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 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 A Novel Infection Protocol in Zebrafish Embryo to Assess Pseudomonas aeruginosa Virulence and Validate Efficacy of a Quorum Sensing Inhibitor In Vivo

Pathogens ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 401
Author(s):  
Pauline Nogaret ◽  
Fatima El El Garah ◽  
Anne-Béatrice Blanc-Potard
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Animal Model ◽  
Quorum Sensing ◽  
Drug Testing ◽  
Drug Efficacy ◽  
Embryo Viability ◽  
Immersion Method ◽  
Opportunistic Human Pathogen

The opportunistic human pathogen Pseudomonas aeruginosa is responsible for a variety of acute infections and is a major cause of mortality in chronically infected cystic fibrosis patients. Due to increased resistance to antibiotics, new therapeutic strategies against P. aeruginosa are urgently needed. In this context, we aimed to develop a simple vertebrate animal model to rapidly assess in vivo drug efficacy against P. aeruginosa. Zebrafish are increasingly considered for modeling human infections caused by bacterial pathogens, which are commonly microinjected in embryos. In the present study, we established a novel protocol for zebrafish infection by P. aeruginosa based on bath immersion in 96-well plates of tail-injured embryos. The immersion method, followed by a 48-hour survey of embryo viability, was first validated to assess the virulence of P. aeruginosa wild-type PAO1 and a known attenuated mutant. We then validated its relevance for antipseudomonal drug testing by first using a clinically used antibiotic, ciprofloxacin. Secondly, we used a novel quorum sensing (QS) inhibitory molecule, N-(2-pyrimidyl)butanamide (C11), the activity of which had been validated in vitro but not previously tested in any animal model. A significant protective effect of C11 was observed on infected embryos, supporting the ability of C11 to attenuate in vivo P. aeruginosa pathogenicity. In conclusion, we present here a new and reliable method to compare the virulence of P. aeruginosa strains in vivo and to rapidly assess the efficacy of clinically relevant drugs against P. aeruginosa, including new antivirulence compounds.

scholarly journals Staphylococcus aureus Serves as an Iron Source for Pseudomonas aeruginosa during In Vivo Coculture

2005 ◽  
Vol 187 (2) ◽  
pp. 554-566 ◽  
Author(s):  
Lauren M. Mashburn ◽  
Amy M. Jett ◽  
Darrin R. Akins ◽  
Marvin Whiteley
Keyword(s):  
Staphylococcus Aureus ◽  
Pseudomonas Aeruginosa ◽  
Pathogenic Bacteria ◽  
Iron Acquisition ◽  
Low Oxygen ◽  
Dialysis Membrane ◽  
Opportunistic Human Pathogen ◽  
The Impact

ABSTRACT Pseudomonas aeruginosa is a gram-negative opportunistic human pathogen often infecting the lungs of individuals with the heritable disease cystic fibrosis and the peritoneum of individuals undergoing continuous ambulatory peritoneal dialysis. Often these infections are not caused by colonization with P. aeruginosa alone but instead by a consortium of pathogenic bacteria. Little is known about growth and persistence of P. aeruginosa in vivo, and less is known about the impact of coinfecting bacteria on P. aeruginosa pathogenesis and physiology. In this study, a rat dialysis membrane peritoneal model was used to evaluate the in vivo transcriptome of P. aeruginosa in monoculture and in coculture with Staphylococcus aureus. Monoculture results indicate that approximately 5% of all P. aeruginosa genes are differentially regulated during growth in vivo compared to in vitro controls. Included in this analysis are genes important for iron acquisition and growth in low-oxygen environments. The presence of S. aureus caused decreased transcription of P. aeruginosa iron-regulated genes during in vivo coculture, indicating that the presence of S. aureus increases usable iron for P. aeruginosa in this environment. We propose a model where P. aeruginosa lyses S. aureus and uses released iron for growth in low-iron environments.

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 Pseudomonas Quorum-Sensing Regulator RsaL Belongs to the Tetrahelical Superclass of H-T-H Proteins

2006 ◽  
Vol 189 (5) ◽  
pp. 1922-1930 ◽  
Author(s):  
Giordano Rampioni ◽  
Fabio Polticelli ◽  
Iris Bertani ◽  
Karima Righetti ◽  
Vittorio Venturi ◽  
...  
Keyword(s):  
Dna Binding ◽  
Quorum Sensing ◽  
In Silico ◽  
Mobility Shift ◽  
Specific Domain ◽  
Signal Molecule ◽  
In Silico Model ◽  
Opportunistic Human Pathogen

