scholarly journals Gaussia Luciferase as a Reporter for Quorum Sensing in Staphylococcus aureus

Sensors ◽  
2020 ◽  
Vol 20 (15) ◽  
pp. 4305
Author(s):  
Isobel Blower ◽  
Carmen Tong ◽  
Xiaohui Sun ◽  
Ewan Murray ◽  
Jeni Luckett ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Quorum Sensing ◽  
Quantitative Assessment ◽  
Chemical Agent ◽  
Secreted Protein ◽  
Human Pathogen ◽  
Gaussia Luciferase ◽  
Bacterial Pathogenicity ◽  
Autoinducing Peptides

Gaussia luciferase (GLuc) is a secreted protein with significant potential for use as a reporter of gene expression in bacterial pathogenicity studies. To date there are relatively few examples of its use in bacteriology. In this study we show that GLuc can be functionally expressed in the human pathogen Staphylococcus aureus and furthermore show that it can be used as a biosensor for the agr quorum sensing (QS) system which employs autoinducing peptides to control virulence. GLuc was linked to the P3 promoter of the S. aureusagr operon. Biosensor strains were validated by evaluation of chemical agent-mediated activation and inhibition of agr. Use of GLuc enabled quantitative assessment of agr activity. This demonstrates the utility of Gaussia luciferase for in vitro monitoring of agr activation and inhibition.

scholarly journals Canine Bone Marrow Mesenchymal Stem Cell Conditioned Media Affect Bacterial Growth, Biofilm-Associated Staphylococcus aureus and AHL-Dependent Quorum Sensing

2020 ◽  
Vol 8 (10) ◽  
pp. 1478 ◽  
Author(s):  
Dobroslava Bujňáková ◽  
Anna Čuvalová ◽  
Milan Čížek ◽  
Filip Humenik ◽  
Michel Salzet ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Bone Marrow ◽  
Stem Cell ◽  
Quorum Sensing ◽  
Mesenchymal Stem Cell ◽  
Bacterial Growth ◽  
Conditioned Media ◽  
Amyloid Β Peptide

The present study investigated the in vitro antibacterial, antibiofilm and anti-Quorum Sensing (anti-QS) activities of canine bone marrow mesenchymal stem cell-conditioned media (cBM MSC CM) containing all secreted factors <30 K, using a disc diffusion test (DDT), spectrophotometric Crystal Violet Assay (SCVA) and Bioluminescence Assay (BA) with QS-reporter Escherichia coli JM109 pSB1142. The results show a sample-specific bacterial growth inhibition (zones varied between 7–30 mm), statistically significant modulation of biofilm-associated Staphylococcus aureus and Escherichia coli bioluminescence (0.391 ± 0.062 in the positive control to the lowest 0.150 ± 0.096 in the experimental group, cf. 11,714 ± 1362 to 7753 ± 700, given as average values of absorbance A550 ± SD versus average values of relative light units to growth RLU/A550 ± SD). The proteomic analysis performed in our previous experiment revealed the presence of several substances with documented antibacterial, antibiofilm and immunomodulatory properties (namely, apolipoprotein B and D; amyloid-β peptide; cathepsin B; protein S100-A4, galectin 3, CLEC3A, granulin, transferrin). This study highlights that cBM MSC CM may represent an important new approach to managing biofilm-associated and QS signal molecule-dependent bacterial infections. To the best of our knowledge, there is no previous documentation of canine BM MSC CM associated with in vitro antibiofilm and anti-QS activity.

scholarly journals Candida albicans Impacts Staphylococcus aureus Alpha-Toxin Production via Extracellular Alkalinization

mSphere ◽  
2019 ◽  
Vol 4 (6) ◽  
Author(s):  
Olivia A. Todd ◽  
Mairi C. Noverr ◽  
Brian M. Peters
Keyword(s):  
Staphylococcus Aureus ◽  
Candida Albicans ◽  
Quorum Sensing ◽  
Bacterial Pathogen ◽  
Toxin Production ◽  
Extracellular Ph ◽  
Morbidity And Mortality ◽  
Alpha Toxin ◽  
Content Type

