Knock‐out of multidrug efflux pump MexXY‐OprM results in increased susceptibility to antimicrobial peptides in Pseudomonas aeruginosa

Multidrug efflux systems of the resistance-nodulation-cell division family play a crucial role in resistance of Pseudomonas aeruginosa to a large variety of antibiotics. Here, we investigated the role of clinically relevant efflux pumps MexAB$^−$OprM, MexCD$^−$OprJ, and MexXY$^−$OprM in resistance against different cationic antimicrobial peptides (AMPs). Our results indicate that a knock-out in efflux pump MexXY-OprM increased susceptibility to some AMPs by two- to eightfold. Our data suggest a contribution of MexXY-OprM in resistance to certain AMPs in P. aeruginosa, which should be considered in the future development of new and highly active antimicrobial peptides to fight multidrug resistant infections

The oxidative stress agent hypochlorite stimulates c-di-GMP synthesis and biofilm formation in Pseudomonas aeruginosa

The opportunistic human pathogen Pseudomonas aeruginosa is able to survive under a variety of often harmful environmental conditions due to a multitude of intrinsic and adaptive resistance mechanisms, including biofilm formation as one important survival strategy. Here, we investigated the adaptation of P. aeruginosa PAO1 to hypochlorite (HClO), a phagocyte-derived host defense compound and frequently used disinfectant. In static biofilm assays, we observed a significant enhancement in initial cell attachment in the presence of sublethal HClO concentrations. Subsequent LC-MS analyses revealed a strong increase in cyclic-di-GMP (c-di-GMP) levels suggesting a key role of this second messenger in HClO-induced biofilm development. Using DNA microarrays, we identified a 26-fold upregulation of ORF PA3177 coding for a putative diguanylate cyclase (DGC), which catalyzes the synthesis of the second messenger c-di-GMP – an important regulator of bacterial motility, sessility and persistence. This DGC PA3177 was further characterized in more detail demonstrating its impact on P. aeruginosa motility and biofilm formation. In addition, cell culture assays attested a role for PA3177 in the response of P. aeruginosa to human phagocytes. Using a subset of different mutants, we were able to show that both Pel and Psl exopolysaccharides are effectors in the PA3177-dependent c-di-GMP network

Enzyme-Mediated Quenching of the Pseudomonas Quinolone Signal (PQS) Promotes Biofilm Formation of Pseudomonas aeruginosa by Increasing Iron Availability

The 2-alkyl-3-hydroxy-4(1H)-quinolone 2,4-dioxygenase HodC was previously described to cleave the Pseudomonas quinolone signal, PQS, which is exclusively used in the complex quorum sensing (QS) system of Pseudomonas aeruginosa, an opportunistic pathogen employing QS to regulate virulence and biofilm development. Degradation of PQS by exogenous addition of HodC to planktonic cells of P.aeruginosa attenuated production of virulence factors, and reduced virulence inplanta. However, proteolytic cleavager educed the efficacy of HodC. Here, we identified the secreted protease LasB of P.aeruginosa to be responsible for HodC degradation. In static biofilms of the P.aeruginosa PA14 lasB::Tn mutant, the catalytic activity of HodC led to an increase in viable biomass in newly formed but also in established biofilms, and reduced the expression of genes involved in iron metabolism and siderophore production, such as pvdS, pvdL, pvdA, and pvdQ. This is likely due to an increase in the levels of bioavailable iron by degradation of PQS, which is able to sequester iron from the surrounding environment. Thus, HodC, despite ist ability to quench the production of virulence factors, is contraindicated for combating P.aeruginosa biofilms

TypA is involved in virulence, antimicrobial resistance and biofilm formation in Pseudomonas aeruginosa

Background: Pseudomonas aeruginosa is an important opportunistic human pathogen and is extremely difficult to treat due to its high intrinsic and adaptive antibiotic resistance, ability to form biofilms in chronic infections and broad arsenal of virulence factors, which are finely regulated. TypA is a GTPase that has recently been identified to modulate virulence in enteric Gram-negative pathogens. Results: Here, we demonstrate that mutation of typA in P. aeruginosa resulted in reduced virulence in phagocytic amoebae and human macrophage models of infection. In addition, the typA mutant was attenuated in rapid cell attachment to surfaces and biofilm formation, and exhibited reduced antibiotic resistance to ß-lactam, tetracycline and antimicrobial peptide antibiotics. Quantitative RT-PCR revealed the down-regulation, in a typA mutant, of important virulence-related genes such as those involved in regulation and assembly of the Type III secretion system, consistent with the observed phenotypes and role in virulence of P. aeruginosa. Conclusions: These data suggest that TypA is a newly identified modulator of pathogenesis in P. aeruginosa and is involved in multiple virulence-related characteristics.Other UBCNon UBCReviewedFacult

Human host defense peptide LL-37 stimulates virulence factor production and adaptive resistance in Pseudomonas aeruginosa.

