Evaluation of a microarray-hybridization based method applicable for discovery of single nucleotide polymorphisms (SNPs) in the Pseudomonas aeruginosa genome

<p>Abstract</p> <p>Background</p> <p>Whole genome sequencing techniques have added a new dimension to studies on bacterial adaptation, evolution and diversity in chronic infections. By using this powerful approach it was demonstrated that <it>Pseudomonas aeruginosa </it>undergoes intense genetic adaptation processes, crucial in the development of persistent disease. The challenge ahead is to identify universal infection relevant adaptive bacterial traits as potential targets for the development of alternative treatment strategies.</p> <p>Results</p> <p>We developed a microarray-based method applicable for discovery of single nucleotide polymorphisms (SNPs) in <it>P. aeruginosa </it>as an easy and economical alternative to whole genome sequencing. About 50% of all SNPs theoretically covered by the array could be detected in a comparative hybridization of PAO1 and PA14 genomes at high specificity (> 0.996). Variations larger than SNPs were detected at much higher sensitivities, reaching nearly 100% for genetic differences affecting multiple consecutive probe oligonucleotides. The detailed comparison of the <it>in silico </it>alignment with experimental hybridization data lead to the identification of various factors influencing sensitivity and specificity in SNP detection and to the identification of strain specific features such as a large deletion within the PA4684 and PA4685 genes in the Washington Genome Center PAO1.</p> <p>Conclusion</p> <p>The application of the genome array as a tool to identify adaptive mutations, to depict genome organizations, and to identify global regulons by the "ChIP-on-chip" technique will expand our knowledge on <it>P. aeruginosa </it>adaptation, evolution and regulatory mechanisms of persistence on a global scale and thus advance the development of effective therapies to overcome persistent disease.</p

Fitness of Isogenic Colony Morphology Variants of Pseudomonas aeruginosa in Murine Airway Infection

Chronic lung infections with Pseudomonas aeruginosa are associated with the diversification of the persisting clone into niche specialists and morphotypes, a phenomenon called ‘dissociative behaviour’. To explore the potential of P. aeruginosa to change its morphotype by single step loss-of–function mutagenesis, a signature-tagged mini-Tn5 plasposon library of the cystic fibrosis airway isolate TBCF10839 was screened for colony morphology variants under nine different conditions in vitro. Transposon insertion into 1% of the genome changed colony morphology into eight discernable morphotypes. Half of the 55 targets encode features of primary or secondary metabolism whereby quinolone production was frequently affected. In the other half the transposon had inserted into genes of the functional categories transport, regulation or motility/chemotaxis. To mimic dissociative behaviour of isogenic strains in lungs, pools of 25 colony morphology variants were tested for competitive fitness in an acute murine airway infection model. Six of the 55 mutants either grew better or worse in vivo than in vitro, respectively. Metabolic proficiency of the colony morphology variant was a key determinant for survival in murine airways. The most common morphotype of self-destructive autolysis did unexpectedly not impair fitness. Transposon insertions into homologous genes of strain PAO1 did not reproduce the TBCF10839 mutant morphotypes for 16 of 19 examined loci pointing to an important role of the genetic background on colony morphology. Depending on the chosen P. aeruginosa strain, functional genome scans will explore other areas of the evolutionary landscape. Based on our discordant findings of mutant phenotypes in P. aeruginosa strains PAO1, PA14 and TBCF10839, we conclude that the current focus on few reference strains may miss modes of niche adaptation and dissociative behaviour that are relevant for the microevolution of complex traits in the wild

The influence of Pseudomonas Quinolon Signal to interbacterial communication of Pseudomonas aeruginosa

