Lack of AHL-based quorum sensing in Pseudomonas fluorescens isolated from milk

Numerous bacteria coordinate gene expression in response to small signalling molecules in many cases known as acylhomoserine lactones (AHLs), which accumulate as a function of cell density in a process known as quorum sensing. This work aimed to determine if phenotypes that are important to define microbial activity in foods such as biofilm formation, swarming motility and proteolytic activity of two Pseudomonas fluorescens strains, isolated from refrigerated raw milk, are influenced by AHL molecules. The tested P. fluorescens strains did not produce AHL molecules in none of the evaluated media. We found that biofilm formation was dependent on the culture media, but it was not influenced by AHLs. Our results indicate that biofilm formation, swarming motility and proteolytic activity of the tested P. fluorescens strains are not regulated by acyl-homoserine lactones. It is likely that AHL-dependent quorum sensing system is absent from these strains.

Methylobacterium-plant interaction genes regulated by plant exudate and quorum sensing molecules

Bacteria from the genus Methylobacterium interact symbiotically (endophytically and epiphytically) with different plant species. These interactions can promote plant growth or induce systemic resistance, increasing plant fitness. The plant colonization is guided by molecular communication between bacteria-bacteria and bacteria-plants, where the bacteria recognize specific exuded compounds by other bacteria (e.g. homoserine molecules) and/or by the plant roots (e.g. flavonoids, ethanol and methanol), respectively. In this context, the aim of this study was to evaluate the effect of quorum sensing molecules (N-acyl-homoserine lactones) and plant exudates (including ethanol) in the expression of a series of bacterial genes involved in Methylobacterium-plant interaction. The selected genes are related to bacterial metabolism (mxaF), adaptation to stressful environment (crtI, phoU and sss), to interactions with plant metabolism compounds (acdS) and pathogenicity (patatin and phoU). Under in vitro conditions, our results showed the differential expression of some important genes related to metabolism, stress and pathogenesis, thereby AHL molecules up-regulate all tested genes, except phoU, while plant exudates induce only mxaF gene expression. In the presence of plant exudates there is a lower bacterial density (due the endophytic and epiphytic colonization), which produce less AHL, leading to down regulation of genes when compared to the control. Therefore, bacterial density, more than plant exudate, influences the expression of genes related to plant-bacteria interaction.

Regulatory mechanism underlying pathogen biofilm formation and potential drug targets / 药学学报

Bacterial communities usually develop biofilms abound in nature niche. The development of biofilm is a highly dynamic and complex process coordinated by multiple mechanisms, of which two-component system and quorum sensing are two well-defined systems. Biofilm is involved in the virulence of many pathogens. Therefore, targeting the key factors involved in the biofilm formation represents a novel and promising avenue for developing better antibiotics.

Screening & profiling of quorum sensing signal molecules in Pseudomonas aeruginosa isolates from catheterized urinary tract infection patients.

Background & objectives: Catheter associated urinary tract infections are the second most common nosocomial infections and Pseudomonas aeruginosa is the third most common organism responsible for these infections. In this study P. aeruginosa isolates from catheterized urinary tract infection patients were screened and profiled for the presence of different type of quorum sensing (QS) signal molecules. Methods: Screening and quantitation of AHLs was done by using cross feeding assay and by determining β-galactosidase activity respectively using Escherichia coli MG4 as reporter strain. Further, AHL profiles were determined by separating AHLs on TLC coupled with their detection using Chromobacterium violaceum CV026 and Agrobacterium tumifaciens A136 biosensor strains. Results: All uroisolates from catheterized patients having urinary tract infections were found to be producers of QS signal molecules. There were differences in amounts and type of AHL produced amongst uroisolates of P. aeruginosa. Several AHLs belonging to C4-HSL, C6-HSL, oxo-C6-HSL, C8-HSL, C10-HSL and C12-HSL were determined in these strains. Interpretation & conclusions: Simultaneous use of more than one reporter strain and assay method proved useful in determining the AHLs profile in uroisolates of P. aeruginosa. Observed differences in the amounts and types of AHLs may reflect differences in virulence potential of P. aeruginosa to cause UTIs which can be further confirmed by employing animal model system. The present study speculates that production of QS signal molecules may act as a new virulence marker of P. aeruginosa responsible for causing catheter associated UTIs and can be considered as futuristic potential drug targets towards treatment of UTIs.

