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 The 5′ NAD Cap of RNAIII Modulates Toxin Production in Staphylococcus aureus Isolates

2019 ◽  
Vol 202 (6) ◽  
Author(s):  
Hector Gabriel Morales-Filloy ◽  
Yaqing Zhang ◽  
Gabriele Nübel ◽  
Shilpa Elizabeth George ◽  
Natalya Korn ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Secondary Structure ◽  
Quorum Sensing ◽  
Opportunistic Pathogen ◽  
Toxin Production ◽  
Dual Function ◽  
Content Type ◽  
The Stability

ABSTRACT Nicotinamide adenosine dinucleotide (NAD) has been found to be covalently attached to the 5′ ends of specific RNAs in many different organisms, but the physiological consequences of this modification are largely unknown. Here, we report the occurrence of several NAD-RNAs in the opportunistic pathogen Staphylococcus aureus. Most prominently, RNAIII, a central quorum-sensing regulator of this bacterium’s physiology, was found to be 5′ NAD capped in a range from 10 to 35%. NAD incorporation efficiency into RNAIII was found to depend in vivo on the −1 position of the P3 promoter. An increase in RNAIII’s NAD content led to a decreased expression of alpha- and delta-toxins, resulting in reduced cytotoxicity of the modified strains. These effects seem to be caused neither by changes in RNAIII’s secondary structure nor by a different translatability upon NAD attachment, as indicated by unaltered patterns in in vitro chemical probing and toeprinting experiments. Even though we did not observe any effect of this modification on RNAIII’s secondary structure or translatability in vitro, additional unidentified factors might account for the modulation of exotoxins in vivo. Ultimately, the study constitutes a step forward in the discovery of new roles of the NAD molecule in bacteria. IMPORTANCE Numerous organisms, including bacteria, are endowed with a 5′ NAD cap in specific RNAs. While the presence of the 5′ NAD cap modulates the stability of the modified RNA species, a significant biological function and phenotype have not been assigned so far. Here, we show the presence of a 5′ NAD cap in RNAIII from S. aureus, a dual-function regulatory RNA involved in quorum-sensing processes and regulation of virulence factor expression. We also demonstrate that altering the natural NAD modification ratio of RNAIII leads to a decrease in exotoxin production, thereby modulating the bacterium’s virulence. Our work unveils a new layer of regulation of RNAIII and the agr system that might be linked to the redox state of the NAD molecule in the cell.

scholarly journals Quorum sensing et quorum quenching : Comment bloquer la communication des bactéries pour inhiber leur virulence ?

2019 ◽  
Vol 35 (1) ◽  
pp. 31-38 ◽  
Author(s):  
Sonia Mion ◽  
Benjamin Rémy ◽  
Laure Plener ◽  
Éric Chabrière ◽  
David Daudé
Keyword(s):  
Quorum Sensing ◽  
Quorum Quenching

La plupart des bactéries utilisent un système de communication, le quorum sensing, fondé sur la sécrétion et la perception de petites molécules appelées autoinducteurs qui leur permettent d’adapter leur comportement en fonction de la taille de la population. Les bactéries mutualisent ainsi leurs efforts de survie en synchronisant entre elles la régulation de gènes impliqués notamment dans la virulence, la résistance aux antimicrobiens ou la formation du biofilm. Des méthodes ont vu le jour pour inhiber cette communication entre bactéries et limiter leurs effets nocifs. Des inhibiteurs chimiques, des anticorps ou encore des enzymes capables d’interférer avec les autoinducteurs ont été développés et se sont montrés efficaces pour diminuer la virulence des bactéries à la fois in vitro et in vivo. Cette stratégie, appelée quorum quenching, a également montré des effets synergiques avec des traitements antibactériens classiques. Il permettrait notamment d’augmenter la sensibilité des bactéries aux antibiotiques. Ceci constitue une piste thérapeutique prometteuse pour lutter contre les infections bactériennes et limiter les conséquences de l’antibiorésistance.

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.

scholarly journals The 5’-NAD cap of RNAIII modulates toxin production in Staphylococcus aureus isolates

2019 ◽  
Author(s):  
Hector Gabriel Morales-Filloy ◽  
Yaqing Zhang ◽  
Gabriele Nübel ◽  
Shilpa Elizabeth George ◽  
Natalya Korn ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Quorum Sensing ◽  
Biological Function ◽  
Toxin Production ◽  
Dual Function ◽  
Large Step ◽  
Opportunistic Human Pathogen ◽  
The Stability