Die Produktion der meisten Virulenzfaktoren und die Ausbildung von Biofilmen werden von P. aeruginosa über 2 Quorum Sensing (QS) Systeme durch Homoserinlakton-Signalmoleküle reguliert. Kürzlich wurde ein drittes bakterielles Signalmolekül, das Pseudomonas Quinolon Signal (PQS), identifiziert, welches einen Teil der durch QS regulierten Gene positiv beeinflusst. In dieser Arbeit wurde der Biosyntheseweg von 4-Hydroxy-2-alkylquinolinen (HAQs) mit Hilfe von Fütterung stabil-markierter Vorstufen und anschließender Analyse der HAQ Extrakte mit GC/MS und NMR aufgeklärt. Außerdem wurde der Einfluss von PQS auf PAO1 mit Hilfe von Transkriptionsanalysen untersucht. Das durch die exogene Zugabe von PQS hervorgerufenen Transkriptionsprofil zeigte eine Induktion von Genen, die zu den Gruppen der eisenregulierten Gene und den Genen, die durch oxidativen Stress reguliert werden, gehören. Bemerkenswerterweise konnten nicht nur die meisten der regulierten Gene, sondern auch die Induktion einer transkriptionellen lacZ-Fusion mit der Promotorregion von rhlR auf einen starken eisenbindenden Effekt von PQS zurückgeführt werden. Nichtsdestotrotz gibt es PQS spezifische Effekte, die unabhängig von dem Eisenhaushalt sind. Darüber hinaus konnten wir zeigen, dass PQS scheinbar die benachbarten P. aeruginosa Zellen angreift, indem es direkt auf die bakterielle DNA wirkt. Als Konsequenz daraus reichern sich große Mengen extrazellulärer DNA in Biofimkulturen an. Die Opferung eines Teils der Population scheint aber das Überleben der Bakterien unter sich ändernden Umweltbedingungen zu sichern. Ein horizontale Gentransfer könnte dabei z.B. die genetischen Veränderungen, die mit sexueller Reproduktion der normalerweise klonalen Lebensweise der Bakterien nicht erreicht werden, kompensieren. Von einem evolutionären Standpunkt aus betrachtet offenbaren diese Ergebnisse eine außergewöhnliche Bedeutung der natürlichen Transformierbarkeit und macht das Phänomen zu einem medizinisch relevanten Thema.Virulence factor production and the development of biofilms in Pseudomonas aeruginosa have been shown to be regulated by 2 quorum sensing (QS) systems via small acyl-homoserine lactone (AHL) signal molecules. Recently, a third bacterial signal molecule, the Pseudomonas quinolone signal (PQS), has been identified, which regulates a subset of genes dependent on the QS systems. In this study we have unraveled the biosynthetic pathway of HAQs by analysing extracted HAQs by GC/MS and NMR spectroscopy in feeding experiments with isotope labeled precursors. Moreover, we evaluated the impact of PQS on the QS circuitry using transcriptome analysis of PAO1 cultures supplemented with PQS. The global transcriptional profile in response to PQS revealed a marked up-regulation of genes belonging to the tightly interdependent functional groups of iron acquisition and oxidative stress response. Remarkably, not only most of the differentially regulated genes but also the induction of a lacZ transcriptional fusion of rhlR could be traced back to a iron chelating effect of PQS. Nevertheless, although iron deficiency induced rhlR, there are PQS specific effects that are independent of the PQS effect on iron homeostasis. Moreover we could show that PQS seems to be capable of attacking neighbouring P. aeruginosa cells by directly targeting bacterial DNA. As a result large amounts of extracellular DNA accumulate in biofilm cultures and obviously serve as a genetic pool for horizontal gene transfer. This way the self-sacrificing of parts of the population promotes genetic variability of survivors providing the bacterial population with an evolutionary advantage under changing environmental conditions and compensating for the otherwise clonal mode of prokaryotic life where genetic innovation cannot be archived by sexual reproduction. From an evolutionary perspective this finding adds a new dimension to natural genetic transformation and makes this phenomenon a medical significant event

The Pseudomonas aeruginosa quinolone signal (PQS) has an iron-chelating activity.