Estudo do envolvimento do receptor nuclear PPARy na inflamação pulmonar induzida pelo componente de Quorum Sensing de pseudomonas aeruginosa 3-oxo dodecanoil homoserina lactona / Study of the involvement of the nuclear receptor PPARy in lung inflammation induced component of Quorum Sensing in Pseudomonas aeruginosa 3-oxo homoserine lactone dodecanoil

A bactéria Pseudomonas aeruginosa (PA) é um dos principais agentes etiológicos de pneumonias nosocomiais, cujo tratamento é dificultado por sua resistência a antibiótcos e pela secreção de fatores de virulência. Novas alternativas para o tratamento destas infecções incluem manipular o sistema de omunicação bacteriano conhecido como quorum sensing (QS), reduzindo a expressão destes fatores de virulência e prevenindo seus efeitos deletérios em células e sistemas eucariotos. Os efeitos do componente de QS de PA 3-oxo-dodecanoil homoserina lactona (3-oxo C12 HSL) nos pulmões não são conhecidos; portando, a primeira etapa de nosso trabalho consistiu na caracterização dos efeitos desta molécula no ambiente pulmonar. Para isso, camundongos swiss desafiados com 3-oxo C12 HSL por via intratraqueal tiveram amostras de sangue, lavado bronco-alveolar (BAL) e tecido pulmonar e coletadas seis horas após o procedimento. Nossos resultados demonstram que inflamação pulmonar causada por 3-oxo C12 HSL se caracteriza pela migração de células mononucleares e neutrófilos para o espaço alveolar, níveis elevados de atividade mieloperoxidase no tecido pulmonar e formação de edema. A resposta inflamatória causada por 3-oxo C12 HSL não alterou os níveis de TNF-gama, MIF, IL-10 e IL-12 no tempo analisado. Foram observados aumentos nos níveis de IL-6, CCL2/MCP-1, CXCL1/KC e LTB4 no BAL de animais desafiados, sendo o aumento deste eicosanóide acompanhado por uma indução de corpúsculos lipídicos. Evidências in vitro relatam o envolvimento do receptor nuclear PPARgama nos efeitos pró-inflamatórios atribuídos a 3-oxo C12 HSL; portanto, decidimos estudar os efeitos do tratamento com o agonista da PPARgama rosiglitazona no modelo de inflamação pulmonar causado por este componente de QSNós observamos que o tratamento com rosiglitazona (0,5 mg/kg) uma hora após o estímulo diminuiu a formação de corpúsculos lipídicos e de edema pulmonar, causando também uma redução na migração de células mononucleares e neutrófilos e uma menor atividade mieloperoxidase no pulmão dos animais tratados. A redução da migração de células mononucleares parece estar associada à uma redução dos níveis de CCL2/MCP-1, enquanto o decréscimo nos neutrófilos parece envolver a modulação de CXCL1/KC. A instiliação com 3-oxo C12 HSL diminuiu a expressão da enzima paraoxonase (PON) no tecido pulmonar, fenômeno que não foi revertido pelo tratamento com rosiglitazona nesta dose. Estudos demonstram que o PPARgama está envolvido na expressão de PON e que a superexpressão desta enzima é capaz de proteger animais da mortalidade por PA em função de sua atividade lactonase; por este motivo, decidimos testar uma dose maior de rosiglitazona (5 mg/kg) para alcançar mais um benefício nesta proposta terapêutica. O tratamento com rosiglitazona nesta dose foi capaz de reverter a redução da expressão de paraoxonase causada por 3-oxo C12 HSL, diminuindo a formação de edena, a migração de neutrófilos e os níveis de atividade mieloperoxidase no pulmão de animais desafiados. O número de células mononucleares recuperado no BALde animais estimulados e tratados com a droga não foi reduzido, bem como os níveis de CCL2/MCP-1. De fato, a droga por si só causou um aumento de CCL2/MCP-1 que parece ter contribuído para a indução de corpúsculos lipídicos observada. Devido ao envolvimento de macrófagos e da quimiocina CCL2/MCP-1 no processo de eliminação bacteriana, estudos em modelos de pneumonia por PA precisam ser conduzidos para melhor avaliar esta dose de rosiglitazona e valida esta droga como uma estratégia terapêutica no combate à esta bactéria.