1AbstractNicotinamide adenosine dinucleotide (NAD) has been found to be covalently attached to the 5’-ends of specific RNAs in many different organisms, but the physiological consequences of this modification are largely unknown. Here we report the occurrence of several NAD-RNAs in the opportunistic human pathogen Staphylococcus aureus. Most prominently, RNAIII, a central quorum-sensing regulator of this bacterium’s physiology, was found to be 5’-NAD-capped to a significant extent. NAD incorporation efficiency into RNAIII was found to depend in vivo on the −1 position of the P3 promoter. Reduction of RNAIII’s NAD content led to a decreased expression of alpha- and delta-toxins, resulting in reduced cytotoxicity of the modified strains. These effects to not seem to be due to changes in RNAIII’s secondary structure upon NAD attachment, as indicated by largely unaltered patterns in in vitro chemical probing experiments. Our study represents a large step towards establishing a biological function of the 5’-NAD cap, which for RNAIII in S. aureus is to modulate the expression of virulence factors.2ImportanceNumerous organisms, including bacteria, are endowed with a 5’-NAD cap in specific RNAs. While the presence of the 5’-NAD cap modulates the stability of the modified RNA species, a significant biological function and phenotype have not been assigned so far. Here, we show the presence of a 5’-NAD cap in RNAIII from S. aureus, a dual-function regulatory RNA involved in quorum-sensing processes and regulation of virulence factor expression. We also demonstrate that altering the natural NAD modification ratio of RNAIII leads to a decrease in exotoxin production, thereby modulating bacterium’s virulence. Our work unveils a new layer of regulation of RNAIII and the agr system that might be linked to the redox state of the NAD molecule in the cell.

scholarly journals Plant-Derived Inhibitors of AHL-Mediated Quorum Sensing in Bacteria: Modes of Action

2019 ◽  
Vol 20 (22) ◽  
pp. 5588 ◽  
Author(s):  
Dmitry Deryabin ◽  
Anna Galadzhieva ◽  
Dianna Kosyan ◽  
Galimjan Duskaev
Keyword(s):  
Quorum Sensing ◽  
Communication Systems ◽  
Homoserine Lactone ◽  
Current Data ◽  
In Vivo Studies ◽  
Regulatory Pathways ◽  
Benzoic Acid Derivatives ◽  
Biofilm Development

Numerous gram-negative phytopathogenic and zoopathogenic bacteria utilise acylated homoserine lactone (AHL) in communication systems, referred to as quorum sensing (QS), for induction of virulence factors and biofilm development. This phenomenon positions AHL-mediated QS as an attractive target for anti-infective therapy. This review focused on the most significant groups of plant-derived QS inhibitors and well-studied individual compounds for which in silico, in vitro and in vivo studies provide substantial knowledge about their modes of anti-QS activity. The current data about sulfur-containing compounds, monoterpenes and monoterpenoids, phenylpropanoids, benzoic acid derivatives, diarylheptanoids, coumarins, flavonoids and tannins were summarized; their plant sources, anti-QS effects and bioactivity mechanisms have also been summarized and discussed. Three variants of plant-derived molecules anti-QS strategies are proposed: (i) specific, via binding with LuxI-type AHL synthases and/or LuxR-type AHL receptor proteins, which have been shown for terpenes (carvacrol and l-carvone), phenylpropanoids (cinnamaldehyde and eugenol), flavonoid quercetin and ellagitannins; (ii) non-specific, by affecting the QS-related intracellular regulatory pathways by lowering regulatory small RNA expression (sulphur-containing compounds ajoene and iberin) or c-di-GMP metabolism reduction (coumarin); and (iii) indirect, via alteration of metabolic pathways involved in QS-dependent processes (vanillic acid and curcumin).

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 Modification of In Vivo and In Vitro T- and B-Cell-Mediated Immune Responses by the Pseudomonas aeruginosa Quorum-Sensing Molecule N-(3-Oxododecanoyl)-l-Homoserine Lactone

2003 ◽  
Vol 71 (8) ◽  
pp. 4421-4431 ◽  
Author(s):  
Adam J. Ritchie ◽  
Andrew O. W. Yam ◽  
Kara M. Tanabe ◽  
Scott A. Rice ◽  
Margaret A. Cooley
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Immune Responses ◽  
Cytokine Production ◽  
Homoserine Lactone ◽  
Relative Increase ◽  
Ifn Γ ◽  
Quorum Sensing Molecule

ABSTRACT N-3-(oxododecanoyl)-l-homoserine lactone (OdDHL), a quorum-sensing molecule of Pseudomonas aeruginosa, plays an important role in the pathogenesis of the organism through its control of virulence factor expression. Several reports have suggested that OdDHL can also directly modulate host immune responses. However, the nature of the modulation is controversial, with different reports suggesting promotion of either humoral (Th2-mediated) or inflammatory (Th1-mediated) responses. This report describes a series of studies which demonstrate for the first time that in vivo administration of OdDHL can modulate the course of an antibody response, with an increase in ovalbumin (OVA)-specific immunogloblulin G1 (IgG1) but not IgG2a in OdDHL-treated OVA-immunized BALB/c mice compared to levels for controls. In vitro stimulation of lymphocytes from both Th1-biased C57Bl/6 and T-cell receptor transgenic mice and Th2-biased BALB/c mice in the presence of OdDHL demonstrated that OdDHL inhibits in vitro cytokine production in response to both mitogen and antigen, with gamma interferon (IFN-γ) tending to be more inhibited than interleukin-4 (IL-4). In vitro mitogen or antigen restimulation of cells from mice treated with OdDHL in vivo shows effects on cytokine production which depend on the underlying immune bias of the mouse strain used, with a relative increase of IFN-γ in Th1-biased C57Bl/6 mice and a relative increase of IL-4 in Th2-biased BALB/c mice. Thus, the mode of action of OdDHL on T-cell cytokine production is likely to be a relatively nonspecific one which accentuates an underlying immune response bias rather than one which specifically targets either Th1 or Th2 responses.

