RESUMEN
The pqs quorum sensing (QS) system is crucial for Pseudomonas aeruginosa virulence both in vitro and in animal models of infection and is considered an ideal target for the development of anti-virulence agents. However, the precise role played by each individual component of this complex QS circuit in the control of virulence remains to be elucidated. Key components of the pqs QS system are 2-heptyl-4-hydroxyquinoline (HHQ), 2-heptyl-3-hydroxy-4-quinolone (PQS), 2-heptyl-4-hydroxyquinoline N-oxide (HQNO), the transcriptional regulator PqsR and the PQS-effector element PqsE. To define the individual contribution of each of these components to QS-mediated regulation, transcriptomic analyses were performed and validated on engineered P. aeruginosa strains in which the biosynthesis of 2-alkyl-4-quinolones (AQs) and expression of pqsE and pqsR have been uncoupled, facilitating the identification of the genes controlled by individual pqs system components. The results obtained demonstrate that i) the PQS biosynthetic precursor HHQ triggers a PqsR-dependent positive feedback loop that leads to the increased expression of only the pqsABCDE operon, ii) PqsE is involved in the regulation of diverse genes coding for key virulence determinants and biofilm development, iii) PQS promotes AQ biosynthesis, the expression of genes involved in the iron-starvation response and virulence factor production via PqsR-dependent and PqsR-independent pathways, and iv) HQNO does not influence transcription and hence does not function as a QS signal molecule. Overall this work has facilitated identification of the specific regulons controlled by individual pqs system components and uncovered the ability of PQS to contribute to gene regulation independent of both its ability to activate PqsR and to induce the iron-starvation response.
Asunto(s)
Regulación Bacteriana de la Expresión Génica/fisiología , Pseudomonas aeruginosa/fisiología , Percepción de Quorum/fisiología , Virulencia/fisiología , 4-Quinolonas/metabolismo , Biopelículas/crecimiento & desarrollo , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Transducción de Señal , TranscriptomaRESUMEN
The small RNA ErsA of Pseudomonas aeruginosa, transcribed from the same genomic context of the well-known Escherichia coliâ Spot 42, has been characterized. We show that, different from Spot 42, ErsA is under the transcriptional control of the envelope stress response, which is known to impact the pathogenesis of P. aeruginosa through the activity of the alternative sigma factor σ(22) . The transcriptional responsiveness of ErsA RNA also spans infection-relevant cues that P. aeruginosa can experience in mammalian hosts, such as limited iron availability, temperature shifts from environmental to body temperature and reduced oxygen conditions. Another difference between Spot 42 and ErsA is that ErsA does not seem to be involved in the regulation of carbon source catabolism. Instead, our results suggest that ErsA is linked to anabolic functions for the synthesis of exoproducts from sugar precursors. We show that ErsA directly operates in the negative post-transcriptional regulation of the algC gene that encodes the virulence-associated enzyme AlgC, which provides sugar precursors for the synthesis of several P. aeruginosa polysaccharides. Like ErsA, the activation of algC expression is also dependent on σ(22) . Altogether, our results suggest that ErsA and σ(22) combine in an incoherent feed-forward loop to fine-tune AlgC enzyme expression.
Asunto(s)
Regulación Bacteriana de la Expresión Génica , Fosfoglucomutasa/genética , Fosfotransferasas (Fosfomutasas)/genética , Pseudomonas aeruginosa/genética , ARN Pequeño no Traducido/metabolismo , Regulación Enzimológica de la Expresión Génica , Fosfoglucomutasa/metabolismo , Fosfotransferasas (Fosfomutasas)/metabolismo , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/patogenicidad , ARN Pequeño no Traducido/química , ARN Pequeño no Traducido/genética , Regulón , Factor sigma/metabolismo , Factores de Virulencia/genéticaRESUMEN
Life-long bacterial infections in cystic fibrosis (CF) airways constitute an excellent model both for persistent infections and for microbial adaptive evolution in complex dynamic environments. Using high-resolution transcriptomics applied on CF sputum, we profile transcriptional phenotypes of Pseudomonas aeruginosa populations in patho-physiological conditions. Here we show that the soft-core genome of genetically distinct populations, while maintaining transcriptional flexibility, shares a common expression program tied to the lungs environment. We identify genetically independent traits defining P. aeruginosa physiology in vivo, documenting the connection between several previously identified mutations in CF isolates and some of the convergent phenotypes known to develop in later stages of the infection. In addition, our data highlight to what extent this organism can exploit its extensive repertoire of physiological pathways to acclimate to a new niche and suggest how alternative nutrients produced in the lungs may be utilized in unexpected metabolic contexts.