Quorum sensing signal profile of Acinetobacter strains from nosocomial and environmental sources / Perfil de sensores de quórum en cepas nosocomiales y ambientales de Acinetobacter

A set of 43 strains corresponding to 20 classified and unclassified genomic Acinetobacter species was analyzed for the production of typical N-acyl homoserine lactone quorum sensing molecules in culture broths. A large percentage of the strains (74%) displayed quorum sensing signals that could be separated into three statistically significantly different chromatographic groups (p < 0.001) based on their retention factor in TLC, i.e. Rf1 (0.22 ± 0.02); Rf2 (0.40 ± 0.02) and Rf3 (0.54 ± 0.02). Noteworthy, 63% of the strains tested produced more than one quorum signal. The frequency of signal appearance was Rf3 > Rf2 > Rf1. None of the three signals could be specifically assigned to a particular species in the genus; furthermore, no distinction could be made between the quorum sensing signals secreted by typical opportunistic strains of the A. calcoaceticus-A. baumannii complex, isolated from patients, with respect to the other species of the genus, except for the Rf1 signal which was present in all the QS positive strains belonging to this complex and DNA group 13 TU. In conclusion, quorum sensors in Acinetobacter are not homogenously distributed among species and one of them is present in most of the A. calcoaceticus-baumannii complex.

Se analizó la producción de moléculas típicas de N-acil homoserina lactona con actividad de quorum sensing en cultivos líquidos de un grupo de 43 cepas correspondientes a 20 especies genómicas clasificadas y no clasificadas de Acinetobacter. Un porcentaje alto de las cepas (74%) mostraron señales de quorum sensing que pudieron ser separadas en tres grupos cromatográficos significativamente diferentes entre sí (p < 0,001) sobre la base de sus factores de retención en TLC, a saber: Rf1 (0.22 ± 0.02); Rf2 (0.40 ± 0.02) y Rf3 (0.54 ± 0.02). Es de notar que 63% de las cepas ensayadas produjeron más de una señal de quorum. La frecuencia de aparición de las señales fue Rf3 > Rf2 > Rf1. Ninguna de las tres señales pudo ser asignada a una especie en particular dentro del género; es más, no se encontró diferencia entre las señales producidas por las cepas típicamente oportunistas (complejo A. calcoaceticus-A. baumannii) aisladas de pacientes respecto de las producidas por otras cepas del mismo género, excepto para el caso de Rf1, que se encontró presente en todos los aislamientos quorum sensing positivos del mencionado complejo y en las cepas del grupo de DNA 13TU. En conclusión, los sensores de quórum en Acinetobacter no están homogéneamente distribuidos entre especies y uno de ellos (Rf1) está presente en la mayoría de los miembros del complejo calcoaceticus-baumannii.

Cloning of phzIR from the endophytic Pseudomonas sp. G5 and its expression in Escherichia coli / 生物工程学报

