Many bacterial histidine kinases work in two-component systems that combine into larger multi-kinase networks. NahK is one of the kinases in the GacS Multi-Kinase Network (MKN), which is the MKN that controls biofilm regulation in the opportunistic pathogen
- NSF-PAR ID:
- 10294948
- Date Published:
- Journal Name:
- Frontiers in Microbiology
- Volume:
- 12
- ISSN:
- 1664-302X
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Bondy-Denomy, Joseph (Ed.)more » « less
ABSTRACT Pseudomonas aeruginosa . This network has also been associated with regulating many virulence factorsP. aeruginosa secretes to cause disease. However, the individual role of each kinase is unknown. In this study, we identify NahK as a novel regulator of the phenazine pyocyanin (PYO). Deletion ofnahK leads to a fourfold increase in PYO production, almost exclusively through upregulation of phenazine operon two (phz2 ). We determined that this upregulation is due to mis-regulation of allP. aeruginosa quorum-sensing (QS) systems, with a large upregulation of thePseudomonas quinolone signal system and a decrease in production of the acyl-homoserine lactone-producing system,las . In addition, we see differences in expression of quorum-sensing inhibitor proteins that align with these changes. Together, these data contribute to understanding how the GacS MKN modulates QS and virulence and suggest a mechanism for cell density-independent regulation of quorum sensing.IMPORTANCE Pseudomonas aeruginosa is a Gram-negative bacterium that establishes biofilms as part of its pathogenicity.P. aeruginosa infections are associated with nosocomial infections. As the prevalence of multi-drug-resistantP. aeruginosa increases, it is essential to understand underlying virulence molecular mechanisms. Histidine kinase NahK is one of several kinases inP. aeruginosa implicated in biofilm formation and dispersal. Previous work has shown that the nitric oxide sensor, NosP, triggers biofilm dispersal by inhibiting NahK. The data presented here demonstrate that NahK plays additional important roles in theP. aeruginosa lifestyle, including regulating bacterial communication mechanisms such as quorum sensing. These effects have larger implications in infection as they affect toxin production and virulence. -
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Abstract Objectives To assess the ability of oxyclozanide to enhance tobramycin killing of Pseudomonas aeruginosa biofilms and elucidate its mechanism of action.
Methods Twenty-four hour biofilms formed by the P. aeruginosa strain PAO1 and cystic fibrosis (CF) isolates were tested for susceptibility to oxyclozanide and tobramycin killing using BacTiter-Glo™ and cfu. Biofilm dispersal was measured using crystal violet staining. Membrane potential and permeabilization were quantified using DiOC2(3) and TO-PRO-3, respectively.
Results Here we show that the ionophore anthelmintic oxyclozanide, combined with tobramycin, significantly increased killing of P. aeruginosa biofilms over each treatment alone. This combination also significantly accelerated the killing of cells within biofilms and stationary phase cultures and it was effective against 4/6 CF clinical isolates tested, including a tobramycin-resistant strain. Oxyclozanide enhanced the ability of additional aminoglycosides and tetracycline to kill P. aeruginosa biofilms. Finally, oxyclozanide permeabilized cells within the biofilm, reduced the membrane potential and increased tobramycin accumulation within cells of mature P. aeruginosa biofilms.
Conclusions Oxyclozanide enhances aminoglycoside and tetracycline activity against P. aeruginosa biofilms by reducing membrane potential, permeabilizing cells and enhancing tobramycin accumulation within biofilms. We propose that oxyclozanide counteracts the adaptive resistance response of P. aeruginosa to aminoglycosides, increasing both their maximum activity and rate of killing. As oxyclozanide is widely used in veterinary medicine for the treatment of parasitic worm infections, this combination could offer a new approach for the treatment of biofilm-based P. aeruginosa infections, repurposing oxyclozanide as an anti-biofilm agent.
