RESUMO
The opportunistic bacterial pathogen Pseudomonas aeruginosa causes chronic lung infections in cystic fibrosis (CF) patients. Importantly, virulence factor expression and biofilm formation in P. aeruginosa is coordinated by quorum sensing (QS) and one of the key QS signaling molecules is 3-oxo-C12-HSL. Remarkably, a tetramic acid, (C12-TA), with antibacterial properties is formed spontaneously from 3-oxo-C12-HSL under physiological conditions. Seeking to better understand this relationship, we sought to investigate whether 3-oxo-C12-HSL and C12-TA may be contributing factors to the overall pathogenicity of P. aeruginosa in CF individuals and if their detection and quantitation in sputum samples might be used as an indicator to assess disease states and monitor therapy success in CF patients. To this end, 3-oxo-C12-HSL and C12-TA concentrations were initially analyzed in P. aeruginosa flow cell biofilms using liquid chromatography coupled with mass spectrometry (LC-MS). A liquid chromatography tandem mass spectrometry (LC-MS/MS)-based method was then developed and validated for their detection and quantification in the sputa of CF patients. To the best of our knowledge, this is the first report to show the presence of both the quorum sensing molecule (3-oxo-C12-HSL) and its rearranged product (C12-TA) in human clinical samples such as sputum. A total of 47 sputum samples from 20 CF and 2 non-CF individuals were analyzed. 3-Oxo-C12-HSL was detected and quantified in 45 samples with concentrations ranging from 20 to >1000 nM; C12-TA was found in 14 samples (13-900 nM). On the basis of our findings, quorum sensing autoinducers merit further investigation as biomarkers for infectious disease states.
Assuntos
Pseudomonas aeruginosa/isolamento & purificação , Percepção de Quorum , Espectrometria de Massas por Ionização por Electrospray/métodos , Escarro/química , Adolescente , Adulto , Biomarcadores/análise , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Infecções por Pseudomonas/diagnóstico , Adulto JovemRESUMO
Quorum sensing (QS) allows bacteria to communicate with one another by means of QS signaling molecules and control certain behaviors in a group-based manner, including pathogenicity and biofilm formation. Bacterial gut microflora may play a role in inflammatory bowel disease pathogenesis, and antibiotics are one of the available therapeutic options for Crohn's disease. In the present study, we employed genetically engineered bioluminescent bacterial whole-cell sensing systems as a tool to evaluate the ability of antibiotics commonly employed in the treatment of chronic inflammatory conditions to interfere with QS. We investigated the effect of ciprofloxacin, metronidazole, and tinidazole on quorum sensing. Several concentrations of individual antibiotics were allowed to interact with two different types of bacterial sensing cells, in both the presence and absence of a fixed concentration of N-acylhomoserine lactone (AHL) QS molecules. The antibiotic effect was then determined by monitoring the biosensor's bioluminescence response. Ciprofloxacin, metronidazole, and tinidazole exhibited a dose-dependent augmentation in the response of both bacterial sensing systems, thus showing an AHL-like effect. Additionally, such an augmentation was observed, in both the presence and absence of AHL. The data obtained indicate that ciprofloxacin, metronidazole, and tinidazole may interfere with bacterial communication systems. The results suggest that these antibiotics, at the concentrations tested, may themselves act as bacterial signaling molecules. The beneficial effect of these antibiotics in the treatment of intestinal inflammation may be due, at least in part, to their effect on QS-related bacterial behavior in the gut.
Assuntos
Antibacterianos/farmacologia , Técnicas Biossensoriais/instrumentação , Escherichia coli/efeitos dos fármacos , Escherichia coli/fisiologia , Percepção de Quorum/efeitos dos fármacos , Antibacterianos/química , Técnicas Biossensoriais/métodos , Escherichia coli/genética , Viabilidade Microbiana/efeitos dos fármacosRESUMO
The triphenyl amide/ester 12 was originally reported to be a potent mimic of the natural 3-oxo-dodecanoyl homoserine lactone quorum sensing molecule in Pseudomonas aeruginosa. However, explicit synthesis/chemical characterization was lacking, and a later report providing protein crystallographic data inferred 12 to be incorrect, with 9 now being the surmised structure. Because of these inconsistencies and our interest in quorum sensing molecules utilized by gram-negative bacteria, we found it necessary to synthesize 9 and 12 to test for agonistic activity in a P. aeruginosa reporter assay. Despite distinct regiochemical differences, both 9 and 12 were found to have comparable EC(50) values. To reconcile these unanticipated findings, modeling studies were conducted, and both compounds were revealed to have comparable properties for binding to the LasR receptor.
