Role of quorum sensing in the pathogenicity of Burkholderia pseudomallei

2004 ◽  
Vol 53 (11) ◽  
pp. 1053-1064 ◽  
Author(s):  
Ricky L Ulrich ◽  
David DeShazer ◽  
Ernst E Brueggemann ◽  
Harry B Hines ◽  
Petra C Oyston ◽  
...  
Keyword(s):  
Quorum Sensing ◽  
Burkholderia Pseudomallei ◽  
Homoserine Lactone ◽  
Mass Spectrometry Analysis ◽  
Syrian Hamsters ◽  
Spectrometry Analysis ◽  
Time To Death ◽  
Signalling Molecules

Burkholderia pseudomallei is the causative agent of human and animal melioidosis. The role of quorum sensing (QS) in the in vivo pathogenicity of B. pseudomallei via inhalational exposure of BALB/c mice and intraperitoneal challenge of Syrian hamsters has not been reported. This investigation demonstrates that B. pseudomallei encodes a minimum of three luxI and five luxR homologues that are involved in animal pathogenicity. Mass spectrometry analysis of culture supernatants revealed that wild-type B. pseudomallei and the luxI mutants synthesized numerous signalling molecules, including N-octanoyl-homoserine lactone, N-decanoyl-homoserine lactone, N-(3-hydroxyoctanoyl)-l-homoserine lactone, N-(3-hydroxydecanoyl)-l-homoserine lactone and N-(3-oxotetradecanoyl)-l-homoserine lactone, which was further confirmed by heterologous expression of the B. pseudomallei luxI alleles in Escherichia coli. Mutagenesis of the B. pseudomallei QS system increased the time to death and reduced organ colonization of aerosolized BALB/c mice. Further, intraperitoneal challenge of Syrian hamsters with the B. pseudomallei QS mutants resulted in a significant increase in the LD50. Using semi-quantitative plate assays, preliminary analysis suggests that QS does not affect lipase, protease and phospholipase C biosynthesis/secretion in B. pseudomallei. The findings of the investigation demonstrate that B. pseudomallei encodes multiple luxIR genes, and disruption of the QS alleles reduces animal pathogenicity, but does not affect exoproduct secretion.

scholarly journals DNA–PK facilitates piggyBac transposition by promoting paired-end complex formation

2017 ◽  
Vol 114 (28) ◽  
pp. 7408-7413 ◽  
Author(s):  
Yan Jin ◽  
Yaohui Chen ◽  
Shimin Zhao ◽  
Kun-Liang Guan ◽  
Yuan Zhuang ◽  
...  
Keyword(s):  
Complex Formation ◽  
Germ Line ◽  
Mechanistic Explanation ◽  
Mass Spectrometry Analysis ◽  
Physical Interaction ◽  
Spectrometry Analysis ◽  
Culture Cells ◽  
Underlying Mechanisms

The involvement of host factors is critical to our understanding of underlying mechanisms of transposition and the applications of transposon-based technologies. Modified piggyBac (PB) is one of the most potent transposon systems in mammals. However, varying transposition efficiencies of PB among different cell lines have restricted its application. We discovered that the DNA–PK complex facilitates PB transposition by binding to PB transposase (PBase) and promoting paired-end complex formation. Mass spectrometry analysis and coimmunoprecipitation revealed physical interaction between PBase and the DNA–PK components Ku70, Ku80, and DNA-PKcs. Overexpression or knockdown of DNA–PK components enhances or suppresses PB transposition in tissue culture cells, respectively. Furthermore, germ-line transposition efficiency of PB is significantly reduced in Ku80 heterozygous mutant mice, confirming the role of DNA–PK in facilitating PB transposition in vivo. Fused dimer PBase can efficiently promote transposition. FRET experiments with tagged dimer PBase molecules indicated that DNA–PK promotes the paired-end complex formation of the PB transposon. These data provide a mechanistic explanation for the role of DNA–PK in facilitating PB transposition and suggest a transposition-promoting manipulation by enhancing the interaction of the PB ends. Consistent with this, deletions shortening the distance between the two PB ends, such as PB vectors with closer ends (PB-CE vectors), have a profound effect on transposition efficiency. Taken together, our study indicates that in addition to regulating DNA repair fidelity during transposition, DNA–PK also affects transposition efficiency by promoting paired-end complex formation. The approach of CE vectors provides a simple practical solution for designing efficient transposon vectors.

scholarly journals Microdomain protein Nce102 is a local sensor of plasma membrane sphingolipid balance