We isolated a new strain of endophytic Pseudomonas G5 from the stems of Chinese parsley (Coriandrum sativum L.), and it is tentatively identified as Pseudomonas aurantiaca according to analysis of the entire substrate utilization profiles using BIOLOG Microstation system (BIOLOG, Inc, Hayward CA). An array of evidence established that many Gram-negative bacteria employ Quorum sensing (QS) system to regulate gene expression in response to cell density using small diffusible signal molecules, N-acyl homoserine lactones (AHLs), and control diverse phenotypic traits in plant-associated bacteria. In this study, we showed that Pseudomonas sp. strain G5 can produce several types of AHLs at a detectable level using Thin Layer Chromatography (TLC) analysis combined with bioreporter Chromobacterium violaceum CV026 bioassay, and N-hexanoyl-homoserine lactone (HHL, C6-HSL) with Rf value 0.4 is the major signal molecule. Furthermore, we have identified its quorum sensing system composed of PhzI and PhzR by cloning and sequencing of phzI-phzR. PhzI is responsible for synthesis of AHLs signal molecules, and PhzR is a transcriptional regulator. Finally, we heterologously expressed the recombinant plasmid pMD-phzIR in Escherichia coli JM109 and verified it using C. violaceum CV026 bioassay. The phylogenetic analysis using MEGA4 revealed highly similarities exist among the phzIR homologs, suggesting it is evolutionary well conserved in the genus Pseudomonas.

Biofilm formation and acyl homoserine lactone production in Hafnia alvei isolated from raw milk

The objective of this study was to detect the presence of acyl homoserine lactones (AHLs), signal molecules of the quorum sensing system in biofilm formed by Hafnia alvei strains. It also evaluated the effect of synthetic quorum sensing inhibitors in biofilm formation. AHLs were assayed using well diffusion techniques, thin layer chromatography (TLC) and detection directly in biofilm with biomonitors. The extracts obtained from planktonic and sessile cell of H. alvei induced at least two of three monitor strains evaluated. The presence of AHLs with up to six carbon atoms was confirmed by TLC. Biofilm formation by H. alvei was inhibited by furanone, as demonstrated by 96-well assay of crystal violet in microtitre plates and by scanning electron microscopy. The H. alvei 071 hall mutant was deficient in biofilm formation. All these results showed that the quorum sensing system is probably involved in the regulation of biofilm formation by H. alvei.

Expression of gene aiiA carrying the promoter of gene cry3Aa in Bacillus thuringiensis / 生物工程学报

N-acyl-homoserine lactones (AHLs), are widely conserved signal molecules present in quorum-sensing systems of many Gram-negative bacteria. AHLs molecules mediate the expression of virulence genes of a range of bacterial pathogens. Recently, it has been reported that AiiA protein, which widely exists in Bacillus species, can inactivate the AHLs by hydrolyzing the lactone bond of AHLs, thus attenuate the diseases caused by the expression of virulence genes of bacterial pathogens. Bacillus thuringiensis, a type of Gram-positive bacteria, has been used extensively as a microbial insecticide in the last few decades. However, most of important insecticidal B. thuringiensis strains have not been exploited for bacterial disease control because they usually do not produce antibiotics that are effective against bacteria and fungi. The discovery of AiiA protein in B. thuringiensis shows the application potential of B. thuringiensis on biocontrol against bacterial diseases. In this study, in order to construct the B. thuringiensis recombinant strain that has high expression of AiiA protein, the promoter of insecticidal crystal protein coding gene cry3Aa of B. thuringiensis was selected. The promoter of gene cry3Aa is a non-sporulation promoter, it promotes the transcription earlier and longer than the promoters of other cry genes. The promoter of AiiA protein coding gene aiiA was replaced with the promoter of gene cry3Aa by overlapping PCR, resulting fusion gene pro3A-aiiA. The gene pro3A-aiiA was inserted into shuttle vector pHT304 at site BamH I / Sph I , resulting recombinant plasmid pBMB686. The plasmid pBMB686 was introduced into B. thuringiensis acrystalliferous strain BMB171, the resulting strain BMB686 had a higher and more stable expression level of protein AiiA comparing with the parental strain BMB171. Furthermore, the strain BMB686 exhibited stronger ability of AHLs inactivation and much more effective restraint to the potato's soft rot disease caused by Erwinia carotovora than those of the parental strain BMB171. From these results, it was concluded that the B. thuringiensis strain harvesting the fusion gene pro3A-aiiA may be utilized in the future to control bacterial diseases which are mediated by the AHL quorum-sensing signals.