Assuntos
4-Butirolactona/análogos & derivados , Modelos Moleculares , Pseudomonas aeruginosa/fisiologia , Percepção de Quorum , 4-Butirolactona/síntese químicaRESUMO
As a guide for chemical probe design, focused analogue synthetic studies were undertaken upon the lactone ring of 3-oxo-C(12)-homoserine lactone. We have concluded that hydrolytic instability of the heterocyclic ring is pivotal for its ability to modulate immune signaling and probe preparation was aligned with these findings.
Assuntos
4-Butirolactona/análogos & derivados , Homosserina/análogos & derivados , Percepção de Quorum/fisiologia , 4-Butirolactona/síntese química , 4-Butirolactona/química , 4-Butirolactona/metabolismo , Animais , Células da Medula Óssea/citologia , Estresse do Retículo Endoplasmático , Homosserina/síntese química , Homosserina/química , Homosserina/metabolismo , Imunomodulação , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , NF-kappa B/metabolismo , Oxirredução , Poli(ADP-Ribose) Polimerases/metabolismo , Pseudomonas aeruginosa/fisiologiaRESUMO
Tropolone emerged from the screening of a chelator fragment library (CFL) as an inhibitor of the Zn(2+)-dependent virulence factor, Pseudomonas aeruginosa elastase (LasB). Based on this initial hit, a series of substituted tropolone-based LasB inhibitors was prepared, and a compound displaying potent activity in vitro and in a bacterial swarming assay was identified. Importantly, this inhibitor was found to be specific for LasB over other metalloenzymes, validating the usage of tropolone as a viable scaffold for identifying first-in-class LasB inhibitors.
Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Quelantes/química , Elastase Pancreática/antagonistas & inibidores , Tropolona/química , Zinco/química , Proteínas de Bactérias/metabolismo , Quelantes/metabolismo , Cinética , Elastase Pancreática/metabolismo , Ligação Proteica , Pseudomonas aeruginosa/enzimologia , Tropolona/metabolismoRESUMO
Botulinum neurotoxins (BoNTs) are among the most deadly poisons known though ironically, they also are of great therapeutic utility. A number of research programs have been initiated to discover small molecule inhibitors of BoNTs metalloprotease activity. Many, though not all of these programs have screened against a truncated and more stable form of the enzyme, that possess comparable catalytic properties to the full length enzyme. Interestingly, several classes of inhibitors notably the hydroxamates, display a large shift in potency between the two enzyme forms. In this report we compare the kinetics of active-site, alpha-exosite and beta-exosite inhibitors versus truncated and full length enzyme. Molecular dynamics simulations conducted with the truncated and homology models of the fully length BoNT LC/A indicate the flexibility of the C-terminus of the full length enzyme is responsible for the potency shifts of active-site proximally binding inhibitors while distal binding (alpha-exosite) inhibitors remain equipotent.
RESUMO
Bacterial resistance coupled to our current arsenal of antibiotics presents us with a growing threat to public health, thus warranting the exploration of alternative antibacterial strategies. In particular, the targeting of virulence factors has been regarded as a "second generation" antibiotic approach. In Pseudomonas aeruginosa, a Zn(2+) metalloprotease virulence factor, LasB or P. aeruginosa elastase, has been implicated in the development of P. aeruginosa-related keratitis, pneumonia and burn infection. Moreover, the enzyme also plays a critical role in swarming and biofilm formation, both of which are processes that have been linked to antibiotic resistance. To further validate the importance of LasB in P. aeruginosa infection, we describe our efforts toward the discovery of non-peptidic small molecule inhibitors of LasB. Using identified compounds, we have confirmed the role that LasB plays in P. aeruginosa swarming and demonstrate the potential for LasB-targeted small molecules in studying antimicrobial resistant P. aeruginosa phenotypes.
RESUMO
Bacteria use small diffusible molecules to exchange information in a process called quorum sensing (QS). An important class of quorum sensing molecules used by Gram-negative bacteria is the family of N-acylhomoserine lactones (HSL). It was recently discovered that a degradation product of the QS molecule 3-oxo-C(12)-homoserine lactone, the tetramic acid 3-(1-hydroxydecylidene)-5-(2-hydroxyethyl)pyrrolidine-2,4-dione, is a potent antibacterial agent, thus implying roles for QS outside of simply communication. Because these tetramic acids also appear to bind iron with appreciable affinity it was suggested that metal binding might contribute to their biological activity. Here, using a variety of spectroscopic tools, we describe the coordination chemistry of both the methylidene and decylidene tetramic acid derivatives with Fe(III) and Ga(III) and discuss the potential biological significance of such metal binding.