Virulence factor production and the development of biofilms in Pseudomonas aeruginosa have been shown to be regulated by two hierarchically organized quorum-sensing systems activated by two types of small acyl-homoserine lactone signal molecules. Recently, a third type of bacterial signal molecule, the Pseudomonas quinolone signal (PQS), has been identified, which positively regulates a subset of genes dependent on the quorum-sensing systems. However, the molecular mechanism underlying PQS signalling has remained poorly understood. In this study the global transcriptional profile of P. aeruginosa in response to PQS revealed a marked upregulation of genes belonging to the tightly interdependent functional groups of the iron acquisition and the oxidative stress response. Remarkably, most of the differentially regulated genes, as well as the induction of rhlR, could be traced back to an iron-chelating effect of PQS. Our results amount to the elucidation of how PQS affects P. aeruginosa and have important implications for the understanding of the complex regulatory circuits involved in P. aeruginosa gene regulation

Quorum-Sensing Antagonistic Activities of Azithromycin in Pseudomonas aeruginosa PAO1: a Global Approach

The administration of macrolides such as azithromycin for chronic pulmonary infection of cystic fibrosis patients has been reported to be of benefit. Although the mechanisms of action remain obscure, anti-inflammatory effects as well as interference of the macrolide with Pseudomonas aeruginosa virulence factor production have been suggested to contribute to an improved clinical outcome. In this study we used a systematic approach and analyzed the impact of azithromycin on the global transcriptional pattern and the protein expression profile of P. aeruginosa PAO1 cultures versus those in untreated controls. The most remarkable result of this study is the finding that azithromycin exhibited extensive quorum-sensing antagonistic activities. In accordance with the inhibition of the quorum-sensing systems, virulence factor production was diminished and the oxidative stress response was impaired, whereas the type III secretion system was strongly induced. Moreover, P. aeruginosa motility was reduced, which probably accounts for the previously observed impaired biofilm formation capabilities of azithromycin-treated cultures. The interference of azithromycin with quorum-sensing-dependent virulence factor production, biofilm formation, and oxidative stress resistance in P. aeruginosa holds great promise for macrolide therapy in cystic fibrosis. Clearly quorum-sensing antagonist macrolides should be paid more attention in the management of chronic P. aeruginosa infections, and as quorum-sensing antagonists, macrolides might gain vital importance for more general application against chronic infections

Biosynthetic Pathway of Pseudomonas aeruginosa 4-Hydroxy-2-Alkylquinolines

The role of intercellular communication in the regulation of bacterial multicellular behavior has received widespread attention, and a variety of signal molecules involved in bacterial communication have been discovered. In addition to the N-acyl-homoserine lactones, 4-hydroxy-2-alkylquinolines (HAQs), including the Pseudomonas quinolone signal, have been shown to function as signal molecules in Pseudomonas aeruginosa. In this study we unraveled the biosynthetic pathway of HAQs using feeding experiments with isotope-labeled precursors and analysis of extracted HAQs by gas chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy. Our results show that the biosynthesis of various HAQ metabolites is directed via a common metabolic pathway involving a “head-to-head” condensation of anthranilic acid and β-keto fatty acids. Moreover, we provide evidence that the β-keto-(do)decanoic acids, crucial for the biosynthesis of the heptyl and nonyl derivatives of the 4-hydroxyquinolines in P. aeruginosa, are at least in part derived from a common pool of β-hydroxy(do)decanoic acids involved in rhamnolipid biosynthesis

Inter- and Intraclonal Diversity of the Pseudomonas aeruginosa Proteome Manifests within the Secretome

The proteomes of cultured Pseudomonas aeruginosa isolates from chronically infected cystic fibrosis (CF) lungs were compared by using genetically divergent clones and isogenic morphotypes of one strain. Cellular extracts gave very similar protein patterns in two-dimensional gels, suggesting that the conserved species-specific core genome encodes proteins that are expressed under standard culture conditions in vitro. In contrast, the protein profiles of extracts of culture supernatants were dependent on the growth phase, and there were significant differences between clones. The profiles also varied within clonally related morphotypes from one CF patient, including a hyperpiliated small-colony variant. Mass spectrometry revealed that this variant overexpressed proteins secreted by the type I secretion system (including proteins involved in iron acquisition) and by the type III secretion system. Furthermore, the proteins in the supernatant extracts from the small-colony variant which were recognized by sera from different CF patients varied greatly. We concluded that the secretome expression is a sensitive measure of P. aeruginosa strain variation