Multiple antibiotic susceptibility of polyphosphate kinase mutants (ppk1 and ppk2) from Pseudomonas aeruginosa PAO1 as revealed by global phenotypic analysis
© Ortiz-Severin et al.; licensee BioMed Central. 2015
Received: 2 February 2015
Accepted: 8 April 2015
Published: 25 April 2015
Pseudomonas aeruginosa is known to be a multidrug resistant opportunistic pathogen. Particularly, P. aeruginosa PAO1 polyphosphate kinase mutant (ppk1) is deficient in motility, quorum sensing, biofilm formation and virulence.
By using Phenotypic Microarrays (PM) we analyzed near 2000 phenotypes of P. aeruginosa PAO1 polyP kinase mutants (ppk1 and ppk2). We found that both ppk mutants shared most of the phenotypic changes and interestingly many of them related to susceptibility toward numerous and different type of antibiotics such as Ciprofloxacin, Chloramphenicol and Rifampicin.
Combining the fact that ppk1 mutants have reduced virulence and are more susceptible to antibiotics, polyP synthesis and particularly PPK1, is a good target for the design of molecules with anti-virulence and anti-persistence properties.
KeywordsAntibiotic susceptibility Polyphosphate kinase Pseudomonas Phenome Phenotypic microarrays Multidrug resistance
Pseudomonas aeruginosa is a major opportunistic pathogen frequently involved in hospital-acquired infections and can produce severe pneumonia, burn wound infections, and sepsis. Particularly, multidrug resistant (MDR) variants are emerging rapidly in the clinic for this pathogen. In addition, P. aeruginosa resistance rates have increased to available antimicrobial agents, limiting the choice of available anti-infective chemicals . Looking for alternatives with economic and human health impact, new antimicrobial agents with novel biological targets or strategies are desperately needed to combat highly resistant P. aeruginosa infections [2,3]. Current original strategies combine the reduction of bacterial virulence with a simultaneous increase of animal host defence, instead of eradicating the pathogen [4,5].
Inorganic polyphosphate (polyP) are essential for bacterial resilience during stress and stringencies, cellular motility, biofilm formation and virulence . Many bacterial pathogens knockout of polyP synthesis gene (ppk1) result in cellular defects, particularly in the context of virulence toward the host they invade [6,7]. Specifically, a Pseudomonas aeruginosa PAO1 mutant of polyP synthesis (Pappk1) was impaired in motility, biofilm development, quorum sensing and virulence in ocular and burned-mouse models [8,9]. Moreover, this mutant exhibited reduced viability after exposure to a β-lactam antibiotic . Similar results were reported in ppk1 mutants from S. typhimurium and S. dublin that used Polymyxin B15 antibiotic . In addition to homologues of PPK1, another widely conserved polyP enzyme is PPK2, which, in contrast to the ATP-dependent polyP synthetic activity of PPK1, preferentially catalyses the reverse reaction, polyP-driven synthesis of GTP from GDP . The fact that polyP is involved in bacterial virulence and resilience processes makes it an attractive target for antimicrobial agents .
To provide a more complete analysis of P. aeruginosa polyP synthesis mutants (ppk1 and ppk2) phenotypes and to obtain greater insight into the physiological changes and particularly chemical susceptibility we used Phenotype Microarray (PM) technology . Biolog Phenotype MicroArrays studies in Pseudomonas aeruginosa PAO1 have been used to facilitate further characterization of known mutation strains and for testing bioinformatic predictions for mutations in hypothetical or unknown genes . We used P. aeruginosa polyphosphate synthesis knockout mutants ppk1 (PA5242) and ppk2 (PA0141) from a P. aeruginosa Mini- Tn5-Tcr gene knockout mutants collection . We confirmed by PCR both mutations and bacterial cell suspensions from all strains were inoculated into each of the 20 PM plates for full metabolic profiling according to standard protocols recommended by Biolog Inc. for Pseudomonas strains [14,16]. The PM plates were located in an aerobic OmniLog incubator reader set at 30°C which collected data every 15 min over a 72-h period. PM tests were conducted in duplicate, and the plates were also examined visually at the end of each incubation period for independent confirmation. The OmniLog® V. 1.5 comparison module and the average height parameter were used for data analysis with standard thresholds for detection. A consensus graphical profiles for all metabolic and sensitivity tests for each mutant were generated using two independent runs (Additional file 1: Figure S1).
For visualization and clustering data analysis, Multiexperiment Viewer (MeV version 4.6) software was used. MeV is part of the TM4 Microarray Software Suite, an open source system for statistical and clustering analysis of omics data .