ABSTRACT Candida albicans and Staphylococcus aureus are common causes of nosocomial infections with severe morbidity and mortality. Murine polymicrobial intra-abdominal infection (IAI) with C. albicans and S. aureus results in acute mortality dependent on the secreted cytolytic effector alpha-toxin. Here, we confirmed that alpha-toxin is elevated during polymicrobial growth compared to monomicrobial growth in vitro. Therefore, this study sought to unravel the mechanism by which C. albicans drives enhanced staphylococcal alpha-toxin production. Using a combination of functional and genetic approaches, we determined that an intact agr quorum sensing regulon is necessary for enhanced alpha-toxin production during coculture and that a secreted candidal factor likely is not implicated in elevating agr activation. As the agr system is pH sensitive, we observed that C. albicans raises the pH during polymicrobial growth and that this correlates with increased agr activity and alpha-toxin production. Modulation of the pH could predictably attenuate or activate agr activity during coculture. By using a C. albicans mutant deficient in alkalinization (stp2Δ/Δ), we confirmed that modulation of the extracellular pH by C. albicans can drive agr expression and toxin production. Additionally, the use of various Candida species (C. glabrata, C. dubliniensis, C. tropicalis, C. parapsilosis, and C. krusei) demonstrated that those capable of raising the extracellular pH correlated with elevated agr activity and alpha-toxin production during coculture. Overall, we demonstrate that alkalinization of the extracellular pH by the Candida species leads to sustained activation of the staphylococcal agr system. IMPORTANCE Candida albicans and Staphylococcus aureus are commonly coisolated from central venous catheters and deep-seated infections, including intra-abdominal sepsis. Thus, they represent a significant cause of nosocomial morbidity and mortality. Yet how these organisms behave in the context of polymicrobial growth remains poorly understood. In this work, we set out to determine the mechanism by which activation of the staphylococcal agr quorum sensing system and production of its major virulence effector alpha-toxin is enhanced during coculture with C. albicans. Surprisingly, we likely ruled out that a secreted candidal factor drives this process. Instead, we demonstrated that alkalinization of the extracellular milieu by C. albicans and other Candida species correlated with elevated agr activity. Thus, we propose a mechanism where modulation of the extracellular pH by fungal opportunists can indirectly alter virulence of a bacterial pathogen. Uncovering molecular events that drive interkingdom pathogenicity mechanisms may enhance surveillance and treatment for devastating polymicrobial infections.

scholarly journals The Streptococcal Protease SpeB Antagonizes the Biofilms of the Human Pathogen Staphylococcus aureus USA300 through Cleavage of the Staphylococcal SdrC Protein

2020 ◽  
Vol 202 (11) ◽  
Author(s):  
Katelyn E. Carothers ◽  
Zhong Liang ◽  
Jeffrey Mayfield ◽  
Deborah L. Donahue ◽  
Mijoon Lee ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Proteolytic Activity ◽  
Streptococcus Pyogenes ◽  
Human Pathogen ◽  
Content Type ◽  
Functional Studies ◽  
Human Proteins ◽  
Group A ◽  
Biofilm Disruption

