Competition Studies Confirm Two Major Barriers That Can Preclude the Spread of Resistance to Quorum-Sensing Inhibitors in Bacteria

The growing threat of antibiotic resistance necessitates the development of novel antimicrobial therapies. Antivirulence agents that target group-beneficial traits in microorganisms (i.e., phenotypes that help the cells surrounding the producer cell instead of selfishly benefiting only the producer cell) represent a new antimicrobial approach that may be robust against the spread of resistant mutants. One prominent group-beneficial antivirulence target in bacteria is quorum sensing (QS). While scientists are producing new QS inhibitors (QSIs) at an increasing pace for use as research tools and potential therapeutic leads, substantial work remains in empirically demonstrating a robustness against resistance. Herein we report the results of <i>in vitro</i> competition studies in <i>Pseudomonas aeruginosa</i> that explicitly confirm that <i>two separate barriers</i> can impede the spread of resistance to QSIs: (1) insufficient native QS signal levels prevent rare QSI-resistant bacteria from expressing their QS regulon, and (2) group-beneficial QS-regulated phenotypes produced by resistant bacteria are susceptible to cheating by QSI-sensitive neighbors, even when grown on a solid substrate with limited mixing to mimic infected tissue. These results underscore the promise of QSIs and other antivirulence molecules that target group beneficial traits as resistance-robust antimicrobial treatments and provide support for their further development

Structure-Based Design and Biological Evaluation of Triphenyl Scaffold-Based Hybrid Compounds as Hydrolytically Stable Modulators of a LuxR-Type Quorum Sensing Receptor

Many common bacterial pathogens utilize quorum sensing to coordinate group behaviors and initiate virulence at high cell densities. The use of small molecules to block quorum sensing provides a means of abrogating pathogenic phenotypes, but many known quorum sensing modulators have limitations, including hydrolytic instability and displaying non-monotonic dose curves (indicative of additional targets and/or modes of action). To address these issues, we undertook a structure-based scaffold-hopping approach to develop new chemical modulators of the LasR quorum sensing receptor in Pseudomonas aeruginosa. We combined components from a triphenyl derivative known to strongly agonize LasR with chemical moieties known for LasR antagonism and generated potent LasR antagonists that are hydrolytically stable across a range of pH values. Additionally, many of these antagonists do not exhibit non-monotonic dose effects, delivering probes that inhibit LasR across a wider range of assay conditions relative to known lactone-based ligands

Nonwoven Polymer Nanofiber Coatings That Inhibit Quorum Sensing in Staphylococcus aureus: Toward New Nonbactericidal Approaches to Infection Control

We report the fabrication and biological evaluation of nonwoven polymer nanofiber coatings that inhibit quorum sensing (QS) and virulence in the human pathogen Staphylococcus aureus. Our results demonstrate that macrocyclic peptide <b>1</b>, a potent and synthetic nonbactericidal quorum sensing inhibitor (QSI) in S. aureus, can be loaded into degradable polymer nanofibers by electrospinning and that this approach can deposit QSI-loaded nanofiber coatings onto model nonwoven mesh substrates. The QSI was released over ∼3 weeks when these materials were incubated in physiological buffer, retained its biological activity, and strongly inhibited agr-based QS in a GFP reporter strain of S. aureus for at least 14 days without promoting cell death. These materials also inhibited production of hemolysins, a QS-controlled virulence phenotype, and reduced the lysis of erythrocytes when placed in contact with wild-type S. aureus growing on surfaces. This approach is modular and can be used with many different polymers, active agents, and processing parameters to fabricate nanofiber coatings on surfaces important in healthcare contexts. S. aureus is one of the most common causative agents of bacterial infections in humans, and strains of this pathogen have developed significant resistance to conventional antibiotics. The QSI-based strategies reported here thus provide springboards for the development of new anti-infective materials and novel treatment strategies that target virulence as opposed to growth in S. aureus. This approach also provides porous scaffolds for cell culture that could prove useful in future studies on the influence of QS modulation on the development and structure of bacterial communities

AHL degradation by bacteria.

<p>(A) AHL standards were added at 1 μM concentration to exponentially growing cultures and AHL degradation was measured after 24 hours. The amount of AHL degradation at 24 hours is shown as: +++, >90% degradation; ++ 60–90% degradation; +, 30–60% degradation; -, less than 30% degradation. (B) Dynamic AHL degradation in selected bacteria. AHLs (10 μM each) were added to exponentially growing cultures and AHL degradation was monitored over time. Data represents the average of three biological replicates.</p

Identification of Unanticipated and Novel <i>N</i>-Acyl L-Homoserine Lactones (AHLs) Using a Sensitive Non-Targeted LC-MS/MS Method