Summary of P. aeruginosa PAO1 polyP synthesis mutants ( ppk1 and ppk2 ) common gain and lost phenotypes
Mode of action
C-Source, carboxylic acid
Respiration, mitochondrial Ca2+ porter
Protein synthesis, 50S ribosomal subunit, macrolide
C-Source, carboxylic acid
N-Source, amino acid
Microtubulin polymerization inhibitor, antifungal
Toxic anion, P04 analog
Protein synthesis, amphenicol
Protein synthesis, amphenicol
Oxidizes sulfhydryls, depletes glutathione
Antimony (III) chloride
Folate antagonist, PABA analog
Respiration, ionophore, H+
Membrane, detergent, cationic
Membrane, nonspecific binding
Folate antagonist, PABA analog
DNA & RNA synthesis, polymerase inhibitor
Tyrosine phosphatase inhibitor
Respiration, ionophore, H+
The respiration patterns of P. aeruginosa PAO1 ppk1 and ppk2 associated with the metabolism of carbon (PM1-PM2), nitrogen (PM3), phosphorous and sulfur (PM4) sources were relatively moderate as compared to previous unpublished PM results from our laboratory with Δppk1 mutant from Escherichia coli (Additional file 1: Figures S2 and S3). We speculate that the apparent metabolic robustness in Pseudomonas ppk1 mutant is due to in the E. coli genome there is only one gene orthologous to the PPK1 protein . In contrast, in Pseudomonas aeruginosa PAO1 the presence of another ortholog (PPK2) can compensate the lost of ppk1 gene, in fact, despite the absence of detectable PPK1 activity (<1% of wild type), Δppk1 mutants still possess as much as 20% of polyP of the wild type levels .
Interestingly, we observed susceptibility to various antibiotic families with different mechanisms of action such as Penicillins (Nafcillin, Oxacillin), Ansamycins (Streptomycin), Sulfonamides (Sulfadiazine, Sulfathiazole) and Others (Chloramphenicol, Thiamphenicol). To confirm this finding, we used two additional well-established methods for antimicrobial susceptibility testing. The results from the M.I.C.Evaluator™ (Oxoid) and Etest® (Biomeriux) corroborated the susceptibility of ppk mutants observed in the PM results (Additional file 2: Table S5). The minimal inhibitory concentration (MIC) parameters for both ppk mutants were particularly interesting for the antibiotics Rifampicin, Imipinen and Ciprofloxacin, either because Pseudomonas is intrinsically resistant or because recent strains highly resistant to these antibiotics have been found recently . It should be noted that concentrations of antibiotics used in the PM experiments are set according to the minimal inhibitory concentration for E. coli. It is possible that other antibiotic susceptibilities have not been detected since both the control strain and the mutants were resistant.
Taking into account that various bacterial regulatory genes that participate in complex regulatory networks have been reported to influence both virulence and antibiotic resistance , polyP levels could affect regulators that control both antibiotic resistance and virulence.
Finally, our results support the recent finding that bacterial persistence, a phenomenon in which isogenic populations of antibiotic-sensitive bacteria produce rare cells that transiently become multidrug tolerant, is affected in ppk1 deficient strain . This small fraction of cells that grow slowly explains bacterial antibiotic tolerance because the cellular targets disturbed by lethal antibiotics are much less vulnerable in slow-growing than in fast-growing cells. This is important because the presence of persister cells has been suggested to be one of the main reasons for failures in the treatment of these chronic diseases. Indeed, clinical isolates of Pseudomonas aeruginosa from cystic fibrosis patients show an increase in high persistence mutants the longer that these isolates remain in the host. This indicates that persistence plays a major role in the failure to remove these bacterial populations from the cystic fibrosis lung .
Consequently, polyP synthesis, and particularly PPK1, in bacterial pathogens exhibits a potential target for antimicrobial drug design because combines the reduction of bacterial virulence and persistence, while simultaneously increasing susceptibility to antibiotics as we describe here by using PM technology. This could positively impact the host health for clearing the bacterial infection. Considering that no PPK1 homologs have been identified in higher-order eukaryotes , our phenotypic sensibility results highlight the importance of polyP synthesis as a putative target for the treatment of this opportunistic bacterium.
Phenotype Microarray (Biolog)
Polymerase chain reaction
Minimal inhibitory concentration
Polyphosphate kinase 1 from Pseudomonas aeruginosa PAO1
We gratefully acknowledge Dr. Michael Ziman (Biolog, Inc.) for all the support in the phenotypic microarray experiments. We thank Nicole Molina and members of SysmicroLab for helpful technical assistance. This work was supported by a Fondecyt Grant (1120209) from Conicyt. JOS, MV and CBT are Conicyt fellows for postgraduate studies.
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