Asunto(s)
Fibrosis Quística/genética , Pulmón/metabolismo , Mutación/genética , Pseudomonas aeruginosa/genética , Transcriptoma/genética , Adulto , Femenino , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Fenotipo , Secuenciación Completa del Genoma/métodosRESUMEN
The small RNA ErsA of Pseudomonas aeruginosa was previously suggested to be involved in biofilm formation via negative post-transcriptional regulation of the algC gene that encodes the virulence-associated enzyme AlgC, which provides sugar precursors for the synthesis of several polysaccharides. In this study, we show that a knock-out ersA mutant strain forms a flat and uniform biofilm, not characterized by mushroom-multicellular structures typical of a mature biofilm. Conversely, the knock-out mutant strain showed enhanced swarming and twitching motilities. To assess the influence of ErsA on the P. aeruginosa transcriptome, we performed RNA-seq experiments comparing the knock-out mutant with the wild-type. More than 160 genes were found differentially expressed in the knock-out mutant. Parts of these genes, important for biofilm formation and motility regulation, are known to belong also to the AmrZ transcriptional regulator regulon. Here, we show that ErsA binds in vitro and positively regulates amrZ mRNA at post-transcriptional level in vivo suggesting an interesting contribution of the ErsA-amrZ mRNA interaction in biofilm development at several regulatory levels.
RESUMEN
In 474 genome sequenced Pseudomonas aeruginosa isolates from 34 cystic fibrosis (CF) patients, 40% of these harbor mutations in the mexZ gene encoding a negative regulator of the MexXY-OprM efflux pump associated with aminoglycoside and fluoroquinolone resistance. Surprisingly, resistance to aminoglycosides and fluoroquinolones of mexZ mutants was far below the breakpoint of clinical resistance. However, the fitness increase of the mutant bacteria in presence of the relevant antibiotics, as demonstrated in competition experiments between mutant and ancestor bacteria, showed that 1) very small phenotypic changes cause significant fitness increase with severe adaptive consequences, and 2) standardized phenotypic tests fail to detect such low-level variations. The frequent appearance of P. aeruginosa mexZ mutants in CF patients is directly connected to the intense use of the target antibiotics, and low-level antibiotic resistance, if left unnoticed, can result in accumulation of additional genetic changes leading to high-level resistance.
Asunto(s)
Proteínas Bacterianas/genética , Fibrosis Quística/microbiología , Farmacorresistencia Microbiana , Mutación , Pseudomonas aeruginosa/genética , Adaptación Fisiológica , Aminoglicósidos/farmacología , Antibacterianos/farmacología , Fibrosis Quística/tratamiento farmacológico , Fluoroquinolonas/farmacología , Aptitud Genética , Humanos , Fenotipo , Infecciones por Pseudomonas/tratamiento farmacológico , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/crecimiento & desarrollo , Análisis de Secuencia de ARN , Secuenciación Completa del Genoma/métodosRESUMEN
Small non-coding RNAs (sRNAs) are post-transcriptional regulators of gene expression that have been recognized as key contributors to bacterial virulence and pathogenic mechanisms. In this study, we characterized the sRNA PesA of the opportunistic human pathogen Pseudomonas aeruginosa. We show that PesA, which is transcribed within the pathogenicity island PAPI-1 of P. aeruginosa strain PA14, contributes to P. aeruginosa PA14 virulence. In fact, pesA gene deletion resulted in a less pathogenic strain, showing higher survival of cystic fibrosis human bronchial epithelial cells after infection. Moreover, we show that PesA influences positively the expression of pyocin S3 whose genetic locus comprises two structural genes, pyoS3A and pyoS3I, encoding the killing S3A and the immunity S3I proteins, respectively. Interestingly, the deletion of pesA gene results in increased sensitivity to UV irradiation and to the fluoroquinolone antibiotic ciprofloxacin. The degree of UV sensitivity displayed by the PA14 strain lacking PesA is comparable to that of a strain deleted for pyoS3A-I. These results suggest an involvement of pyocin S3 in DNA damage repair and a regulatory role of PesA on this function.