A multitude of different virulence factors as well as the ability to rapidly adapt to adverse environmental conditions are important features for the high pathogenicity of Pseudomonas aeruginosa. Both virulence and adaptive resistance are tightly controlled by a complex regulatory network and respond to external stimuli, such as host signals or antibiotic stress, in a highly specific manner. Here, we demonstrate that physiological concentrations of the human host defense peptide LL-37 promote virulence factor production as well as an adaptive resistance against fluoroquinolone and aminoglycoside antibiotics in P. aeruginosa PAO1. Microarray analyses of P. aeruginosa cells exposed to LL-37 revealed an upregulation of gene clusters involved in the production of quorum sensing molecules and secreted virulence factors (PQS, phenazine, hydrogen cyanide (HCN), elastase and rhamnolipids) and in lipopolysaccharide (LPS) modification as well as an induction of genes encoding multidrug efflux pumps MexCD-OprJ and MexGHI-OpmD. Accordingly, we detected significantly elevated levels of toxic metabolites and proteases in bacterial supernatants after LL-37 treatment. Pre-incubation of bacteria with LL-37 for 2 h led to a decreased susceptibility towards gentamicin and ciprofloxacin. Quantitative Realtime PCR results using a PAO1-pqsE mutant strain present evidence that the quinolone response protein and virulence regulator PqsE may be implicated in the regulation of the observed phenotype in response to LL-37. Further experiments with synthetic cationic antimicrobial peptides IDR-1018, 1037 and HHC-36 showed no induction of pqsE expression, suggesting a new role of PqsE as highly specific host stress sensor

A new data processing routine facilitating the identification of surface adhered proteins from bacterial conditioning films via QCM-D/MALDI-ToF/MS

Conditioning films are an important factor in the initiation and development of microbial biofilms, which are the leading cause of chronic infections associated with medical devices. Here, we analyzed the protein content of conditioning films formed after exposure to supernatants of cultures of the human pathogen Pseudomonas aeruginosa PAO1. Adhesion of substances from the supernatant was monitored using quartz crystal microbalance with dissipation monitoring (QCM-D) sensor chips modified with the commonly used implant material titanium dioxide (TiO2). Attached proteins were identified after on-chip digestion using matrix-assisted laser desorption/ionization (MALDI) time of flight (ToF) mass spectrometry (MS), and a new data processing tool consisting of an XML-database with theoretical tryptic peptides of every PAO1 protein and PHP scripts. Sub-databases containing only proteins, that we found in all replicates, were created and used for MS/MS precursor selection. The obtained MS/MS peaklists were then matched against theoretical fragmentations of the expected peptide sequences to verify protein identification. Using this approach we were able to identify 40 surface-associated proteins. In addition to extracellular proteins such as adhesins, a number of intra-cellular proteins were identified which may be involved in conditioning film formation, suggesting an as-yet unidentified role for these proteins, possibly after cell lysis. [Figure not available: see fulltext.

Time-killing of <i>P. aeruginosa</i> PAO1 by antibiotics ciprofloxacin (A) or gentamicin (B) in the absence or presence of LL-37.

<p>Mid-log phase bacterial cultures were incubated with either 20 µg/ml LL-37 (filled circles) or without LL-37 (open squares) for 2 h. Following dilution of bacterial cultures to 10⁷ cells/ml and addition of 3-fold MIC concentrations of antibiotics ciprofloxacin (0.18 µg/ml) or gentamicin (1.5 µg/ml), colony forming units at indicated time points were determined using the optimized drop plate method <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082240#pone.0082240-Herigstad1" target="_blank">[27]</a>. Experiments were performed in triplicate. The figure shows representative results of one experiment. Error bars indicate standard deviations of 10 spots per sample plated out on two different agar plates (n = 10).</p

Summarized microarray data of dysregulated <i>P. aeruginosa</i> genes in response to LL-37.

<p>Mid-log phase cultures of <i>P. aeruginosa</i> PAO1 were grown in MH broth containing either 20 µg/ml LL-37 or no LL-37 for 2 h at 37°C following RNA extraction and microarray analysis. The graph shows functions of more than 1.5-fold up- or downregulated genes according to the <i>Pseudomonas</i> Genome Database <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082240#pone.0082240-Gooderham2" target="_blank">[28]</a>. Hypothetical genes are not shown.</p

Antimicrobial peptides used in this study.

<p>^a Minimal inhibitory concentrations (MIC) of PAO1 WT against different antimicrobial peptides were determined in MH broth using a standard two-fold serial dilution protocol for microtiter plates. Data represent mode MIC values of three independent experiments for each strain.</p

Microarray results of selected dysregulated genes of <i>P. aeruginosa</i> PAO1 WT in response to 2 h of incubation with LL-37 (20 µg/ml) compared to untreated controls.

<p>Microarray results of selected dysregulated genes of <i>P. aeruginosa</i> PAO1 WT in response to 2 h of incubation with LL-37 (20 µg/ml) compared to untreated controls.</p