2021 ◽  
Author(s):  
Jakub Zahumensky ◽  
Caroline Mota Fernandes ◽  
Petra Vesela ◽  
Maurizio Del Poeta ◽  
James Bernard Konopka ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Physiological Role ◽  
Building Blocks ◽  
Mass Spectrometry Analysis ◽  
Spectrometry Analysis ◽  
Signalling Molecules ◽  
Human Fungal Pathogen ◽  
Sphingolipid Biosynthesis ◽  
Complex Sphingolipids

Sphingolipids are essential building blocks of eukaryotic membranes and important signalling molecules, tightly regulated in response to environmental and physiological inputs. Mechanism of sphingolipid level perception at the plasma membrane remains unclear. In Saccharomyces cerevisiae, Nce102 protein has been proposed to function as sphingolipid sensor as it changes its plasma membrane distribution in response to sphingolipid biosynthesis inhibition. We show that Nce102 redistributes specifically in regions of increased sphingolipid demand, e.g., membranes of nascent buds. Furthermore, we report that production of Nce102 increases following sphingolipid biosynthesis inhibition and Nce102 is internalized when excess sphingolipid precursors are supplied. This suggests that the total amount of Nce102 in the plasma membrane is a measure of the current need for sphingolipids, whereas its local distribution marks sites of high sphingolipid demand. Physiological role of Nce102 in regulation of sphingolipid synthesis is demonstrated by mass spectrometry analysis showing reduced levels of complex sphingolipids and long-chain bases in nce102? deletion mutant. Nce102 behaves analogously in human fungal pathogen Candida albicans, suggesting a conserved principle of local sphingolipid control across species.

scholarly journals Enterobacter cancerogenus produces short chain N-3-oxo-acylhomoserine lactones quorum sensing molecules

2015 ◽  
Author(s):  
Kok-Gan Chan ◽  
Wen-Si Tan
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
High Resolution ◽  
Quorum Sensing ◽  
Homoserine Lactone ◽  
Mass Spectrometry Analysis ◽  
Liquid Chromatography Mass Spectrometry ◽  
Spectrometry Analysis ◽  
Synthesis Activity

Enterobacter cancerogenus strain M004 genome size is 5.67 Mb. Here, its luxI homologue, designated as ecnI which is ecnI gene (633 bp) was cloned and overexpressed. Its AHL synthesis activity was verified using the high-resolution liquid chromatography-mass spectrometry analysis revealed the production of N-(3-oxo-hexanoyl)-L-homoserine lactone (3-oxo-C6-HSL) and N-(3-oxo-hexanoyl)-L-homoserine lactone (3-oxo-C8-HSL). The cloning and characterization of luxI homologue of E. cancerogenus strain M004 was firstly reported here.

Impact and mechanistic role of MicroRNA-1291 on pancreatic tumorigenesis.

2016 ◽  
Vol 34 (4_suppl) ◽  
pp. 243-243 ◽  
Author(s):  
Mei-Juan Tu ◽  
Yu-Zhuo Pan ◽  
Jing-Xin Qiu ◽  
Edward Jae-hoon Kim ◽  
Aiming Yu
Keyword(s):  
Cell Cycle ◽  
Cancer Biology ◽  
Critical Role ◽  
Experimental Studies ◽  
Mass Spectrometry Analysis ◽  
Ductal Adenocarcinoma ◽  
Luciferase Reporter ◽  
Spectrometry Analysis

243 Background: Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer death. Better understanding of pancreatic cancer biology and identification of new targets are highly warranted. MicroRNAs (miRs or miRNAs) play a critical role in the control of tumor progression via crosstalk with cancer signaling pathways. Our recent studies showed that miR-1291 improved chemosensitivity through targeting of efflux transporter ABCC1. This current study investigated the mechanistic role of miR-1291 in the suppression of pancreatic tumorigenesis. Methods: PANC-1 and AsPC-1 cell lines were stably transfected with miR-1291. Cell cycle status and apoptosis of stable miR-1291-expressing cells were tested against control cells using flow cytometry. Cells were injected subcutaneously into nude mice and tumorigenesis was measured in vivo. Proteomic studies were performed by two-dimensional difference gel electrophoresis, matrix-assisted laser desorption/ionization time of flight mass spectrometry analysis. Computationally predicted miR-1291 targets were assessed by luciferase reporter assay and Western blot. Primary PDAC and control samples were tested for miR-1291 and target gene expression levels. Results: Our data showed that stable miR-1291-expressing PANC-1 and AsPC-1 cells both showed a significantly lower rate of proliferation than the control cells, which was associated with a cell cycle arrest and enhanced apoptosis. Furthermore, miR-1291 suppressed the tumorigenesis of PANC-1 cells in mouse models in vivo. Proteomic studies revealed the protein level of several cancer-related genes were downregulated by miR-1291, including a pancreatic tumor promoting protein AGR2 which was reduced ~10-fold. Through computational and experimental studies we further identified that FOXA2, a transcription factor governing AGR2 expression, was a direct target of miR-1291. In addition, we found a significant down-regulation of miR-1291 in a set of PDAC patient tumor samples overexpressing AGR2. Conclusions: These results indicate that miR-1291 suppresses pancreatic tumorigenesis via targeting of FOXA2-AGR regulatory pathway providing new insight supporting development of miR-1291-based therapy for PDAC.

