Generation of attenuated Mycobacterium bovis strains by signature-tagged mutagenesis for discovery of novel vaccine candidates.

Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex, has a particularly wide host range and causes tuberculosis in most mammals, including humans. A signature tag mutagenesis approach, which employed illegitimate recombination and infection of guinea pigs, was applied to M. bovis to discover genes important for virulence and to find potential vaccine candidates. Fifteen attenuated mutants were identified, four of which produced no lesions when inoculated separately into guinea pigs. One of these four mutants had nine deleted genes including mmpL4 and sigK and, in guinea pigs with aerosol challenge, provided protection against tuberculosis at least equal to that of M. bovis BCG. Seven mutants had mutations near the esxA (esat-6) locus, and immunoblot analysis of these confirmed the essential role of other genes at this locus in the secretion of EsxA (ESAT-6) and EsxB (CFP10). Mutations in the eight other attenuated mutants were widely spread through the chromosome and included pks1, which is naturally inactivated in clinical strains of M. tuberculosis. Many genes identified were different from those found by signature tag mutagenesis of M. tuberculosis by use of a mouse infection model and illustrate how the use of different approaches enables identification of a wider range of attenuating mutants.

Siderophore-mediated cell signalling in Pseudomonas aeruginosa: divergent pathways regulate virulence factor production and siderophore receptor synthesis.

Under iron-limiting conditions, Pseudomonas aeruginosa produces a siderophore called pyoverdine. Pyoverdine is secreted into the extracellular environment where it chelates iron, and the resulting ferri-pyoverdine complexes are transported back into the bacteria by a cell surface receptor protein FpvA. Pyoverdine also acts as a signalling molecule inducing the production of three secreted virulence factors. Binding of ferri-pyoverdine to FpvA transduces a signal to the periplasmic part of the membrane-spanning antisigma factor FpvR. The signal is transmitted to the cytoplasmic part of FpvR, which controls the activity of an extracytoplasmic family (ECF) sigma factor protein PvdS. This results in the production of the virulence factors pyoverdine, exotoxin A and PrpL endoprotease. Here, we show that a second divergent branch of this signalling pathway regulates the production of the FpvA protein. FpvR negatively regulates the activity of a second ECF sigma factor, FpvI, which is required for the synthesis of FpvA, and the presence of ferri-pyoverdine greatly increases the activity of FpvI so that production of FpvA is induced. To the best of our knowledge, this is the first example of a branched signalling system of this sort and the first example of an antisigma factor protein (FpvR) that directly regulates the activities of two different ECF sigma factor proteins (PvdS and FpvI).