PhoP–PhoQ homologues in Pseudomonas aeruginosa regulate expression of the outer‐membrane protein OprH and polymyxin B resistance

Abstract

Rapid adaptation to environmental challenge is essential for the survival of many bacterial species, and is often effectively mediated by two‐component regulatory systems. Part of the adaptive response of Pseudomonas aeruginosa to Mg2+ starvation is overexpression of the outer‐membrane protein OprH and increased resistance to the polycationic antibiotic polymyxin B. Two overlapping open reading frames that encoded proteins with high similarities to the PhoP–PhoQ two‐component regulatory system of Salmonella typhimurium were identified downstream of the oprH gene. A P. aeruginosa PhoP‐null mutant, H851, was constructed by means of a phoP::xylE‐GmR transcriptional fusion, and shown to be deficient in OprH expression. In contrast, an analogous PhoQ‐null mutant, H854 (phoQ::xylE‐GmR), exhibited constitutive overexpression of OprH. Normal Mg2+‐regulated OprH expression could be restored in both mutants by complementation with a plasmid carrying the phoP and phoQ genes. Measurement of the catechol‐2,3‐dioxygenase activity, expressed from the xylE transcriptional fusion in strains H851 and H854, indicated that PhoP–PhoQ is involved in the regulation of phoP–phoQ as well as oprH. Reverse transcription polymerase chain reaction experiments and Northern blot analysis revealed linkage of oprH, phoP and phoQ into an operon that was demonstrated to be under the joint control of PhoP–PhoQ and Mg2+ ion concentration. In addition, studies of the polymyxin B resistance of the two mutant strains, H851 and H854, indicated that PhoP–PhoQ is involved in regulating P. aeruginosa polymyxin resistance in response to external Mg2+ concentrations.

Proteomic analysis of Pseudomonas aeruginosa grown under magnesium limitation

“…These included the transcriptional regulator PhoP and magnesium transporter homologue MgtA (Table 2). PhoP orthologues regulate expression of genes essential for virulence and magnesium acquisition in several Gram-negative bacteria [14,20,11]. Some of the PhoP-induced enzymes promote increased bacterial resistance to antimicrobial peptides, a key component of host's innate immunity [10,17,21,22].…”

“…Significant Gram-negative envelope remodeling is part of bacterial adaptation to magnesium limitation [10,23,11]. To define proteins that contribute to the envelope remodeling either structurally or by providing an enzymatic function to this process by mediating LPS modifications, total membrane protein of P. aeruginosa was also analyzed by quantitative proteomic analysis.…”

“…Furthermore, transcriptional profiling analysis confirmed increased expression of genes encoding OprH and PA4824 during growth of P. aeruginosa in low magnesium. OprH is an outer membrane protein previously shown to be induced by magnesium stress and regulated by PhoP [11]. Although highly abundant, OprH was not identified in the course of ICAT analysis due to its lack of cysteine.…”

“…One of the earliest reported adaptations of P. aeruginosa isolates from airways of infants with CF is the synthesis of specific lipopolysaccharide (LPS) at the bacterial surface. The result of such envelope modification is increased bacterial resistance to antimicrobial peptides [10,11] and increased proinflammatory signaling through the human Tlr4 receptor [12]. P. aeruginosa was also isolated from CF patients' airways in the form of biofilms, antibiotic-resistant bacterial communities [13].…”

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

“…One striking feature of the predicted protein complement of P. aeruginosa is the presence of a disproportionately large number of regulatory proteins. Recently we identified the PhoP-PhoQ homologues in P. aeruginosa (Macfarlane et al, 1999). The genes for this regulatory system lie immediately downstream of the oprH gene, which encodes a small outer-membrane protein OprH that is highly expressed under Mg# + -starvation conditions.…”