scholarly journals Engineering quorum quenching enzymes: progress and perspectives

2019 ◽  
Vol 47 (3) ◽  
pp. 793-800 ◽  
Author(s):  
Shereen A. Murugayah ◽  
Monica L. Gerth
Keyword(s):  
Quorum Sensing ◽  
Quorum Quenching ◽  
Homoserine Lactone ◽  
Microbial Pathogens ◽  
Human Pathogens ◽  
New Approach ◽  
Signalling Molecules ◽  
The Stability

Abstract Quorum sensing is a key contributor to the virulence of many important plant, animal and human pathogens. The disruption of this signalling—a process referred to as ‘quorum quenching’—is a promising new approach for controlling microbial pathogens. In this mini-review, we have focused on efforts to engineer enzymes that disrupt quorum sensing by inactivating acyl-homoserine lactone signalling molecules. We review different approaches for protein engineering and provide examples of how these engineering approaches have been used to tailor the stability, specificity and activities of quorum quenching enzymes. Finally, we grapple with some of the issues around these approaches—including the disconnect between in vitro biochemistry and potential in vivo applications.

scholarly journals circPDE4B prevents articular cartilage degeneration and promotes repair by acting as a scaffold for RIC8A and MID1

2021 ◽  
pp. annrheumdis-2021-219969
Author(s):  
Shuying Shen ◽  
Yute Yang ◽  
Panyang Shen ◽  
Jun Ma ◽  
Bin Fang ◽  
...  
Keyword(s):  
Rna Binding ◽  
Cartilage Degeneration ◽  
Mitogen Activated Protein Kinase ◽  
Mass Spectrometry Analysis ◽  
Circular Rnas ◽  
Nucleotide Exchange ◽  
Spectrometry Analysis

ObjectivesCircular RNAs (circRNAs) have emerged as significant biological regulators. Herein, we aimed to elucidate the role of an unidentified circRNA (circPDE4B) that is reportedly downregulated in osteoarthritis (OA) tissues.MethodsThe effects of circPDE4B were explored in human and mouse chondrocytes in vitro. Specifically, RNA pull-down (RPD)-mass spectrometry analysis (MS), immunoprecipitation, glutathione-S-transferase (GST) pull-down, RNA immunoprecipitation and RPD assays were performed to verify the interactions between circPDE4B and the RIC8 guanine nucleotide exchange factor A (RIC8A)/midline 1 (MID1) complex. A mouse model of OA was also employed to confirm the role of circPDE4B in OA pathogenesis in vivo.ResultscircPDE4B regulates chondrocyte cell viability and extracellular matrix metabolism. Mechanistically, FUS RNA binding protein (FUS) was found to promote the splicing of circPDE4B, while downregulation of circPDE4B in OA is partially caused by upstream inhibition of FUS. Moreover, circPDE4B facilitates the association between RIC8A and MID1 by acting as a scaffold to promote RIC8A degradation through proteasomal degradation. Furthermore, ubiquitination of RIC8A at K415 abrogates RIC8A degradation. The circPDE4B–RIC8A axis was observed to play an important role in regulating downstream p38 mitogen-activated protein kinase (MAPK) signalling. Furthermore, delivery of a circPDE4B adeno-associated virus (AAV) abrogates the breakdown of cartilage matrix by medial meniscus destabilisation in mice, whereas a RIC8A AAV induces the opposite effect.ConclusionThis work highlights the function of the circPDE4B–RIC8A axis in OA joints, as well as its regulation of MAPK-p38, suggesting this axis as a potential therapeutic target for OA.

scholarly journals Role of the C-Terminal Domain of the Alpha Subunit of RNA Polymerase in LuxR-Dependent Transcriptional Activation of the lux Operon during Quorum Sensing

