RESUMO
The recent finding that high numbers of strict anaerobes are present in the respiratory tract of cystic fibrosis (CF) patients has drawn attention to the pathogenic contribution of the CF microbiome to airway disease. In this study, we investigated the specific interactions of the most dominant bacterial CF pathogen, Pseudomonas aeruginosa, with the anaerobic bacterium Veillonella parvula, which has been recovered at comparable cell numbers from the respiratory tract of CF patients. In addition to growth competition experiments, transcriptional profiling, and analyses of biofilm formation by in vitro studies, we used our recently established in vivo murine tumor model to investigate mutual influences of the two pathogens during a biofilm-associated infection process. We found that P. aeruginosa and V. parvula colonized distinct niches within the tumor. Interestingly, significantly higher cell numbers of P. aeruginosa could be recovered from the tumor tissue when mice were coinfected with both bacterial species than when mice were monoinfected with P. aeruginosa. Concordantly, the results of in vivo transcriptional profiling implied that the presence of V. parvula supports P. aeruginosa growth at the site of infection in the host, and the higher P. aeruginosa load correlated with clinical deterioration of the host. Although many challenges must be overcome to dissect the specific interactions of coinfecting bacteria during an infection process, our findings exemplarily demonstrate that the complex interrelations between coinfecting microorganisms and the immune responses determine clinical outcome to a much greater extent than previously anticipated.
Assuntos
Interações Microbianas , Neoplasias/microbiologia , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/patogenicidade , Veillonella/patogenicidade , Animais , Carga Bacteriana , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Camundongos Endogâmicos BALB C , Neoplasias/complicaçõesRESUMO
Bacterial populations co-ordinate gene expression collectively through quorum sensing (QS), a cell-to-cell communication mechanism employing diffusible signal molecules. The LysR-type transcriptional regulator (LTTR) protein PqsR (MvfR) is a key component of alkyl-quinolone (AQ)-dependent QS in Pseudomonas aeruginosa. PqsR is activated by 2-alkyl-4-quinolones including the Pseudomonas quinolone signal (PQS; 2-heptyl-3-hydroxy-4(1H)-quinolone), its precursor 2-heptyl-4-hydroxyquinoline (HHQ) and their C9 congeners, 2-nonyl-3-hydroxy-4(1H)-quinolone (C9-PQS) and 2-nonyl-4-hydroxyquinoline (NHQ). These drive the autoinduction of AQ biosynthesis and the up-regulation of key virulence determinants as a function of bacterial population density. Consequently, PqsR constitutes a potential target for novel antibacterial agents which attenuate infection through the blockade of virulence. Here we present the crystal structures of the PqsR co-inducer binding domain (CBD) and a complex with the native agonist NHQ. We show that the structure of the PqsR CBD has an unusually large ligand-binding pocket in which a native AQ agonist is stabilized entirely by hydrophobic interactions. Through a ligand-based design strategy we synthesized and evaluated a series of 50 AQ and novel quinazolinone (QZN) analogues and measured the impact on AQ biosynthesis, virulence gene expression and biofilm development. The simple exchange of two isosteres (OH for NH2) switches a QZN agonist to an antagonist with a concomitant impact on the induction of bacterial virulence factor production. We also determined the complex crystal structure of a QZN antagonist bound to PqsR revealing a similar orientation in the ligand binding pocket to the native agonist NHQ. This structure represents the first description of an LTTR-antagonist complex. Overall these studies present novel insights into LTTR ligand binding and ligand-based drug design and provide a chemical scaffold for further anti-P. aeruginosa virulence drug development by targeting the AQ receptor PqsR.
Assuntos
Proteínas de Bactérias/metabolismo , Pseudomonas aeruginosa/fisiologia , Quinolonas/metabolismo , Percepção de Quorum , Transdução de Sinais , Fatores de Transcrição/metabolismo , Alquilação , Antibacterianos/química , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/agonistas , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Sítios de Ligação , Biofilmes/efeitos dos fármacos , Desenho de Fármacos , Regulação Bacteriana da Expressão Gênica , Ligantes , Conformação Molecular , Proteínas Mutantes/agonistas , Proteínas Mutantes/antagonistas & inibidores , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Fragmentos de Peptídeos/agonistas , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/patogenicidade , Quinolonas/química , Quinolonas/farmacologia , Percepção de Quorum/efeitos dos fármacos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Fatores de Transcrição/agonistas , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/química , Virulência/efeitos dos fármacosRESUMO
Regulation of gene expression plays a key role in bacterial adaptability to changes in the environment. An integral part of this gene regulatory network is achieved via quorum sensing (QS) systems that coordinate bacterial responses under high cellular densities. In the nosocomial pathogen Pseudomonas aeruginosa, the 2-alkyl-4-quinolone (pqs) signaling pathway is crucial for bacterial survival under stressful conditions. Biosynthesis of the Pseudomonas quinolone signal (PQS) is dependent on the pqsABCDE operon, which is positively regulated by the LysR family regulator PqsR and repressed by the transcriptional regulator protein RhlR. However, the molecular mechanisms underlying this inhibition have remained elusive. Here, we demonstrate that not only PqsR but also RhlR activates transcription of pqsA. The latter uses an alternative transcriptional start site and induces expression of a longer transcript that forms a secondary structure in the 5' untranslated leader region. As a consequence, access of the ribosome to the Shine-Dalgarno sequence is restricted and translation efficiency reduced. We propose a model of a novel posttranscriptional regulation mechanism that fine-tunes PQS biosynthesis, thus highlighting the complexity of quorum sensing in P. aeruginosa.
Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Quinolonas/metabolismo , Isoformas de RNA/metabolismo , Proteínas de Bactérias/genética , ÓperonRESUMO
Pseudomonas aeruginosa employs both N-acylhomoserine lactone and 2-alkyl-4(1H)-quinolone (AQ)-mediated interbacterial signalling for the orchestration of a genome-wide gene regulatory network. Despite the many advances that have been made in understanding the target genes of quorum sensing regulation, little is known on how quorum sensing systems are influenced by environmental cues. In this study, we show that AQ production is modulated by an orphan P. aeruginosa sensor kinase. Transcriptional studies of the sensor kinase (MxtR) mutant demonstrated that an induced expression of MexT, a LysR-type transcriptional regulator, largely determined the global transcriptional profile. Thereby, overexpression of the MexT-regulated MexEF-OprN efflux pump led to a delayed expression of the AQ biosynthetic genes and of AQ-dependent virulence factors. Furthermore, we demonstrated that autophosphorylation of MxtR was inhibited by ubiquinone, the central electron carrier of respiration in in vitro experiments. Our results elucidate on a mechanism by which P. aeruginosa senses environmental conditions and adapts by controlling the production of interbacterial AQ signal molecules. A regulatory function of a sensor kinase may indicate that there is a pre-emptive role of adaptation mechanisms that are turned on under distinct environmental conditions and that are important for efficient colonization and pathogenesis.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas Quinases/metabolismo , Pseudomonas aeruginosa/enzimologia , Quinolonas/metabolismo , Percepção de Quorum , Transdução de Sinais , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Teste de Complementação Genética , Glicolipídeos/biossíntese , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação , Regiões Promotoras Genéticas , Proteínas Quinases/genética , Pseudomonas aeruginosa/genética , Piocianina/biossíntese , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcriptoma , Ubiquinona/metabolismo , Fatores de Virulência/metabolismoRESUMO
Acute and chronic infections caused by the opportunistic pathogen Pseudomonas aeruginosa pose a serious threat to human health worldwide, and its increasing resistance to antibiotics requires alternative treatments that are more effective than available strategies. Clinical studies have clearly demonstrated that cystic fibrosis (CF) patients with chronic P. aeruginosa infections benefit from long-term low-dose azithromycin (AZM) treatment. Immunomodulating activity, the impact of AZM on the expression of quorum-sensing-dependent virulence factors, type three secretion, and motility in P. aeruginosa seem to contribute to the therapeutic response. However, to date, the molecular mechanisms underlying these AZM effects have remained elusive. Our data indicate that the AZM-mediated phenotype is caused by a depletion of the intracellular pools of tRNAs available for protein synthesis. Overexpression of the P. aeruginosa peptidyl-tRNA hydrolase, which recycles the tRNA from peptidyl-tRNA drop-off during translation, counteracted the effects of AZM on stationary-phase cell killing, cytotoxicity, and the production of rhamnolipids and partially restored swarming motility. Intriguingly, the exchange of a rare for a frequent codon in rhlR also explicitly diminished the AZM-mediated decreased production of rhamnolipids. These results indicate that depletion of the tRNA pools by AZM seems to affect the translation of genes that use rare aminoacyl-tRNA isoacceptors to a great extent and might explain the selective activity of AZM on the P. aeruginosa proteome and possibly also on the protein expression profiles of other bacterial pathogens.