ABSTRACT Streptococcus pyogenes, or group A Streptococcus (GAS), is both a pathogen and an asymptomatic colonizer of human hosts and produces a large number of surface-expressed and secreted factors that contribute to a variety of infection outcomes. The GAS-secreted cysteine protease SpeB has been well studied for its effects on the human host; however, despite its broad proteolytic activity, studies on how this factor is utilized in polymicrobial environments are lacking. Here, we utilized various forms of SpeB protease to evaluate its antimicrobial and antibiofilm properties against the clinically important human colonizer Staphylococcus aureus, which occupies niches similar to those of GAS. For our investigation, we used a skin-tropic GAS strain, AP53CovS+, and its isogenic ΔspeB mutant to compare the production and activity of native SpeB protease. We also generated active and inactive forms of recombinant purified SpeB for functional studies. We demonstrate that SpeB exhibits potent biofilm disruption activity at multiple stages of S. aureus biofilm formation. We hypothesized that the surface-expressed adhesin SdrC in S. aureus was cleaved by SpeB, which contributed to the observed biofilm disruption. Indeed, we found that SpeB cleaved recombinant SdrC in vitro and in the context of the full S. aureus biofilm. Our results suggest an understudied role for the broadly proteolytic SpeB as an important factor for GAS colonization and competition with other microorganisms in its niche. IMPORTANCE Streptococcus pyogenes (GAS) causes a range of diseases in humans, ranging from mild to severe, and produces many virulence factors in order to be a successful pathogen. One factor produced by many GAS strains is the protease SpeB, which has been studied for its ability to cleave and degrade human proteins, an important factor in GAS pathogenesis. An understudied aspect of SpeB is the manner in which its broad proteolytic activity affects other microorganisms that co-occupy niches similar to that of GAS. The significance of the research reported herein is the demonstration that SpeB can degrade the biofilms of the human pathogen Staphylococcus aureus, which has important implications for how SpeB may be utilized by GAS to successfully compete in a polymicrobial environment.

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 Characterization of structural elements in native autoinducing peptides and non-native analogues that permit the differential modulation of AgrC-type quorum sensing receptors in Staphylococcus aureus

2016 ◽  
Vol 14 (1) ◽  
pp. 113-121 ◽  
Author(s):  
Yftah Tal-Gan ◽  
Monika Ivancic ◽  
Gabriel Cornilescu ◽  
Helen E. Blackwell
Keyword(s):  
Staphylococcus Aureus ◽  
Quorum Sensing ◽  
Structural Elements ◽  
Differential Modulation ◽  
Autoinducing Peptides

Structural analyses of autoinducing peptides and analogues thereof reveal motifs critical for modulation of quorum sensing receptors inS. aureus.

scholarly journals Prevalence of Agr phase variants in Staphylococcus aureus

2020 ◽  
Vol 2 (7A) ◽  
Author(s):  
Vishal Gor ◽  
Mitsuaki Hoshi ◽  
Aya Takemura ◽  
Masato Higashide ◽  
Veronica Romero ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Quorum Sensing ◽  
Virulence Factors ◽  
Human Pathogen ◽  
Accessory Gene ◽  
Accessory Gene Regulator ◽  
Inversion Event ◽  
First Time ◽  
Phase Variants ◽  
Phenotypic Reversion

Staphylococcus aureus is an important human pathogen whose success is largely attributed to its vast arsenal of virulence factors that facilitate its invasion into, and survival within, the human host. The expression of these virulence factors is controlled by the quorum sensing Accessory Gene Regulator (Agr) system. However, a large proportion of clinical S. aureus isolates are consistently found to have a mutationally inactivated Agr system. These mutants have a survival advantage in the host but are considered irreversible mutants. Here we show, for the first time, that a fraction of Agr-negative mutants can revert their Agr activity. By serially passaging Agr negative strains and screening for phenotypic reversion of haemolysis and subsequent sequencing, we identified two mutational events responsible for reversion: a genetic duplication plus inversion event and a poly(A) tract alteration. Additionally, we demonstrate that one clinical Agr-negative MRSA isolate could reproducibly generate Agr-revertant colonies with a poly(A) tract genetic mechanism. We also show that these revertants activate their Agr system upon phagocytosis. To assess the significance of our findings we screened a series of primary clinical isolates, which had undergone minimal handling post-isolation, and successfully identified a fraction which were Agr phase variants. Taken together, we propose a model where some Agr-negative S. aureus strains are phase variants who can revert their Agr activity and may act as a cryptic insurance strategy against host-mediated stress.