2002 ◽  
Vol 184 (16) ◽  
pp. 4520-4528 ◽  
Author(s):  
Angela H. Finney ◽  
Robert J. Blick ◽  
Katsuhiko Murakami ◽  
Akira Ishihama ◽  
Ann M. Stevens
Keyword(s):  
Quorum Sensing ◽  
Rna Polymerase ◽  
Transcriptional Activation ◽  
Homoserine Lactone ◽  
Dependent Manner ◽  
Α Subunit ◽  
Terminal Domain

ABSTRACT During quorum sensing in Vibrio fischeri, the luminescence, or lux, operon is regulated in a cell density-dependent manner by the activator LuxR in the presence of an acylated homoserine lactone autoinducer molecule [N-(3-oxohexanoyl) homoserine lactone]. LuxR, which binds to the lux operon promoter at a position centered at −42.5 relative to the transcription initiation site, is thought to function as an ambidextrous activator making multiple contacts with RNA polymerase (RNAP). The specific role of the α-subunit C-terminal domain (αCTD) of RNAP in LuxR-dependent transcriptional activation of the lux operon promoter has been investigated. The effects of 70 alanine substitution variants of the α subunit were determined in vivo by measuring the rate of transcription of the lux operon via luciferase assays in recombinant Escherichia coli. The mutant RNAPs from strains exhibiting at least twofold-increased or -decreased activity in comparison to the wild type were further examined by in vitro assays. Since full-length LuxR has not been purified, an autoinducer-independent N-terminally truncated form of LuxR, LuxRΔN, was used for in vitro studies. Single-round transcription assays were performed using reconstituted mutant RNAPs in the presence of LuxRΔN, and 14 alanine substitutions in the αCTD were identified as having negative effects on the rate of transcription from the lux operon promoter. Five of these 14 α variants were also involved in the mechanisms of both LuxR- and LuxRΔN-dependent activation in vivo. The positions of these residues lie roughly within the 265 and 287 determinants in α that have been identified through studies of the cyclic AMP receptor protein and its interactions with RNAP. This suggests a model where residues 262, 265, and 296 in α play roles in DNA recognition and residues 290 and 314 play roles in α-LuxR interactions at the lux operon promoter during quorum sensing.

AhlD, an N-acylhomoserine lactonase in Arthrobacter sp., and predicted homologues in other bacteria

Microbiology ◽  
2003 ◽  
Vol 149 (6) ◽  
pp. 1541-1550 ◽  
Author(s):  
Sun-Yang Park ◽  
Sang Jun Lee ◽  
Tae-Kwang Oh ◽  
Jong-Won Oh ◽  
Bon-Tag Koo ◽  
...  
Keyword(s):  
Quorum Sensing ◽  
Pathogenic Bacteria ◽  
Erwinia Carotovora ◽  
Homoserine Lactone ◽  
Amino Acid Sequences ◽  
Mass Spectrometry Analysis ◽  
Signal Molecules ◽  
Genome Database ◽  
Spectrometry Analysis ◽  
A Genome

Quorum sensing is a signalling mechanism that controls diverse biological functions, including virulence, via N-acylhomoserine lactone (AHL) signal molecules in Gram-negative bacteria. With the aim of isolating strains or enzymes capable of blocking quorum sensing by inactivating AHL, bacteria were screened for AHL degradation by their ability to utilize N-3-oxohexanoyl-l-homoserine lactone (OHHL) as the sole carbon source. Among four isolates, strain IBN110, identified as Arthrobacter sp., was found to grow rapidly on OHHL, and to degrade various AHLs with different lengths and acyl side-chain substitutions. Co-culture of Arthrobacter sp. IBN110 and the plant pathogen Erwinia carotovora significantly reduced both the AHL amount and pectate lyase activity in co-culture medium, suggesting the possibility of applying Arthrobacter sp. IBN110 in the control of AHL-producing pathogenic bacteria. The ahlD gene from Arthrobacter sp. IBN110 encoding the enzyme catalysing AHL degradation was cloned, and found to encode a protein of 273 amino acids. A mass spectrometry analysis showed that AhlD probably hydrolyses the lactone ring of N-3-hexanoyl-l-homoserine lactone, indicating that AhlD is an N-acylhomoserine lactonase (AHLase). A comparison of AhlD with other known AHL-degrading enzymes, Bacillus sp. 240B1 AiiA, a Bacillus thuringiensis subsp. kyushuensis AiiA homologue and Agrobacterium tumefaciens AttM, revealed 25, 26 and 21 % overall identities, respectively, in the deduced amino acid sequences. Although these identities were relatively low, the HXDH≈H≈D motif was conserved in all the AHLases, suggesting that this motif is essential for AHLase activity. From a genome database search based on the conserved motif, putative AhlD-like lactonase genes were found in several other bacteria, and AHL-degrading activities were observed in Klebsiella pneumoniae and Bacillus stearothermophilus. Furthermore, it was verified that ahlK, an ahlD homologue, encodes an AHL-degrading enzyme in K. pneumoniae. Accordingly, the current results suggest the possibility that AhlD-like AHLases could exist in many other micro-organisms.

