Role of Pseudomonas aeruginosa PhoP-PhoQ in resistance to antimicrobial cationic peptides and aminoglycosides

Resistance to the polycationic antibiotic polymyxin B and expression of the outer-membrane protein OprH in the opportunistic pathogen Pseudomonas aeru ginosa both involve the PhoP-PhoQ two-component regulatory system. The gene s for this system form an operon with oprH, oprH-phoP-phoQ, that responds t o Mg2+ starvation and PhoP levels. In this study, the Mg2+-regulated promot er for this operon was mapped upstream of oprH by primer-extension experime nts. An oprH::xylE-Gm(R) mutant H855 was constructed and measurement of the catechol 2,3-dioxygenase activity expressed from this transcriptional fusi on provided evidence for a second, weak promoter for phoP-phoQ, Wild-type P . aeruginosa PAO1 strain H103 was found to exhibit Mg2+-regulated resistanc e to the ct-helical antimicrobial cationic peptide CP28 in addition to its previously characterized resistance to polymyxin B. Resistance to this pept ide was unchanged in the OprH-null mutant H855 and a PhoP-null mutant H851. In contrast, PhoQ-null mutant H854 demonstrated constitutive CP28 resistan ce. Northern blot analysis revealed constitutive expression of phoP in this strain, implicating PhoP-PhoQ in the resistance of P. aeruginosa to cation ic peptides. Furthermore, all three null-mutant strains demonstrated increa sed resistance to the aminoglycoside antibiotics streptomycin, kanamycin an d amikacin. Two additional mutant strains, H895 and H896, were constructed that carried unmarked deletions in oprH and were found to exhibit aminoglyc oside susceptibility equivalent to that of the wildtype. This result provid ed definitive evidence that OprH is not involved in P. aeruginosa aminoglyc oside resistance and that the changes in resistance in strain H855 and a pr eviously reported oprH mutant were due to polar effects on phoP-phoQ rather than loss of OprH expression. A role for PhoP-PhoQ in resistance to aminog lycosides is envisaged that is distinct from that in resistance to cationic peptides and polymyxin B.