<div><p><i>N</i>-acyl L-homoserine lactones (AHLs) constitute a predominant class of quorum-sensing signaling molecules used by Gram-negative bacteria. Here, we report a sensitive and non-targeted HPLC-MS/MS method based on parallel reaction monitoring (PRM) to identify and quantitate known, unanticipated, and novel AHLs in microbial samples. Using a hybrid quadrupole-high resolution mass analyzer, this method integrates MS scans and all-ion fragmentation MS/MS scans to allow simultaneous detection of AHL parent-ion masses and generation of full mass spectra at high resolution and high mass accuracy in a single chromatographic run. We applied this method to screen for AHL production in a variety of Gram-negative bacteria (i.e. <i>B</i>. <i>cepacia</i>, <i>E</i>. <i>tarda</i>, <i>E</i>. <i>carotovora</i>, <i>E</i>. <i>herbicola</i>, <i>P</i>. <i>stewartii</i>, <i>P</i>. <i>aeruginosa</i>, <i>P</i>. <i>aureofaciens</i>, and <i>R</i>. <i>sphaeroides</i>) and discovered that nearly all of them produce a larger set of AHLs than previously reported. Furthermore, we identified production of an uncommon AHL (i.e. 3-oxo-C7-HL) in <i>E</i>. <i>carotovora</i> and <i>P</i>. <i>stewartii</i>, whose production has only been previously observed within the genera <i>Serratia</i> and <i>Yersinia</i>. Finally, we used our method to quantitate AHL degradation in <i>B</i>. <i>cepacia</i>, <i>E</i>. <i>carotovora</i>, <i>E</i>. <i>herbicola</i>, <i>P</i>. <i>stewartii</i>, <i>P</i>. <i>aeruginosa</i>, <i>P</i>. <i>aureofaciens</i>, the non-AHL producer <i>E</i>. <i>coli</i>, and the Gram-positive bacterium <i>B</i>. <i>subtilis</i>. We found that AHL degradation ability varies widely across these microbes, of which <i>B</i>. <i>subtilis</i> and <i>E</i>. <i>carotovora</i> are the best degraders, and observed that there is a general trend for AHLs containing long acyl chains (≥10 carbons) to be degraded at faster rates than AHLs with short acyl chains (≤6 carbons).</p></div

Extracted Ion chromatographs of AHL standards.

<p>The ion counts of each pseudo-molecular ion [M + H]⁺ are shown against retention time. Retention time axis is broken to expand the scale after five minutes, where most AHLs elute. Range for concentration of AHLs was between 0.04 μM to 3.70 μM. Compound structures are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163469#pone.0163469.s006" target="_blank">S1 Table</a>.</p

Small Molecule Disruption of Quorum Sensing Cross-Regulation in <i>Pseudomonas aeruginosa</i> Causes Major and Unexpected Alterations to Virulence Phenotypes

The opportunistic pathogen <i>Pseudomonas aeruginosa</i> uses three interwoven quorum-sensing (QS) circuitsLas, Rhl, and Pqsto regulate the global expression of myriad virulence-associated genes. Interception of these signaling networks with small molecules represents an emerging strategy for the development of anti-infective agents against this bacterium. In the current study, we applied a chemical approach to investigate how the Las-Rhl-Pqs QS hierarchy coordinates key virulence phenotypes in wild-type <i>P. aeruginosa</i>. We screened a focused library of synthetic, non-native <i>N</i>-acyl l-homoserine lactones and identified compounds that can drastically alter production of two important virulence factors: pyocyanin and rhamnolipid. We demonstrate that these molecules act by targeting RhlR in <i>P. aeruginosa</i>, a QS receptor that has seen far less scrutiny to date relative to other circuitry. Unexpectedly, modulation of RhlR activity by a single compound induces <i>inverse</i> regulation of pyocyanin and rhamnolipid, a result that was not predicted using genetic approaches to interrogate QS in <i>P. aeruginosa</i>. Further, we show that certain RhlR agonists strongly repress Pqs signaling, revealing disruption of Rhl-Pqs cross-regulation as a novel mechanism for QS inhibition. These compounds significantly expand the known repertoire of chemical probes available to study RhlR in <i>P. aeruginosa</i>. Moreover, our results suggest that designing chemical agents to disrupt Rhl-Pqs crosstalk could be an effective antivirulence strategy to fight this common pathogen

Limits of detection and retention times of AHL standards.^*

<p>Limits of detection and retention times of AHL standards.^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163469#t001fn001" target="_blank">*</a>}</p

Relative abundance of characteristic lactone ring fragments obtained from MS/MS spectra of AHL strandards.

<p>Relative abundance of characteristic lactone ring fragments obtained from MS/MS spectra of AHL strandards.</p

Characteristic MS/MS fragmentation of AHLs.

<p>A) Characteristic fragments originating from the lactone ring. These fragments were used to identify AHLs in bacterial samples. Labeled masses for carbon (¹³C) and nitrogen (¹⁵N) isotopes are indicated in parenthesis. B) Characteristic fragmentation observed when no substitution is present at the third carbon of the acyl chain. C) Characteristic fragmentation observed when a 3-oxo substitution is present. In B) and C), black wavy lines indicate the bonds that are broken during fragmentation, the measured fragment is highlighted in red. D) Fragmentation spectra of selected AHL standards. Blue lines indicate peaks for characteristic fragments of the lactone ring.</p