Elena B. M. Breidenstein

Inhibition of bacterial biofilm formation and swarming motility by a small synthetic cationic peptide

ABSTRACT Biofilms 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.

Complex Ciprofloxacin Resistome Revealed by Screening a Pseudomonas aeruginosa Mutant Library for Altered Susceptibility

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.

The Lon Protease Is Essential for Full Virulence in Pseudomonas aeruginosa

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.

Role of intracellular proteases in the antibiotic resistance, motility, and biofilm formation of Pseudomonas aeruginosa.

ABSTRACT 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.

Role of Lon, an ATP-Dependent Protease Homolog, in Resistance of Pseudomonas aeruginosa to Ciprofloxacin

ABSTRACT With few novel antimicrobials in the pharmaceutical pipeline, resistance to the current selection of antibiotics represents a significant therapeutic challenge. Microbial persistence in subinhibitory antibiotic environments has been proposed to contribute to the development of resistance. Pseudomonas aeruginosa cultures pretreated with subinhibitory concentrations of ciprofloxacin were found to exhibit an adaptive resistance phenotype when cultures were subsequently exposed to suprainhibitory ciprofloxacin concentrations. Microarray experiments revealed candidate genes involved in such adaptive resistance. Screening of 10,000 Tn5-luxCDABE mutants identified several mutants with increased or decreased ciprofloxacin susceptibilities, including mutants in PA1803, a close homolog of the ATP-dependent lon protease, which were found to exhibit ≥4-fold-increased susceptibilities to ciprofloxacin and other fluoroquinolones, but not to gentamicin or imipenem, as well as a characteristic elongated morphology. Complementation of the lon mutant restored wild-type antibiotic susceptibility and cell morphology. Expression of the lon mutant, as monitored through a luciferase reporter fusion, was found to increase over time in the presence of subinhibitory ciprofloxacin concentrations. The data are consistent with the hypothesis that the induction of Lon by ciprofloxacin is involved in adaptive resistance.

Mutator Genes Giving Rise to Decreased Antibiotic Susceptibility in Pseudomonas aeruginosa

ABSTRACT Screening of the PA14 genomic transposon mutant library for resistance to ceftazidime, tobramycin, and ciprofloxacin led to the discovery of several mutants that appeared in more than one screen. Testing of the frequency of mutation to ciprofloxacin resistance revealed previously known mutator genes, including mutS and mutL, as well as mutators that have not yet been described for P. aeruginosa, including PA3958 and RadA (PA4609).

Anti-Biofilm and Immunomodulatory Activities of Peptides That Inhibit Biofilms Formed by Pathogens Isolated from Cystic Fibrosis Patients

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.

Involvement of the Lon Protease in the SOS Response Triggered by Ciprofloxacin in Pseudomonas aeruginosa PAO1

ABSTRACT Pseudomonas aeruginosa PAO1 lon mutants have phenotypes of deficiencies in cell division, swarming, twitching, and biofilm formation as well as a phenotype of ciprofloxacin supersusceptibility. In this study, we demonstrated that a lon mutant was also supersensitive to the DNA-damaging agent UV light. To understand the influence of lon in causing these phenotypes, global gene expression was characterized by performing microarrays on the lon mutant and the PAO1 wild type grown in the presence of subinhibitory concentrations of ciprofloxacin. This revealed major differences in the expression of genes involved in the SOS response and DNA repair. Real-time quantitative PCR confirmed that these genes were highly upregulated upon ciprofloxacin exposure in the wild type but were significantly less induced in the lon mutant, indicating that Lon modulates the SOS response and consequentially ciprofloxacin susceptibility. As the known Lon target SulA is a member of the SOS response regulon, the influence of mutating or overexpressing this gene, and the negative regulator of the SOS response, LexA, was examined. Overexpression of lexA had no effect on the Lon-related phenotypes, but sulA overexpression recapitulated certain lon mutant phenotypes, including altered motility and cell division, indicating that Lon regulates these phenotypes through SulA. However, sulA overexpression did not affect ciprofloxacin susceptibility or biofilm formation, indicating that these properties were independently determined. Lon protease was also demonstrated to strongly influence RecA protein accumulation in the presence of ciprofloxacin. A model of DNA repair involving the Lon protease is proposed.

Armand-Frappier outstanding student award -- role of ATP-dependent proteases in antibiotic resistance and virulence.

ATP-dependent proteases are found in nearly all living organisms and are known to play important roles in protein quality control, including protein degradation and protein refolding. ATP-dependent proteases have been well characterized in Escherichia coli. However, in the opportunistic human pathogen Pseudomonas aeruginosa, the role of these proteases is only starting to be understood. This review will discuss the most recent research regarding the role of ATP-dependent proteases, particularly Lon and ClpP, in P. aeruginosa. These studies have revealed that despite the fact that they are not traditional regulators, these proteases are involved in regulating a multitude of processes, including antibiotic resistance and virulence, implicating a broad array of functions that these intracellular proteases have in Pseudomonas. These results are also relevant in the context of drug therapy, since ClpP and Lon are good candidates to become novel therapeutic targets to combat Pseudomonas infections.

Interconnection of post-transcriptional regulation: The RNA-binding protein Hfq is a novel target of the Lon protease in Pseudomonas aeruginosa

Besides being a major opportunistic human pathogen, Pseudomonas aeruginosa can be found in a wide range of environments. This versatility is linked to complex regulation, which is achieved through the action of transcriptional regulators, and post-transcriptional regulation by intracellular proteases including Lon. Indeed, lon mutants in this species show defects in motility, biofilm formation, pathogenicity and fluoroquinolone resistance. Here, the proteomic approach stable isotope labeling by amino acids in cell culture (SILAC) was used to search for novel proteolytic targets. One of the proteins that accumulated in the lon mutant was the RNA-binding protein Hfq. Further experiments demonstrated the ability of Lon to degrade Hfq in vitro. Also, overexpression of the hfq gene in the wild-type strain led to partial inhibition of swarming, swimming and twitching motilities, indicating that Hfq accumulation could contribute to the phenotypes displayed by Lon mutants. Hfq overexpression also led to the upregulation of the small regulatory RNA PhrS. Analysis of the phenotypes of strains lacking or overexpressing this sRNA indicated that the Lon protease might be indirectly regulating the levels and activity of sRNAs via Hfq. Overall, this study revealed new links in the complex regulatory chain that controls multicellular behaviours in P. aeruginosa.