Mechanism of transcriptional activation by Pseudomonas aeruginosa ExsA.

ExsA is a transcriptional activator of the Pseudomonas aeruginosa type III secretion system (T3SS). The T3SS consists of >40 genes organized within 10 transcriptional units, each of which is controlled by the transcriptional activator ExsA. ExsA-dependent promoters contain two adjacent ExsA binding sites that when occupied protect the -30 to -70 region from DNase I cleavage. The promoters also possess regions bearing strong resemblance to the consensus -10 and -35 regions of sigma(70)-dependent promoters. The spacing distance between the putative -10 and -35 regions of ExsA-dependent promoters, however, is increased by 4 to 5 bp compared to that in typical sigma(70)-dependent promoters. In the present study, we demonstrate that ExsA-dependent transcriptional activation requires a 21- or 22-bp spacer length between the -10 and -35 regions. Despite the atypical spacing in this region, in vitro transcription assays using sigma(70)-saturated RNA polymerase holoenzyme (RNAP-sigma(70)) confirm that ExsA-dependent promoters are indeed sigma(70) dependent. Potassium permanganate footprinting experiments indicate that ExsA facilitates an early step in transcriptional initiation. Although RNAP-sigma(70) binds to the promoters with low affinity in the absence of ExsA, the activator stimulates transcription by enhancing recruitment of RNAP-sigma(70) to the P(exsC) and P(exsD) promoters. Abortive initiation assays confirm that ExsA enhances the equilibrium binding constant for RNAP while having only a modest effect on the isomerization rate constant.

The potential of desferrioxamine-gallium as an anti-Pseudomonas therapeutic agent.

The opportunistic pathogen Pseudomonas aeruginosa causes infections that are difficult to treat by antibiotic therapy. This bacterium can cause biofilm infections where it shows tolerance to antibiotics. Here we report the novel use of a metallo-complex, desferrioxamine-gallium (DFO-Ga) that targets P. aeruginosa iron metabolism. This complex kills free-living bacteria and blocks biofilm formation. A combination of DFO-Ga and the anti-Pseudomonas antibiotic gentamicin caused massive killing of P. aeruginosa cells in mature biofilms. In a P. aeruginosa rabbit corneal infection, topical administration of DFO-Ga together with gentamicin decreased both infiltrate and final scar size by about 50% compared to topical application of gentamicin alone. The use of DFO-Ga as a Trojan horse delivery system that interferes with iron metabolism shows promise as a treatment for P. aeruginosa infections.

Characterization of ExsA and of ExsA-dependent promoters required for expression of the Pseudomonas aeruginosa type III secretion system.

Expression of the Pseudomonas aeruginosa type III secretion system (T3SS) is activated by ExsA, a member of the AraC/XylS family of transcriptional regulators. In the present study we examine the DNA-binding properties of ExsA. ExsA was purified as a histidine-tagged fusion protein (ExsA(His)) and found to be monomeric in solution. ExsA(His) specifically bound T3SS promoters with high affinity as determined by electrophoretic mobility shift assays (EMSA). For each promoter tested two distinct ExsA-DNA complexes were detected. Biochemical analyses indicate that the higher-mobility complex consists of a single ExsA(His) molecule bound to DNA while the lower-mobility complex results from the binding of two ExsA(His) molecules. DNase I protection assays demonstrate that the ExsA(His) binding site overlaps the -35 RNA polymerase binding site and extends upstream an additional approximately 34 bp. An alignment of all 10 ExsA-dependent promoters revealed a number of highly conserved nucleotides within the footprinted region. We find that most of the highly conserved nucleotides are required for transcription in vivo; EMSA-binding assays confirm that several of these nucleotides are essential determinants of ExsA(His) binding. The combined data support a model in which two ExsA(His) molecules bind adjacent sites on the promoter to activate T3SS gene transcription.

Enhancement of antimicrobial activity against pseudomonas aeruginosa by coadministration of G10KHc and tobramycin.

Pseudomonas aeruginosa is a common opportunistic human pathogen that is associated with life-threatening acute infections and chronic airway colonization during cystic fibrosis. Previously, we converted the wide-spectrum antimicrobial peptide novispirin G10 into a selectively-targeted antimicrobial peptide (STAMP), G10KHc. Compared to novispirin G10, the STAMP had an enhanced ability to kill Pseudomonas mendocina. In this study, we explored the activity of G10KHc against P. aeruginosa. G10KHc was found to be highly active (as active as tobramycin) against P. aeruginosa clinical isolates. Most interestingly, we observed a synergistic-like enhancement in killing activity when biofilms and planktonic cultures of P. aeruginosa were cotreated with G10KHc and tobramycin. The data indicate that the mechanism of enhanced activity may involve increased tobramycin uptake due to G10KHc-mediated cell membrane disruption. These results suggest that G10KHc may be useful against P. aeruginosa during acute and chronic infection states, especially when it is coadministered with tobramycin.

Chelator-induced dispersal and killing of Pseudomonas aeruginosa cells in a biofilm.

Biofilms consist of groups of bacteria attached to surfaces and encased in a hydrated polymeric matrix. Bacteria in biofilms are more resistant to the immune system and to antibiotics than their free-living planktonic counterparts. Thus, biofilm-related infections are persistent and often show recurrent symptoms. The metal chelator EDTA is known to have activity against biofilms of gram-positive bacteria such as Staphylococcus aureus. EDTA can also kill planktonic cells of Proteobacteria like Pseudomonas aeruginosa. In this study we demonstrate that EDTA is a potent P. aeruginosa biofilm disrupter. In Tris buffer, EDTA treatment of P. aeruginosa biofilms results in 1,000-fold greater killing than treatment with the P. aeruginosa antibiotic gentamicin. Furthermore, a combination of EDTA and gentamicin results in complete killing of biofilm cells. P. aeruginosa biofilms can form structured mushroom-like entities when grown under flow on a glass surface. Time lapse confocal scanning laser microscopy shows that EDTA causes a dispersal of P. aeruginosa cells from biofilms and killing of biofilm cells within the mushroom-like structures. An examination of the influence of several divalent cations on the antibiofilm activity of EDTA indicates that magnesium, calcium, and iron protect P. aeruginosa biofilms against EDTA treatment. Our results are consistent with a mechanism whereby EDTA causes detachment and killing of biofilm cells.