scholarly journals Key driving forces in the biosynthesis of autoinducing peptides required for staphylococcal virulence

2015 ◽  
Vol 112 (34) ◽  
pp. 10679-10684 ◽  
Author(s):  
Boyuan Wang ◽  
Aishan Zhao ◽  
Richard P. Novick ◽  
Tom W. Muir
Keyword(s):  
Quorum Sensing ◽  
Driving Forces ◽  
Side Chain ◽  
Accessory Gene ◽  
Accessory Gene Regulator ◽  
C Terminus ◽  
In Vitro Reconstitution ◽  
Intimate Link ◽  
Autoinducing Peptides

Staphylococci produce autoinducing peptides (AIPs) as quorum-sensing signals that regulate virulence. These AIPs feature a thiolactone macrocycle that connects the peptide C terminus to the side chain of an internal cysteine. AIPs are processed from ribosomally synthesized precursors [accessory gene regulator D (AgrD)] through two proteolytic events. Formation of the thiolactone is coupled to the first of these and involves the activity of the integral membrane protease AgrB. This step is expected to be thermodynamically unfavorable, and therefore, it is unclear how AIP-producing bacteria produce sufficient amounts of the thiolactone-containing intermediate to drive quorum sensing. Herein, we present the in vitro reconstitution of the AgrB-dependent proteolysis of an AgrD precursor from Staphylococcus aureus. Our data show that efficient thiolactone production is driven by two unanticipated features of the system: (i) membrane association of the thiolactone-containing intermediate, which stabilizes the macrocycle, and (ii) rapid degradation of the C-terminal proteolysis fragment AgrDC, which affects the reaction equilibrium position. Cell-based studies confirm the intimate link between AIP production and intracellular AgrDC levels. Thus, our studies explain the chemical principles that drive AIP production, including uncovering a hitherto unknown link between quorum sensing and peptide turnover.

scholarly journals Synthesis and biological activity of methylated derivatives of the Pseudomonas metabolites HHQ, HQNO and PQS

2019 ◽  
Vol 15 ◽  
pp. 187-193 ◽  
Author(s):  
Sven Thierbach ◽  
Max Wienhold ◽  
Susanne Fetzner ◽  
Ulrich Hennecke
Keyword(s):  
Cystic Fibrosis ◽  
Staphylococcus Aureus ◽  
Pseudomonas Aeruginosa ◽  
Biological Activity ◽  
Quorum Sensing ◽  
Cellular Respiration ◽  
Metal Exchange ◽  
Human Pathogen ◽  
Naturally Occurring ◽  
Derivatives Of

Selectively methylated analogues of naturally occurring 2-heptyl-4(1H)-quinolones, which are alkaloids common within the Rutaceae family and moreover are associated with quorum sensing and virulence of the human pathogen Pseudomonas aeruginosa, have been prepared. While the synthesis by direct methylation was successful for 3-unsubstituted 2-heptyl-4(1H)-quinolones, methylated derivatives of the Pseudomonas quinolone signal (PQS) were synthesized from 3-iodinated quinolones by methylation and iodine–metal exchange/oxidation. The two N- and O-methylated derivatives of the PQS showed strong quorum sensing activity comparable to that of PQS itself. Staphylococcus aureus, another pathogenic bacterium often co-occurring with P. aeruginosa especially in the lung of cystic fibrosis patients, was inhibited in planktonic growth and cellular respiration by the 4-O-methylated derivatives of HQNO and HHQ, respectively.

scholarly journals Biofilm-Associated Agr and Sar Quorum Sensing Systems of Methicillin-Resistant Staphylococcus Aureus are Inhibited by Fruit Extracts of Illicium Verum

