Identification of the essential Brucella melitensis porin Omp2b as a suppressor of Bax-induced cell death in yeast in a genome-wide screening.

BACKGROUND: Inhibition 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 FINDINGS: In 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/SIGNIFICANCE: We 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

Regulatory (pan-)genome of an obligate intracellular pathogen in the PVC superphylum.

Like other obligate intracellular bacteria, the Chlamydiae feature a compact regulatory genome that remains uncharted owing to poor genetic tractability. Exploiting the reduced number of transcription factors (TFs) encoded in the chlamydial (pan-)genome as a model for TF control supporting the intracellular lifestyle, we determined the conserved landscape of TF specificities by ChIP-Seq (chromatin immunoprecipitation-sequencing) in the chlamydial pathogen Waddlia chondrophila. Among 10 conserved TFs, Euo emerged as a master TF targeting >100 promoters through conserved residues in a DNA excisionase-like winged helix-turn-helix-like (wHTH) fold. Minimal target (Euo) boxes were found in conserved developmentally-regulated genes governing vertical genome transmission (cytokinesis and DNA replication) and genome plasticity (transposases). Our ChIP-Seq analysis with intracellular bacteria not only reveals that global TF regulation is maintained in the reduced regulatory genomes of Chlamydiae, but also predicts that master TFs interpret genomic information in the obligate intracellular α-proteobacteria, including the rickettsiae, from which modern day mitochondria evolved

Identification and characterization of potential Brucella abortus effectors interacting with Rab GTPases and endoplasmic reticulum exit sites associated proteins

Brucellae are intracellular facultative pathogenic bacteria which are able to invade professional and non-professional phagocytes. Once inside the cell, Brucella resides in a vacuole (BCV, Brucella containing vacuole), which successively interacts with endocytic compartements, i.e. early endosomes, late endosomes and lysosomes. Finally, Brucella reaches endoplasmic reticulum (ER) at particular sites, ER exit sites (ERES), to extensively proliferate in the ER. Type IV secretion system (T4SS) VirB, was shown to be required to sustain interaction with ER and consequently to proliferate. It was suggested that T4SS translocates proteins to hijack membrane trafficking processes such as endocytic and secretory pathways, regulated by Rab GTPases. Up to now, at least three T4SS substrates were identified but their function remains unkown. In order to identify Brucella effectors, we took advantage of the availability of the Brucella ORFeome in our laboratory to perform a genome-wide yeast two-hybrid (Y2H) screening between Brucella predicted proteins and human phagosomal proteins, Rab GTPases and ERES associated proteins. In the current study, we report the identification of three specific interactions between Rab2 and RicA (Rab2 interacting conserved protein A), Rab34 and PitF (protein interacting with Rab thirty four), and Sec24A and CostA (conserved Sec24A-targeted protein A). We showed that RicA and PitF are translocated during infection of macrophages and these translocations are T4SS-dependent. RicA and CostA are secreted in bacteriological culture and the secretion of these two proteins is kept in the ΔvirB mutant strain, suggesting that another secretion system is functional in these conditions. An in frame deletion was constructed for each Brucella gene coding for a potential effector and mutants were characterized. The ΔricA mutant strain recruits less GTP-locked myc-Rab2 on its BCVs. This mutant has also an altered trafficking and a faster proliferation compared to the B. abortus wild type strain. The ΔpitF mutant has an internalization defect. Finally, the costA mutant is impaired for its association to the Sec23 ERES marker and displayed a particular trafficking, with a lower association to LAMP1 and calnexin at 12 h post-infection in HeLa cells, but its intracellular proliferation kinetics was not affected. In conclusion, this work allows the identification of two Brucella effectors with a proposed function during a model cell infection. Indeed, RicA could act on trafficking by recruiting Rab2 and PitF plays a role during Brucella internalization, perhaps through its interaction with Rab34.(DOCSC03) -- FUNDP, 201