scholarly journals The BpeAB-OprB Efflux Pump of Burkholderia pseudomallei 1026b Does Not Play a Role in Quorum Sensing, Virulence Factor Production, or Extrusion of Aminoglycosides but Is a Broad-Spectrum Drug Efflux System

2010 ◽  
Vol 54 (8) ◽  
pp. 3113-3120 ◽  
Author(s):  
Takehiko Mima ◽  
Herbert P. Schweizer
Keyword(s):  
Quorum Sensing ◽  
Virulence Factor ◽  
Burkholderia Pseudomallei ◽  
Efflux Pump ◽  
Homoserine Lactone ◽  
Genetically Engineered ◽  
Multidrug Efflux ◽  
Efflux System ◽  
Aminoglycoside Resistance

ABSTRACT Most Burkholderia pseudomallei strains are intrinsically aminoglycoside resistant, mainly due to AmrAB-OprA-mediated efflux. Rare naturally occurring or genetically engineered mutants lacking this pump are aminoglycoside susceptible despite the fact that they also encode and express BpeAB-OprB, which was reported to mediate efflux of aminoglycosides in the Singapore strain KHW. To reassess the role of BpeAB-OprB in B. pseudomallei aminoglycoside resistance, we used mutants overexpressing or lacking this pump in either AmrAB-OprA-proficient or -deficient strain 1026b backgrounds. Our data show that BpeAB-OprB does not mediate efflux of aminoglycosides but is a multidrug efflux system which extrudes macrolides, fluoroquinolones, tetracyclines, acriflavine, and, to a lesser extent, chloramphenicol. Phylogenetically, BpeAB-OprB is closely related to Pseudomonas aeruginosa MexAB-OprM, which has a similar substrate spectrum. AmrAB-OprA is most closely related to MexXY, the only P. aeruginosa efflux pump known to extrude aminoglycosides. Since BpeAB-OprB in strain KHW was also implicated in playing a major role in export of acylated homoserine lactone (AHL) quorum-sensing molecules and in expression of diverse virulence factors, we explored whether this was also true in the strain 1026b background. The results showed that BpeAB-OprB was not required for AHL export, and mutants lacking this efflux system exhibited normal swimming motility and siderophore production, which were severely impaired in KHW bpeAB-oprB mutants. Biofilm formation was impaired in 1026b Δ(amrRAB-oprA) and Δ(amrRAB-oprA) Δ(bpeAB-oprB) mutants. At present, we do not know why our BpeAB-OprB susceptibility and virulence factor expression results with 1026b and its derivatives are different from those previously published for Singapore strain KHW.

scholarly journals Extracellular release of two peptidases dominates generation of the trypanosome quorum-sensing signal

2021 ◽  
Author(s):  
Mabel Dedalem Tettey ◽  
Federico Rojas ◽  
Keith R Matthews
Keyword(s):  
Quorum Sensing ◽  
Gene Knockout ◽  
Signal Transduction Pathway ◽  
Mass Spectrometry Analysis ◽  
Spectrometry Analysis ◽  
Oligopeptidase B ◽  
Pathway Gene ◽  
Extracellular Release ◽  
Signal Production

Trypanosomes causing African sleeping sickness use quorum-sensing (QS) to generate transmission-competent stumpy forms in their mammalian hosts. This density-dependent process is signalled by oligopeptides that stimulate the signal transduction pathway leading to stumpy formation. Using mass spectrometry analysis, peptidases released by trypanosomes were identified and, for 12 peptidases, their extracellular delivery was confirmed. Thereafter, the contribution of each peptidase to QS signal production was determined using systematic inducible overexpression in vivo, activity being confirmed to operate through the physiological QS signalling pathway. Gene knockout of the QS-active peptidases identified two enzymes, oligopeptidase B and metallocarboxypeptidase I, that significantly reduced QS when ablated individually. Further, a combinatorial gene knockout of both peptidases confirmed their dominance in the generation of the QS signal, with peptidase release of oligopeptidase B mediated via an unconventional protein secretion pathway. This identifies how the QS signal driving trypanosome virulence and transmission is generated in mammalian hosts.

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