2019 ◽  
Author(s):  
Pitchaipillai Sankar Ganesh ◽  
Veena Krishnamurthy ◽  
Koneti ISwamy ◽  
Suvaiyarasan Suvaithenamudhan ◽  
Murugesan Amuthan ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Molecular Docking ◽  
Quorum Sensing ◽  
Light Microscopy ◽  
Major Constituent ◽  
Antibiofilm Activity ◽  
Systems Methodology ◽  
Biofilm Inhibition ◽  
Swarming Motility

Background: Staphylococcus aureus (S. aureus) is an opportunistic pathogen and a predominant cause of life-threatening nosocomial infections. Drug resistance in S. aureus is attributed to production of biofilm, which is controlled largely by bacterial quorum sensing (QS) systems. Methodology: In vitro analysis of biofilm inhibition assay was performed using crystal violet staining assay, swarming motility, light microscopy and growth curve analyses. Identification of the major constituents of I. verum fruit extract was performed by GC-MS. Ligand-protein interaction was analyzed by molecular docking investigations. Results: The methanol extract of I. verum inhibited the growth of MRSA at the concentration of 4.8 mg/ml. At the sub-inhibitory concentration (2.4mg/ml), the extract showed significant reduction in biofilmogenesis. Light microscopy analysis confirmed the antibiofilm activity as well as the efficacy in disturbing biofilm architecture. A reduced swarming motility was observed at the lowest concentration of 2.4mg/ml. GC-MS analysis revealed anethol (AL) as the major constituent. The molecular docking analysis attributes the antibiofilm activity to an active ligand AL, which strongly interacted with the active site residues of AgrA and SarA proteins of S. aureus. Conclusion: We report the activities of I. verum to be immensely interfering with QS system and biofilm formation in MRSA.

scholarly journals ω-Hydroxyemodin Limits Staphylococcus aureus Quorum Sensing-Mediated Pathogenesis and Inflammation

2015 ◽  
Vol 59 (4) ◽  
pp. 2223-2235 ◽  
Author(s):  
Seth M. Daly ◽  
Bradley O. Elmore ◽  
Jeffrey S. Kavanaugh ◽  
Kathleen D. Triplett ◽  
Mario Figueroa ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Quorum Sensing ◽  
Immune Cell ◽  
Solid Phase ◽  
Response Regulator ◽  
Dependent Manner ◽  
Accessory Gene ◽  
Antibiotic Resistant ◽  
Content Type

ABSTRACTAntibiotic-resistant pathogens are a global health threat. Small molecules that inhibit bacterial virulence have been suggested as alternatives or adjuncts to conventional antibiotics, as they may limit pathogenesis and increase bacterial susceptibility to host killing.Staphylococcus aureusis a major cause of invasive skin and soft tissue infections (SSTIs) in both the hospital and community settings, and it is also becoming increasingly antibiotic resistant. Quorum sensing (QS) mediated by the accessory gene regulator (agr) controls virulence factor production essential for causing SSTIs. We recently identified ω-hydroxyemodin (OHM), a polyhydroxyanthraquinone isolated from solid-phase cultures ofPenicillium restrictum, as a suppressor of QS and a compound sought for the further characterization of the mechanism of action. At concentrations that are nontoxic to eukaryotic cells and subinhibitory to bacterial growth, OHM preventedagrsignaling by all fourS. aureus agralleles. OHM inhibited QS by direct binding to AgrA, the response regulator encoded by theagroperon, preventing the interaction of AgrA with theagrP2 promoter. Importantly, OHM was efficacious in a mouse model ofS. aureusSSTI. Decreased dermonecrosis with OHM treatment was associated with enhanced bacterial clearance and reductions in inflammatory cytokine transcription and expression at the site of infection. Furthermore, OHM treatment enhanced the immune cell killing ofS. aureusin vitroin anagr-dependent manner. These data suggest that bacterial disarmament through the suppression ofS. aureusQS may bolster the host innate immune response and limit inflammation.

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