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Bondy-Denomy, Joseph (Ed.)ABSTRACT Chemical communication between bacteria and between bacteria and the bacteriophage (phage) viruses that prey on them can shape the outcomes of phage-bacterial encounters. Quorum sensing (QS) is a bacterial cell-to-cell communication process that promotes collective undertaking of group behaviors. QS relies on the production, release, accumulation, and detection of signal molecules called autoinducers. Phages can exploit QS-mediated communication to manipulate their hosts and maximize their own survival. In the opportunistic pathogen Pseudomonas aeruginosa , the LasI/R QS system induces the RhlI/R QS system, and in opposing manners, these two systems control the QS system that relies on the autoinducer called PQS. A P. aeruginosa Δ lasI mutant is impaired in PQS synthesis, leading to accumulation of the precursor molecule HHQ, and HHQ suppresses growth of the P. aeruginosa Δ lasI strain. We show that, in response to a phage infection, the P. aeruginosa Δ lasI mutant reactivates QS, which, in turn, restores pqsH expression, enabling conversion of HHQ into PQS. Moreover, downstream QS target genes encoding virulence factors are induced. Additionally, phage-infected P. aeruginosa Δ lasI cells transiently exhibit superior growth compared to uninfected cells. IMPORTANCE Clinical isolates of P. aeruginosa frequently harbor mutations in particular QS genes. Here, we show that infection by select temperate phages restores QS, a cell-to-cell communication mechanism in a P. aeruginosa QS mutant. Restoration of QS increases expression of genes encoding virulence factors. Thus, phage infection of select P. aeruginosa strains may increase bacterial pathogenicity, underscoring the importance of characterizing phage-host interactions in the context of bacterial mutants that are relevant in clinical settings.more » « less
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Kulig, Waldemar (Ed.)Quorum sensing (QS) is a bacterial communication process mediated by both native and non-native small-molecule quorum sensing modulators (QSMs), many of which have been synthesized to disrupt QS pathways. While structure-activity relationships have been developed to relate QSM structure to the activation or inhibition of QS receptors, less is known about the transport mechanisms that enable QSMs to cross the lipid membrane and access intracellular receptors. In this study, we used atomistic MD simulations and an implicit solvent model, called COSMOmic, to analyze the partitioning and translocation of QSMs across lipid bilayers. We performed umbrella sampling at atomistic resolution to calculate partitioning and translocation free energies for a set of naturally occurring QSMs, then used COSMOmic to screen the water-membrane partition and translocation free energies for 50 native and non-native QSMs that target LasR, one of the LuxR family of quorum-sensing receptors. This screening procedure revealed the influence of systematic changes to head and tail group structures on membrane partitioning and translocation free energies at a significantly reduced computational cost compared to atomistic MD simulations. Comparisons with previously determined QSM activities suggest that QSMs that are least likely to partition into the bilayer are also less active. This work thus demonstrates the ability of the computational protocol to interrogate QSM-bilayer interactions which may help guide the design of new QSMs with engineered membrane interactions.more » « less
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Background Chronic rhinosinusitis (CRS) is a chronic inflammatory disease characterized by persistent inflammation and bacterial infection. Ciprofloxacin and azithromycin are commonly prescribed antibiotics for CRS, but the ability to provide targeted release in the sinuses could mitigate side effects and improve drug concentrations at the infected site. This study was aimed to evaluate the efficacy of the novel ciprofloxacin‐azithromycin sinus stent (CASS) in vitro.
Methods The CASS was created by coating ciprofloxacin (hydrophilic, inner layer) and azithromycin (hydrophobic, outer layer) onto a biodegradable poly‐
l ‐lactic acid (PLLA) stent. In‐vitro evaluation included: (1) assessment of drug‐coating stability within the stent using scanning electron microscopy (SEM); (2) determination of ciprofloxacin and azithromycin release kinetics; and (3) assessment of anti‐biofilm activities againstPseudomonas aeruginosa .Results The ciprofloxacin nanoparticle suspension in the inner layer was confirmed by zeta potential. Both ciprofloxacin (60 µg) and azithromycin (3 mg) were uniformly coated on the surface of the PLLA stents. The CASS showed ciprofloxacin/azithromycin sustained release patterns, with 80.55 ± 11.61% of ciprofloxacin and 93.85 ± 6.9% of azithromycin released by 28 days. The CASS also significantly reduced
P aeruginosa biofilm mass compared with bare stents and controls (relative optical density units at 590‐nm optical density: CASS, 0.037 ± 0.006; bare stent, 0.911 ± 0.015; control, 1.000 ± 0.000;p < 0.001; n = 3).Conclusion The CASS maintains a uniform coating and sustained delivery of ciprofloxacin and azithromycin, providing anti‐biofilm activities against
P aeruginosa . Further studies evaluating the efficacy of CASS in a preclinical model are planned.
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3389/fmicb.2021.598498