Assuntos
Azitromicina/farmacologia , Proteínas de Bactérias/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/metabolismo , Aminoacil-RNA de Transferência/metabolismo , Fenômenos Fisiológicos Bacterianos/efeitos dos fármacos , Linhagem Celular , Humanos , Pseudomonas aeruginosa/enzimologiaRESUMO
Pseudomonas aeruginosa pathogenicity and its capability to adapt to multiple environments are dependent on the production of diverse virulence factors, controlled by the sophisticated quorum sensing (QS) network of P. aeruginosa. To better understand the molecular mechanisms that underlie this adaptation we searched for novel key regulators of virulence factor production by screening a PA14 transposon mutant library for potential candidates acting downstream of the unique 2-alkyl-4-quinolone (AQ) QS system of P. aeruginosa. We focused the work on a protein named HemK with high homology to PrmC of Escherichia coli displaying a similar enzymatic activity (therefore also referred to as PrmC). In this study, we demonstrate that PrmC is an S-adenosyl-l-methionine (AdoMet)-dependent methyltransferase of peptide chain release factors (RFs) essential for the expression of several virulence factors, such as pyocyanin, rhamnolipids and the type III-secreted toxin ExoT. Furthermore, the PA14_prmC mutant strain is unable to grow under anoxic conditions and has a significantly reduced pathogenicity in the infection model Galleria mellonella. Along with transcriptomic and proteomic analyses, the presented data indicate that the methylation of RFs in P. aeruginosa seems to have a global effect on cellular processes related to the virulence of this nosocomial pathogen.
Assuntos
Adaptação Fisiológica/genética , Metiltransferases/metabolismo , Pseudomonas aeruginosa/enzimologia , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Larva/microbiologia , Metiltransferases/genética , Mariposas/microbiologia , Mutação , Proteômica , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/patogenicidade , Percepção de Quorum/genética , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
The quorum sensing (QS) system of Pseudomonas aeruginosa constitutes a sophisticated genome-wide gene regulatory network employing both N-acylhomoserine lactone and 2-alkyl-4-quinolone (AQ) signal molecules. AQ signalling utilizes 2-heptyl-3-hydroxy-4-quinolone (PQS) and its immediate precursor, 2-heptyl-4-quinolone (HHQ). AQ biosynthesis requires the first four genes of the pqsABCDE operon and while the biochemical function of pqsE is not known, it is required for the production of secondary metabolites such as pyocyanin. To gain insights into the relationship between the AQ stimulon, the PqsE stimulon and the regulatory function of PqsE, we constructed a pqsE inducible mutant (pqsEind) and compared the transcriptomes of the induced and uninduced states with a pqsA mutant. Of 158 genes exhibiting altered expression in the pqsA mutant, 51% were also affected in the pqsE mutant. Following induction of pqsE, 237 genes were differentially expressed compared with the wild-type strain. In the pqsEind strain, pqsA was highly expressed but following induction both pqsA expression and AQ biosynthesis were repressed, revealing a negative autoregulatory role for PqsE. Furthermore, pqsE was required for swarming motility and virulence in plant and animal infection models in the absence of AQs, while mature biofilm development required both pqsA and pqsE. Taken together these data reveal that PqsE is a key regulator within the QS circuitry facilitating the environmental adaptation of P. aeruginosa.
Assuntos
Adaptação Fisiológica , Proteínas de Bactérias/metabolismo , Meio Ambiente , Perfilação da Expressão Gênica , Pseudomonas aeruginosa/fisiologia , Quinolonas/metabolismo , Animais , Proteínas de Bactérias/genética , Biofilmes/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Humanos , Análise em Microsséries , Estrutura Molecular , Óperon , Plantas/microbiologia , Pseudomonas aeruginosa/patogenicidade , Piocianina/metabolismo , Quinolonas/química , Percepção de Quorum/fisiologia , Transdução de Sinais/fisiologiaRESUMO
2-Heptyl-3-hydroxy-4(1H)-quinolone (PQS) is a quorum-sensing signal molecule used by Pseudomonas aeruginosa. The structural similarity between 3-hydroxy-2-methyl-4(1H)-quinolone, the natural substrate for the 2,4-dioxygenase, Hod, and PQS prompted us to investigate whether Hod quenched PQS signaling. Hod is capable of catalyzing the conversion of PQS to N-octanoylanthranilic acid and carbon monoxide. In P. aeruginosa PAO1 cultures, exogenously supplied Hod protein reduced expression of the PQS biosynthetic gene pqsA, expression of the PQS-regulated virulence determinants lectin A, pyocyanin, and rhamnolipids, and virulence in planta. However, the proteolytic cleavage of Hod by extracellular proteases, competitive inhibition by the PQS precursor 2-heptyl-4(1H)-quinolone, and PQS binding to rhamnolipids reduced the efficiency of Hod as a quorum-quenching agent. Nevertheless, these data indicate that enzyme-mediated PQS inactivation has potential as an antivirulence strategy against P. aeruginosa.