SummaryIn contrast to many antimicrobial peptides, members of the proline-rich group of antimicrobial peptides inactivate Gram-negative bacteria by a non-lytic mechanism. Several lines of evidence indicate that they are internalized into bacteria and their activity mediated by interaction with unknown cellular components. With the aim of identifying such interactors, we selected mutagenized Escherichia coli clones resistant to the proline-rich Bac7(1-35) peptide and analysed genes responsible for conferring resistance, whose products may thus be involved in the peptide's mode of action. We isolated a number of genomic regions bearing such genes, and one in particular coding for SbmA, an inner membrane protein predicted to be part of an ABC transporter. An E. coli strain carrying a point mutation in sbmA, as well as other sbmA-null mutants, in fact showed resistance to several proline-rich peptides but not to representative membranolytic peptides. Use of fluorescently labelled Bac7(1-35) confirmed that resistance correlated with a decreased ability to internalize the peptide, suggesting that a bacterial protein, SbmA, is necessary for the transport of, and for susceptibility to, proline-rich antimicrobial peptides of eukaryotic origin.
Pseudomonas fuscovaginae is a Gram-negative fluorescent pseudomonad pathogenic towards several plant species. Despite its importance as a plant pathogen, no molecular studies of virulence have thus far been reported. In this study we show that P. fuscovaginae possesses two conserved N-acyl homoserine lactone (AHL) quorum sensing (QS) systems which we designated PfsI/R and PfvI/R. The PfsI/R system is homologous to the BviI/R system of Burkholderia vietnamiensis and produces and responds to C10-HSL and C12-HSL whereas PfvI/R is homologous to the LasI/R system of Pseudomonas aeruginosa and produces several long-chain 3-oxo-HSLs and responds to 3-oxo-C10-HSL and 3-oxo-C12-HSL and at high AHL concentrations can also respond to structurally different long-chain AHLs. Both systems were found to be negatively regulated by a repressor protein which was encoded by a gene located intergenically between the AHL synthase and LuxR-family response regulator. The pfsI/R system was regulated by a novel repressor designated RsaM while the pfvI/R system was regulated by both the RsaL repressor and by RsaM. The two systems are not transcriptionally hierarchically organized but share a common AHL response and both are required for plant virulence. Pseudomonas fuscovaginae has therefore a unique complex regulatory network composed of at least two different repressors which directly regulate the AHL QS systems and pathogenicity.
Antimicrobial peptides (AMPs) are secreted in the airway and contribute to initial defence against inhaled pathogens. Infections of the respiratory tract are a major cause of morbidity and mortality in preterm newborns and in patients with cystic fibrosis (CF). In this latter group, the state of chronic lung infection is due to the ability of bacteria to grow as mucoid biofilm, a condition characterised by overproduction and release of polysaccharides (PSs). In this study, we investigate the effect of PSs produced by lung pathogens such as Pseudomonas aeruginosa, Klebsiella pneumoniae and members of the Burkholderia cepacia complex on the antibacterial activity of structurally different peptides. The AMPs tested in this study include the cathelicidin LL-37 and the beta-defensin hBD-3 from humans, both released at the alveolar level, as well as peptides from other mammals, i.e. SMAP-29, PG-1 and Bac7(1-35). Susceptibility assays, time killing and membrane permeabilization kinetics experiments were carried out to establish whether PSs produced by lung pathogens may be involved in the poor defence reaction of infected lungs and thus explain infection persistence. All the PSs investigated inhibited, albeit to a different extent, the antibacterial activity of the peptides tested, suggesting that their presence in the lungs of patients with CF may contribute to the decreased defence response of this district upon infection by PS-producing microorganisms. The results also show that inhibition of the antibacterial activity is not simply due to ionic interaction between the negatively charged PSs and the cationic AMPs, but it also involves other structural features of both interactors.
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