ABSTRACT In the opportunistic human pathogen Pseudomonas aeruginosa, quorum sensing (QS) is crucial for virulence. The RsaL protein directly represses the transcription of lasI, the synthase gene of the main QS signal molecule. On the basis of sequence homology, RsaL cannot be predicted to belong to any class of characterized DNA-binding proteins. In this study, an in silico model of the RsaL structure was inferred showing that RsaL belongs to the tetrahelical superclass of helix-turn-helix proteins. The overall structure of RsaL is very similar to the N-terminal domain of the lambda cI repressor and to the POU-specific domain of the mammalian transcription factor Oct-1 (Oct-1 POUs). Moreover, residues of Oct-1 POUs important for structural stability and/or DNA binding are conserved in the same positions in RsaL and in its homologs found in GenBank. These residues were independently replaced with Ala, and the activities of the mutated variants of RsaL were compared to that of the wild-type counterpart in vivo by complementation assays and in vitro by electrophoretic mobility shift assays. The results validated the RsaL in silico model and showed that residues Arg 20, Gln 38, Ser 42, Arg 43, and Glu 45 are important for RsaL function. Our data indicate that RsaL could be the founding member of a new protein family within the tetrahelical superclass of helix-turn-helix proteins. Finally, the minimum DNA sequence required for RsaL binding on the lasI promoter was determined, and our data support the hypothesis that RsaL binds DNA as a dimer.

Pseudomonas aeruginosa tolerance to tobramycin, hydrogen peroxide and polymorphonuclear leukocytes is quorum-sensing dependent

Microbiology ◽  
2005 ◽  
Vol 151 (2) ◽  
pp. 373-383 ◽  
Author(s):  
Thomas Bjarnsholt ◽  
Peter Østrup Jensen ◽  
Mette Burmølle ◽  
Morten Hentzer ◽  
Janus A. J. Haagensen ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Polymorphonuclear Leukocytes ◽  
Present Report ◽  
Cell Communication ◽  
A Cell ◽  
Opportunistic Human Pathogen ◽  
Conventional Antibiotics

The opportunistic human pathogen Pseudomonas aeruginosa is the predominant micro-organism of chronic lung infections in cystic fibrosis (CF) patients. P. aeruginosa colonizes the CF lungs by forming biofilm structures in the alveoli. In the biofilm mode of growth the bacteria are highly tolerant to otherwise lethal doses of antibiotics and are protected from bactericidal activity of polymorphonuclear leukocytes (PMNs). P. aeruginosa controls the expression of many of its virulence factors by means of a cell–cell communication system termed quorum sensing (QS). In the present report it is demonstrated that biofilm bacteria in which QS is blocked either by mutation or by administration of QS inhibitory drugs are sensitive to treatment with tobramycin and H2O2, and are readily phagocytosed by PMNs, in contrast to bacteria with functional QS systems. In contrast to the wild-type, QS-deficient biofilms led to an immediate respiratory-burst activation of the PMNs in vitro. In vivo QS-deficient mutants provoked a higher degree of inflammation. It is suggested that quorum signals and QS-inhibitory drugs play direct and opposite roles in this process. Consequently, the faster and highly efficient clearance of QS-deficient bacteria in vivo is probably a two-sided phenomenon: down regulation of virulence and activation of the innate immune system. These data also suggest that a combination of the action of PMNs and QS inhibitors along with conventional antibiotics would eliminate the biofilm-forming bacteria before a chronic infection is established.

scholarly journals Concurrent Local Delivery of Diflunisal Limits Bone Destruction but Fails To Improve Systemic Vancomycin Efficacy during Staphylococcus aureus Osteomyelitis

2020 ◽  
Vol 64 (7) ◽  
Author(s):  
Thomas J. Spoonmore ◽  
Caleb A. Ford ◽  
Jacob M. Curry ◽  
Scott A. Guelcher ◽  
James E. Cassat
Keyword(s):  
Staphylococcus Aureus ◽  
Adjunctive Therapy ◽  
Bone Destruction ◽  
Standard Of Care ◽  
Quorum Quenching ◽  
Toxin Production ◽  
Local Drug Delivery ◽  
Content Type

ABSTRACT Staphylococcus aureus osteomyelitis is a debilitating infection of bone. Treatment of osteomyelitis is impaired by the propensity of invading bacteria to induce pathological bone remodeling that may limit antibiotic penetration to the infectious focus. The nonsteroidal anti-inflammatory drug diflunisal was previously identified as an osteoprotective adjunctive therapy for osteomyelitis, based on the ability of this compound to inhibit S. aureus quorum sensing and subsequent quorum-dependent toxin production. When delivered locally during experimental osteomyelitis, diflunisal significantly limits bone destruction without affecting bacterial burdens. However, because diflunisal’s “quorum-quenching” activity could theoretically increase antibiotic recalcitrance, it is critically important to evaluate this adjunctive therapy in the context of standard-of-care antibiotics. The objective of this study is to evaluate the efficacy of vancomycin to treat osteomyelitis during local diflunisal treatment. We first determined that systemic vancomycin effectively reduces bacterial burdens in a murine model of osteomyelitis and identified a dosing regimen that decreases bacterial burdens without eradicating infection. Using this dosing scheme, we found that vancomycin activity is unaffected by the presence of diflunisal in vitro and in vivo. Similarly, locally delivered diflunisal still potently inhibits osteoblast cytotoxicity in vitro and bone destruction in vivo in the presence of subtherapeutic vancomycin. However, we also found that the resorbable polyester urethane (PUR) foams used to deliver diflunisal serve as a nidus for infection. Taken together, these data demonstrate that diflunisal does not significantly impact standard-of-care antibiotic therapy for S. aureus osteomyelitis, but they also highlight potential pitfalls encountered with local drug delivery.