scholarly journals In Vitro Cellular Toxicity PredictsPseudomonas aeruginosa Virulence in Lung Infections

1998 ◽  
Vol 66 (7) ◽  
pp. 3242-3249 ◽  
Author(s):  
Teiji Sawa ◽  
Maria Ohara ◽  
Kiyoyasu Kurahashi ◽  
Sally S. Twining ◽  
Dara W. Frank ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Quorum Sensing ◽  
Homoserine Lactone ◽  
Lung Epithelial Cells ◽  
Cellular Damage ◽  
Culture Cells ◽  
Invasive Strain ◽  
In Vivo Cytotoxicity

ABSTRACT The role of quorum sensing by Pseudomonas aeruginosa in producing cytotoxicity has not been fully investigated. Strains ofP. aeruginosa have been characterized as having an invasive or a cytotoxic phenotype (S. M. J. Fleiszig et al., Infect. Immun. 65:579–586, 1997). We noted that the application of a large inoculum of the invasive strain 6294 caused cytotoxicity of cultured epithelial cells. To investigate this dose-related cytotoxicity, we compared the behavior of 6294 to that of another invasive strain, PAO1, and determined whether the cytotoxicity could be related to quorum sensing. Both invasive strains, 6294 and PAO1, appear to have quorum-sensing systems that were operative when large doses of bacteria were applied to cultured lung epithelial cells or instilled into the lungs of animals. Nonetheless, only 6294 was cytotoxic. Cytotoxicity induced by 6294 correlated with increased elastase production. These experiments suggest that there are multiple mechanisms for the induction of cytotoxicity, pathology, and mortality in vivo. However, in vivo cytotoxicity and mortality, but not pathology, could be predicted by quantitative in vitro cellular damage experiments utilizing a range of bacteria-to-cell ratios. It appears that quorum sensing may inversely correlate with virulence in that strains that produced PAI [N-(3-oxododecanoyl) homoserine lactone] also appeared to attract more polymorphonuclear leukocytes in vivo and were possibly eliminated more quickly. In addition, exoproduct production in bacteriological medium in vitro may differ significantly from exoproduct expression from infections in vivo or during cocultivation of bacteria with tissue culture cells.

Formation and Structure of Casein Micelles in Lactating Mammary Tissue

1970 ◽  
Vol 28 ◽  
pp. 150-151
Author(s):  
Robert J. Carroll ◽  
Marvin P. Thompson ◽  
Harold M. Farrell
Keyword(s):  
Size Distribution ◽  
G Protein ◽  
Milk Proteins ◽  
Mammary Tissue ◽  
Colloidal System ◽  
Casein Micelles ◽  
The Stability

Milk is an unusually stable colloidal system; the stability of this system is due primarily to the formation of micelles by the major milk proteins, the caseins. Numerous models for the structure of casein micelles have been proposed; these models have been formulated on the basis of in vitro studies. Synthetic casein micelles (i.e., those formed by mixing the purified αsl- and k-caseins with Ca2+ in appropriate ratios) are dissimilar to those from freshly-drawn milks in (i) size distribution, (ii) ratio of Ca/P, and (iii) solvation (g. water/g. protein). Evidently, in vivo organization of the caseins into the micellar form occurs in-a manner which is not identical to the in vitro mode of formation.

Kinetics of Various 99mTc-Sn-Pyrophosphate Compounds in the Rat

1977 ◽  
Vol 16 (04) ◽  
pp. 157-162 ◽  
Author(s):  
C. Schümichen ◽  
B. Mackenbrock ◽  
G. Hoffmann
Keyword(s):  
Normal Saline ◽  
Half Life ◽  
Compound A ◽  
Short Half Life ◽  
Low Concentrations ◽  
The Stability ◽  
Kinetics Of ◽  
In Vitro Stability

SummaryThe bone-seeking 99mTc-Sn-pyrophosphate compound (compound A) was diluted both in vitro and in vivo and proved to be unstable both in vitro and in vivo. However, stability was much better in vivo than in vitro and thus the in vitro stability of compound A after dilution in various mediums could be followed up by a consecutive evaluation of the in vivo distribution in the rat. After dilution in neutral normal saline compound A is metastable and after a short half-life it is transformed into the other 99mTc-Sn-pyrophosphate compound A is metastable and after a short half-life in bone but in the kidneys. After dilution in normal saline of low pH and in buffering solutions the stability of compound A is increased. In human plasma compound A is relatively stable but not in plasma water. When compound B is formed in a buffering solution, uptake in the kidneys and excretion in urine is lowered and blood concentration increased.It is assumed that the association of protons to compound A will increase its stability at low concentrations while that to compound B will lead to a strong protein bond in plasma. It is concluded that compound A will not be stable in vivo because of a lack of stability in the extravascular space, and that the protein bond in plasma will be a measure of its in vivo stability.

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