Molecular basis for the transcriptional regulation of an epoxide-based virulence circuit in Pseudomonas aeruginosa.

The opportunistic pathogen Pseudomonas aeruginosa infects cystic fibrosis (CF) patient airways and produces a virulence factor Cif that is associated with worse outcomes. Cif is an epoxide hydrolase that reduces cell-surface abundance of the cystic fibrosis transmembrane conductance regulator (CFTR) and sabotages pro-resolving signals. Its expression is regulated by a divergently transcribed TetR family transcriptional repressor. CifR represents the first reported epoxide-sensing bacterial transcriptional regulator, but neither its interaction with cognate operator sequences nor the mechanism of activation has been investigated. Using biochemical and structural approaches, we uncovered the molecular mechanisms controlling this complex virulence operon. We present here the first molecular structures of CifR alone and in complex with operator DNA, resolved in a single crystal lattice. Significant conformational changes between these two structures suggest how CifR regulates the expression of the virulence gene cif. Interactions between the N-terminal extension of CifR with the DNA minor groove of the operator play a significant role in the operator recognition of CifR. We also determined that cysteine residue Cys107 is critical for epoxide sensing and DNA release. These results offer new insights into the stereochemical regulation of an epoxide-based virulence circuit in a critically important clinical pathogen.

Intracellular Passage Triggers a Molecular Response in Brucella abortus That Increases Its Infectiousness.

Brucella abortus is a facultatively extracellular-intracellular pathogen that encounters a diversity of environments within the host cell. We report that bacteria extracted from infected cells at late stages (48 h postinfection) of the intracellular life cycle significantly increase their ability to multiply in new target cells. This increase depends on early interaction with the cell surface, since the bacteria become more adherent and penetrate more efficiently than in vitro-grown bacteria. At this late stage of infection, the bacterium locates within an autophagosome-like compartment, facing starvation and acidic conditions. At this point, the BvrR/BvrS two-component system becomes activated, and the expression of the transcriptional regulator VjbR and the type IV secretion system component VirB increases. Using bafilomycin to inhibit BvrR/BvrS activation and using specific inhibitors for VjbR and VirB, we showed that the BvrR/BvrS and VjbR systems correlate with increased interaction with new host cells, while the VirB system does not. Bacteria released from infected cells under natural conditions displayed the same phenotype as intracellular bacteria. We propose a model in which the B. abortus BvrR/BvrS system senses the transition from its replicative niche at the endoplasmic reticulum to the autophagosome-like exit compartment. This activation leads to the expression of VirB, which participates in the release of the bacterium from the cells, and an increase in VjbR expression that results in a more efficient interaction with new host cells.