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dBiofilms cause up to 80% of infections and are difficult to treat due to their substantial multidrug resistance compared to their planktonic counterparts. Based on the observation that human peptide LL-37 is able to block biofilm formation at concentrations below its MIC, we screened for small peptides with antibiofilm activity and identified novel synthetic cationic peptide 1037 of only 9 amino acids in length. Peptide 1037 had very weak antimicrobial activity, but at 1/30th the MIC the peptide was able to effectively prevent biofilm formation (>50% reduction in cell biomass) by the Gram-negative pathogens Pseudomonas aeruginosa and Burkholderia cenocepacia and Gram-positive Listeria monocytogenes. Using a flow cell system and a widefield fluorescence microscope, 1037 was shown to significantly reduce biofilm formation and lead to cell death in biofilms. Microarray and follow-up studies showed that, in P. aeruginosa, 1037 directly inhibited biofilms by reducing swimming and swarming motilities, stimulating twitching motility, and suppressing the expression of a variety of genes involved in biofilm formation (e.g., PA2204). Comparison of microarray data from cells treated with peptides LL-37 and 1037 enabled the identification of 11 common P. aeruginosa genes that have a role in biofilm formation and are proposed to represent functional targets of these peptides. Peptide 1037 shows promise as a potential therapeutic agent against chronic, recurrent biofilm infections caused by a variety of bacteria.

No abstract

Pseudomonas aeruginosa offers substantial therapeutic challenges due to its high intrinsic resistance to many antibiotics and its propensity to develop mutational and/or adaptive resistance. The PA14 comprehensive mutant library was screened for mutants exhibiting either two-to eightfold increased susceptibilities (revealing genes involved in intrinsic resistance) or decreased susceptibilities (mutational resistance) to the fluoroquinolone ciprofloxacin. Thirty-five and 79 mutants with increased and decreased susceptibilities, respectively, were identified, as confirmed by broth dilution.

Caseinolytic peptidases (ClpPs) regulate diverse aspects of cellular physiology in bacteria. Some species have multiple ClpPs, including the opportunistic pathogen Pseudomonas aeruginosa, in which there is an archetypical isoform, ClpP1, and a second isoform, ClpP2, about which little is known. Here, we use phenotypic assays to investigate the biological roles of ClpP1 and ClpP2 and biochemical assays to characterize purified ClpP1, ClpP2, ClpX, and ClpA. Interestingly, ClpP1 and ClpP2 have distinct intracellular roles for motility, pigment production, iron scavenging, and biofilm formation. Of particular interest, ClpP2, but not ClpP1, is required for microcolony organization, where multicellular organized structures first form on the pathway to biofilm production. We found that purified ClpP1 with ClpX or ClpA was enzymatically active, yet to our surprise, ClpP2 was inactive and not fully assembled in vitro; attempts to assist ClpP2 assembly and activation by mixing with the other Clp components failed to turn on ClpP2, as did solution conditions that have helped activate other ClpPs in vitro. We postulate that the active form of ClpP2 has yet to be discovered, and we present several potential models to explain its activation as well as the unique role ClpP2 plays in the development of the clinically important biofilms in P. aeruginosa. IMPORTANCE Pseudomonas aeruginosa is responsible for severe infections of immunocompromised patients. Our work demonstrates that two different isoforms of the Clp peptidase, ClpP1 and ClpP2, control distinct aspects of cellular physiology for this organism. In particular, we identify ClpP2 as being necessary for microcolony organization. Pure active forms of ClpP1 and either ClpX or ClpA were characterized as assembled and active, and ClpP2 was incompletely assembled and inactive. By establishing both the unique biological roles of ClpP1 and ClpP2 and their initial biochemical assemblies, we have set the stage for important future work on the structure, function, and biological targets of Clp proteolytic enzymes in this important opportunistic pathogen.

Pseudomonas aeruginosa PAO1 lon mutants are supersusceptible to ciprofloxacin, and exhibit a defect in cell division and in virulence-related properties, such as swarming, twitching and biofilm formation, despite the fact that the Lon protease is not a traditional regulator. Here we set out to investigate the influence of a lon mutation in a series of infection models. It was demonstrated that the lon mutant had a defect in cytotoxicity towards epithelial cells, was less virulent in an amoeba model as well as a mouse acute lung infection model, and impacted on in vivo survival in a rat model of chronic infection. Using qRT-PCR it was demonstrated that the lon mutation led to a down-regulation of Type III secretion genes. The Lon protease also influenced motility and biofilm formation in a mucin-rich environment. Thus alterations in several virulence-related processes in vitro in a lon mutant were reflected by defective virulence in vivo.

Pseudomonas aeruginosa possesses complex regulatory networks controlling virulence and survival under adverse conditions, including antibiotic pressure, which are interconnected and share common regulatory proteins. Here, we screen a panel of 13 mutants defective in intracellular proteases and demonstrate that, in addition to the known alterations in Lon and AsrA mutants, mutation of three protease-related proteins PfpI, ClpS, and ClpP differentially affected antibiotic resistance, swarming motility, and biofilm formation.

Cystic fibrosis (CF) patients often acquire chronic respiratory tract infections due to Pseudomonas aeruginosa and Burkholderia cepacia complex (Bcc) species. In the CF lung, these bacteria grow as multicellular aggregates termed biofilms. Biofilms demonstrate increased (adaptive) resistance to conventional antibiotics, and there are currently no available biofilm-specific therapies. Using plastic adherent, hydroxyapatite and flow cell biofilm models coupled with confocal and scanning electron microscopy, it was demonstrated that an anti-biofilm peptide 1018 prevented biofilm formation, eradicated mature biofilms and killed biofilms formed by a wide range of P. aeruginosa and B. cenocepacia clinical isolates. New peptide derivatives were designed that, compared to their parent peptide 1018, showed similar or decreased anti-biofilm activity against P. aeruginosa biofilms, but increased activity against biofilms formed by the Gram-positive bacterium methicillin resistant Staphylococcus aureus. In addition, some of these new peptide derivatives retained the immunomodulatory activity of 1018 since they induced the production of the chemokine monocyte chemotactic protein-1 (MCP-1) and suppressed lipopolysaccharide-mediated tumor necrosis factor-α (TNF-α) production by human peripheral blood mononuclear cells (PBMC) and were non-toxic towards these cells. Peptide 1018 and its derivatives provide promising leads for the treatment of chronic biofilm infections and hyperinflammatory lung disease in CF patients.