The two-component system CprRS senses cationic peptides and triggers adaptive resistance in Pseudomonas aeruginosa independently of ParRS.

Cationic antimicrobial peptides pass across the outer membrane by interacting with negatively charged lipopolysaccharide (LPS), leading to outer membrane permeabilization in a process termed self-promoted uptake. Resistance can be mediated by the addition of positively charged arabinosamine through the action of the arnBCADTEF operon. We recently described a series of two-component regulators that lead to the activation of the arn operon after recognizing environmental signals, including low-Mg(2+) (PhoPQ, PmrAB) or cationic (ParRS) peptides. However, some peptides did not activate the arn operon through ParRS. Here, we report the identification of a new two-component system, CprRS, which, upon exposure to a wide range of antimicrobial peptides, triggered the expression of the LPS modification operon. Thus, mutations in the cprRS operon blocked the induction of the arn operon in response to several antimicrobial peptides independently of ParRS but did not affect the response to low Mg(2+). Distinct patterns of arn induction were identified. Thus, the responses to polymyxins were abrogated by either parR or cprR mutations, while responses to other peptides, including indolicidin, showed differential dependency on the CprRS and ParRS systems in a concentration-dependent manner. It was further demonstrated that, following exposure to inducing antimicrobial peptides, cprRS mutants did not become adaptively resistant to polymyxins as was observed for wild-type cells. Our microarray studies demonstrated that the CprRS system controlled a quite modest regulon, indicating that it was quite specific to adaptive peptide resistance. These findings provide greater insight into the complex regulation of LPS modification in Pseudomonas aeruginosa, which involves the participation of at least 4 two-component systems.

Adaptive resistance to the "last hope" antibiotics polymyxin B and colistin in Pseudomonas aeruginosa is mediated by the novel two-component regulatory system ParR-ParS.

As multidrug resistance increases alarmingly, polymyxin B and colistin are increasingly being used in the clinic to treat serious Pseudomonas aeruginosa infections. In this opportunistic pathogen, subinhibitory levels of polymyxins and certain antimicrobial peptides induce resistance toward higher, otherwise lethal, levels of these antimicrobial agents. It is known that the modification of lipid A of lipopolysaccharide (LPS) is a key component of this adaptive peptide resistance, but to date, the regulatory mechanism underlying peptide regulation in P. aeruginosa has remained elusive. The PhoP-PhoQ and PmrA-PmrB two-component systems, which control this modification under low-Mg2+ conditions, do not appear to play a major role in peptide-mediated adaptive resistance, unlike in Salmonella where PhoQ is a peptide sensor. Here we describe the identification and characterization of a novel P. aeruginosa two-component regulator affecting polymyxin-adaptive resistance, ParR-ParS (PA1799-PA1798). This system was required for activation of the arnBCADTEF LPS modification operon in the presence of subinhibitory concentrations of polymyxin, colistin, or the bovine peptide indolicidin, leading to increased resistance to various polycationic antibiotics, including aminoglycosides. This study highlights the complexity of the regulatory network controlling resistance to cationic antibiotics and host peptides in P. aeruginosa, which has major relevance in the development and deployment of cationic antimicrobials.

Induction by cationic antimicrobial peptides and involvement in intrinsic polymyxin and antimicrobial peptide resistance, biofilm formation, and swarming motility of PsrA in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an important opportunistic pathogen that causes infections that can be extremely difficult to treat due to its high intrinsic antibiotic resistance and broad repertoire of virulence factors, both of which are highly regulated. It is demonstrated here that the psrA gene, encoding a transcriptional regulator, was upregulated in response to subinhibitory concentrations of cationic antimicrobial peptides. Compared to the wild type and the complemented mutant, a P. aeruginosa PAO1 psrA::Tn5 mutant displayed intrinsic supersusceptibility to polymyxin B, a last-resort antimicrobial used against multidrug-resistant infections, and the bovine neutrophil antimicrobial peptide indolicidin; this supersusceptibility phenotype correlated with increased outer membrane permeabilization by these agents. The psrA mutant was also defective in simple biofilm formation, rapid attachment, and swarming motility, all of which could be complemented by the cloned psrA gene. The role of PsrA in global gene regulation was studied by comparing the psrA mutant to the wild type by microarray analysis, demonstrating that 178 genes were up- or downregulated >or=2-fold (P

Regulation of virulence and antibiotic resistance by two-component regulatory systems in Pseudomonas aeruginosa.

The Gram-negative opportunistic pathogen Pseudomonas aeruginosa ubiquitously inhabits soil and water habitats and also causes serious, often antibiotic resistant, infections in immunocompromised patients (e.g. cystic fibrosis). This versatility is mediated in part by a large repertoire of two-component regulatory systems that appear instrumental in the regulation of both virulence processes and resistance to antimicrobials. Major two-component regulatory system proteins demonstrated to regulate these diverse processes include PhoP-PhoQ, GacA-GacS, RetS, LadS, and AlgR, among others. Here, we summarize the current body of knowledge of these and other two-component systems that provides insight into the complex regulation of virulence and resistance in P. aeruginosa.

The sensor kinase PhoQ mediates virulence in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a ubiquitous environmental Gram-negative bacterium that is also a major opportunistic human pathogen in nosocomial infections and cystic fibrosis chronic lung infections. PhoP-PhoQ is a two-component regulatory system that has been identified as essential for virulence and cationic antimicrobial peptide resistance in several other Gram-negative bacteria. This study demonstrated that mutation of phoQ caused reduced twitching motility, biofilm formation and rapid attachment to surfaces, 2.2-fold reduced cytotoxicity to human lung epithelial cells, substantially reduced lettuce leaf virulence, and a major, 10 000-fold reduction in competitiveness in chronic rat lung infections. Microarray analysis revealed that PhoQ controlled the expression of many genes consistent with these phenotypes and with its known role in polymyxin B resistance. It was also demonstrated that PhoQ controls the expression of many genes outside the known PhoP regulon.

Construction of a mini-Tn5-luxCDABE mutant library in Pseudomonas aeruginosa PAO1: a tool for identifying differentially regulated genes.

Pseudomonas aeruginosa is a major cause of nosocomial (hospital-derived) infections, is the predominant pathogen in chronic cystic fibrosis lung infections, and remains difficult to treat due to its high intrinsic antibiotic resistance. The completion of the P. aeruginosa PAO1 genome sequence provides the opportunity for genome-wide studies to increase our understanding of the pathogenesis and biology of this important pathogen. In this report, we describe the construction of a mini-Tn5-luxCDABE mutant library and a high-throughput inverse PCR method to amplify DNA flanking the site of insertion for sequencing and insertion site mapping. In addition to producing polar knockout mutations in nonessential genes, the promoterless luxCDABE reporter present in the transposon serves as a real-time reporter of gene expression for the inactivated gene. A total of 2519 transposon insertion sites were mapped, 77% of which were nonredundant insertions. Of the insertions within an ORF, -55% of total and unique insertion sites were transcriptional luxCDABE fusions. A bias toward low insertion-site density in the genome region that surrounds the predicted terminus of replication was observed. To demonstrate the utility of chromosomal lux fusions, we performed extensive regulatory screens to identify genes that were differentially regulated under magnesium or phosphate limitation. This approach led to the discovery of many known and novel genes necessary for these environmental adaptations, including genes involved in resistance to cationic antimicrobial peptides. This dual-purpose mutant library allows for functional and regulation studies and will serve as a resource for the research community to further our understanding of P. aeruginosa biology.