixed bacterial cultures confirmed the role of the EPS and the modulation of the biofilm structure by the PrsD-PrsE secreted proteins. Protein analysis identified several additional proteins secreted by the PrsD-PrsE secretion system, and N-terminal sequencing revealed peptides homologous to the N termini of proteins from the Rap family (Rhizobium adhering proteins), which could have roles in cellular adhesion in R. leguminosarum. We propose a model for R. leguminosarum in which synthesis of the EPS leads the formation of a biofilm and several PrsD-PrsE secreted proteins are involved in different aspects of biofilm maturation, such as modulation of the EPS length or mediating attachment between bacteria

The TolC Homologue of Brucella suis Is Involved in Resistance to Antimicrobial Compounds and Virulence

Brucella spp., like other pathogens, must cope with the environment of diverse host niches during the infection process. In doing this, pathogens evolved different type of transport systems to help them survive and disseminate within the host. Members of the TolC family have been shown to be involved in the export of chemically diverse molecules ranging from large protein toxins to small toxic compounds. The role of proteins from the TolC family in Brucella and other α-2-proteobacteria has been explored little. The gene encoding the unique member of the TolC family from Brucella suis (BepC) was cloned and expressed in an Escherichia coli mutant disrupted in the gene encoding TolC, which has the peculiarity of being involved in diverse transport functions. BepC fully complemented the resistance to drugs such as chloramphenicol and acriflavine but was incapable of restoring hemolysin secretion in the tolC mutant of E. coli. An insertional mutation in the bepC gene strongly affected the resistance phenotype of B. suis to bile salts and toxic chemicals such as ethidium bromide and rhodamine and significantly decreased the resistance to antibiotics such as erythromycin, ampicillin, tetracycline, and norfloxacin. Moreover, the B. suis bepC mutant was attenuated in the mouse model of infection. Taken together, these results suggest that BepC-dependent efflux processes of toxic compounds contribute to B. suis survival inside the host

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

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

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