Identification of the Essential Brucella melitensis Porin Omp2b as a Suppressor of Bax-Induced Cell Death in Yeast in a Genome-Wide Screening

Abstract

BackgroundInhibition of apoptosis is one of the mechanisms selected by numerous intracellular pathogenic bacteria to control their host cell. Brucellae, which are the causative agent of a worldwide zoonosis, prevent apoptosis of infected cells, probably to support survival of their replication niche.Methodology/Principal FindingsIn order to identify Brucella melitensis anti-apoptotic effector candidates, we performed a genome-wide functional screening in yeast. The B. melitensis ORFeome was screened to identify inhibitors of Bax-induced cell death in S. cerevisiae. B. melitensis porin Omp2b, here shown to be essential, prevents Bax lethal effect in yeast, unlike its close paralog Omp2a. Our results based on Omp2b size variants characterization suggest that signal peptide processing is required for Omp2b effect in yeast.Conclusion/SignificanceWe report here the first application to a bacterial genome-wide library of coding sequences of this “yeast-rescue” screening strategy, previously used to highlight several new apoptosis regulators. Our work provides B. melitensis proteins that are candidates for an anti-apoptotic function, and can be tested in mammalian cells in the future. Hypotheses on possible molecular mechanisms of Bax inhibition by the B. melitensis porin Omp2b are discussed.

Intruders below the Radar: Molecular Pathogenesis of Bartonella spp

“…The surface display of fibronectin binding proteins is a common theme among the infection strategies of many bacterial pathogens (reviewed in reference 175) and has been linked to endothelial invasion and inflammation, for example, in infective endocarditis (346) as a well-known complication of Bartonella infections. OMP43 of Bartonella is a porin-like protein homologous to OMP2b of Brucella (which is known to be a potent inhibitor of apoptosis [248]) and further exhibits considerable sequence similarity to the RopA protein of Rhizobium leguminosarum, whose expression is strongly repressed in bacteroids (65,125). In addition to fibronectin binding, OMP43 was shown to bind HUVECs and thus was proposed to serve as a major adhesin during Bartonella infection (65,162).…”

The Sinorhizobium meliloti Essential Porin RopA1 Is a Target for Numerous Bacteriophages

“…The porB gene of Neisseria gonorrhea has also been reported to be essential, but again, no experimental evidence is given (35,36). Since both ropA1 in S. meliloti (this report) and omp2b in Brucella melitensis (21) are believed to be essential, it may be that ropA homologs are essential in most Rhizobiales species which possess them. One possible exception is the single ropA homolog in Bartonella henselae, omp43, which has been successfully disrupted (37).…”

“…Mutations in ropA1 that conferred resistance to bacteriophages were always point mutations or insertions/deletions that were multiples of three base pairs, strongly suggesting that ropA1-null alleles are not tolerated. Furthermore, a ropA1 homolog in Brucella melitensis, omp2b, was reported to be essential (21), though no experimental evidence was provided. To test whether ropA1 might be essential for viability, we first made several failed attempts to create an in-frame deletion of ropA1 in strain Rm1021 using the pJQ200sk sacB vector (22).…”

“…The omp2b gene of Brucella melitensis (a ropA1 homolog) has been reported to be essential, but no experimental evidence is given (21). The porB gene of Neisseria gonorrhea has also been reported to be essential, but again, no experimental evidence is given (35,36).…”

Host-Brucella interactions and the Brucella genome as tools for subunit antigen discovery and immunization against brucellosis

“…Brucellae Omp25 regulates TNF-α secretion and prevents apoptosis of macrophages (Gross et al, 2000; Jubier-Maurin et al, 2001; Billard et al, 2007). Anti-apoptotic properties have been demonstrated for the Omp2b porin in macrophages and yeast (He et al, 2006; Laloux et al, 2010). Interestingly, recent reports describe apoptosis of B. abortus infected T-lymphocytes and astrocytes (Garcia Samartino et al, 2010; Velasquez et al, 2012).…”