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 Staquorsin: A Novel Staphylococcus aureus Agr-Mediated Quorum Sensing Inhibitor Impairing Virulence in vivo Without Notable Resistance Development

2021 ◽  
Vol 12 ◽  
Author(s):  
Norhan H. Mahdally ◽  
Riham F. George ◽  
Mona T. Kashef ◽  
Medhat Al-Ghobashy ◽  
Fathia E. Murad ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Quorum Sensing ◽  
Virulence Factors ◽  
Lipase Production ◽  
Health Concern ◽  
Drug Candidate ◽  
Microbial Infection ◽  
Development Of Resistance

The emergence of microbial resistance to the available antibiotics is a major public health concern, especially with the limited rate of developing new antibiotics. The utilization of anti-virulence agents is a non-conventional approach that can be used to combat microbial infection. In Staphylococcus aureus, many virulence factors are regulated by the Agr-mediated quorum sensing (QS). We developed a chemical compound that acts a potential Agr-inhibitor without reducing bacterial viability. The compound was designated staquorsin for Staphylococcus aureus QS inhibitor. In silico analyses confirmed the binding of staquorsin to the AgrA active site with an absolute binding score comparable to savirin, a previously described AgrA inhibitor. However, staquorsin turned out to be superior over savarin in not affecting the S. aureus viability in concentrations up to 600 μM. On the other hand, savirin inhibited S. aureus growth in concentrations as low as 25 μM. Moreover, staquorsin proved to be a potent inhibitor of the Agr system by inhibiting hemolysins, lipase production, and affecting biofilms formation and detachment. On the molecular level it significantly inhibited the effector transcript RNA III. In vivo testing, using the murine skin abscess model, confirmed the ability of staquorsin to modulate S. aureus virulence by effectively controlling the infection. Twenty passages of S. aureus in the presence of 40 μM staquorsin have not resulted in loss of activity as evidenced by maintaining its ability to reduce hemolysin production and RNA III transcript levels. In conclusion, we hereby describe a novel anti-virulence compound inhibiting the S. aureus Agr-system and its associated virulence factors. It is active both in vitro and in vivo, and its frequent use does not lead to the development of resistance. These findings model staquorsin as a promising drug candidate to join the fierce battle against the formidable pathogen S. aureus.

scholarly journals Modulation of Staphylococcus aureus Response to Antimicrobials by the Candida albicans Quorum Sensing Molecule Farnesol

2017 ◽  
Vol 61 (12) ◽  
Author(s):  
Eric F. Kong ◽  
Christina Tsui ◽  
Sona Kucharíková ◽  
Patrick Van Dijck ◽  
Mary Ann Jabra-Rizk
Keyword(s):  
Staphylococcus Aureus ◽  
Candida Albicans ◽  
Quorum Sensing ◽  
Physiological Role ◽  
Efflux Pumps ◽  
Content Type ◽  
The Impact ◽  
Quorum Sensing Molecule

ABSTRACT In microbial biofilms, microorganisms utilize secreted signaling chemical molecules to coordinate their collective behavior. Farnesol is a quorum sensing molecule secreted by the fungal species Candida albicans and shown to play a central physiological role during fungal biofilm growth. Our pervious in vitro and in vivo studies characterized an intricate interaction between C. albicans and the bacterial pathogen Staphylococcus aureus, as these species coexist in biofilm. In this study, we aimed to investigate the impact of farnesol on S. aureus survival, biofilm formation, and response to antimicrobials. The results demonstrated that in the presence of exogenously supplemented farnesol or farnesol secreted by C. albicans in biofilm, S. aureus exhibited significantly enhanced tolerance to antimicrobials. By using gene expression studies, S. aureus mutant strains, and chemical inhibitors, the mechanism for the enhanced tolerance was attributed to upregulation of drug efflux pumps. Importantly, we showed that sequential exposure of S. aureus to farnesol generated a phenotype of high resistance to antimicrobials. Based on the presence of intracellular reactive oxygen species upon farnesol exposure, we hypothesize that antimicrobial tolerance in S. aureus may be mediated by farnesol-induced oxidative stress triggering the upregulation of efflux pumps, as part of a general stress response system. Hence, in mixed biofilms, C. albicans may influence the pathogenicity of S. aureus through acquisition of a drug-tolerant phenotype, with important therapeutic implications. Understanding interspecies signaling in polymicrobial biofilms and the specific drug resistance responses to secreted molecules may lead to the identification of novel targets for drug development.

Sign in / Sign up

Export Citation Format

Share Document