The Pseudomonas Quinolone Signal (PQS) Balances Life and Death in Pseudomonas aeruginosa Populations

When environmental conditions deteriorate and become inhospitable, generic survival strategies for populations of bacteria may be to enter a dormant state that slows down metabolism, to develop a general tolerance to hostile parameters that characterize the habitat, and to impose a regime to eliminate damaged members. Here, we provide evidence that the pseudomonas quinolone signal (PQS) mediates induction of all of these phenotypes. For individual cells, PQS, an interbacterial signaling molecule of Pseudomonas aeruginosa, has both deleterious and beneficial activities: on the one hand, it acts as a pro-oxidant and sensitizes the bacteria towards oxidative and other stresses and, on the other, it efficiently induces a protective anti-oxidative stress response. We propose that this dual function fragments populations into less and more stress tolerant members which respond differentially to developing stresses in deteriorating habitats. This suggests that a little poison may be generically beneficial to populations, in promoting survival of the fittest, and in contributing to bacterial multi-cellular behavior. It further identifies PQS as an essential mediator of the shaping of the population structure of Pseudomonas and of its response to and survival in hostile environmental conditions

Structural basis for native agonist and synthetic inhibitor recognition by the Pseudomonas aeruginosa quorum sensing regulator PqsR (MvfR)

Bacterial populations co-ordinate gene expression collectively through quorum sensing (QS), a cell-to-cell communication mechanism employing diffusible signal molecules. The LysR-type transcriptional regulator (LTTR) protein PqsR (MvfR) is a key component of alkyl-quinolone (AQ)-dependent QS in Pseudomonas aeruginosa. PqsR is activated by 2-alkyl-4-quinolones including the Pseudomonas quinolone signal (PQS; 2-heptyl-3-hydroxy-4(1H)-quinolone), its precursor 2-heptyl-4- hydroxyquinoline (HHQ) and their C9 congeners, 2-nonyl-3-hydroxy-4(1H)-quinolone (C9-PQS) and 2-nonyl-4-hydroxyquinoline (NHQ). These drive the autoinduction of AQ biosynthesis and the up-regulation of key virulence determinants as a function of bacterial population density. Consequently, PqsR constitutes a potential target for novel antibacterial agents which attenuate infection through the blockade of virulence. Here we present the crystal structures of the PqsR co-inducer binding domain (CBD) and a complex with the native agonist NHQ. We show that the structure of the PqsR CBD has an unusually large ligand-binding pocket in which a native AQ agonist is stabilized entirely by hydrophobic interactions. Through a ligand-based design strategy we synthesized and evaluated a series of 50 AQ and novel quinazolinone (QZN) analogues and measured the impact on AQ biosynthesis, virulence gene expression and biofilm development. The simple exchange of two isosteres (OH for NH2) switches a QZN agonist to an antagonist with a concomitant impact on the induction of bacterial virulence factor production. We also determined the complex crystal structure of a QZN antagonist bound to PqsR revealing a similar orientation in the ligand binding pocket to the native agonist NHQ. This structure represents the first description of an LTTR-antagonist complex. Overall these studies present novel insights into LTTR ligand binding and ligand-based drug design and provide a chemical scaffold for further anti-P. aeruginosa virulence drug development by targeting the AQ receptor PqsR

Live to cheat another day: bacterial dormancy facilitates the social exploitation of beta-lactamases

The breakdown of antibiotics by β-lactamases may be cooperative, since resistant cells can detoxify their environment and facilitate the growth of susceptible neighbours. However, previous studies of this phenomenon have used artificial bacterial vectors or engineered bacteria to increase the secretion of β-lactamases from cells. Here, we investigated whether a broad-spectrum β-lactamase gene carried by a naturally occurring plasmid (pCT) is cooperative under a range of conditions. In ordinary batch culture on solid media, there was little or no evidence that resistant bacteria could protect susceptible cells from ampicillin, although resistant colonies could locally detoxify this growth medium. However, when susceptible cells were inoculated at high densities, late-appearing phenotypically susceptible bacteria grew in the vicinity of resistant colonies. We infer that persisters, cells that have survived antibiotics by undergoing a period of dormancy, founded these satellite colonies. The number of persister colonies was positively correlated with the density of resistant colonies and increased as antibiotic concentrations decreased. We argue that detoxification can be cooperative under a limited range of conditions: if the toxins are bacteriostatic rather than bacteridical; or if susceptible cells invade communities after resistant bacteria; or if dormancy allows susceptible cells to avoid bactericides. Resistance and tolerance were previously thought to be independent solutions for surviving antibiotics. Here, we show that these are interacting strategies: the presence of bacteria adopting one solution can have substantial effects on the fitness of their neighbours

Quorum Sensing Inhibition Selects for Virulence and Cooperation in Pseudomonas aeruginosa

With the rising development of bacterial resistance the search for new medical treatments beyond conventional antimicrobials has become a key aim of public health research. Possible innovative strategies include the inhibition of bacterial virulence. However, consideration must be given to the evolutionary and environmental consequences of such new interventions. Virulence and cooperative social behaviour of the bacterium Pseudomonas aeruginosa rely on the quorum-sensing (QS) controlled production of extracellular products (public goods). Hence QS is an attractive target for anti-virulence interventions. During colonization, non-cooperating (and hence less virulent) P. aeruginosa QS-mutants, benefiting from public goods provided by wild type isolates, naturally increase in frequency providing a relative protection from invasive infection. We hypothesized that inhibition of QS-mediated gene expression removes this growth advantage and selection of less virulent QS-mutants, and maintains the predominance of more virulent QS-wild type bacteria. We addressed this possibility in a placebo-controlled trial investigating the anti-QS properties of azithromycin, a macrolide antibiotic devoid of bactericidal activity on P. aeruginosa, but interfering with QS, in intubated patients colonized by P. aeruginosa. In the absence of azithromycin, non-cooperating (and hence less virulent) lasR (QS)-mutants increased in frequency over time. Azithromycin significantly reduced QS-gene expression measured directly in tracheal aspirates. Concomitantly the advantage of lasR-mutants was lost and virulent wild-type isolates predominated during azithromycin treatment. We confirmed these results in vitro with fitness and invasion experiments. Azithromycin reduced growth rate of the wild-type, but not of the lasR-mutant. Furthermore, the lasR-mutant efficiently invaded wild-type populations in the absence, but not in the presence of azithromycin. These in vivo and in vitro results demonstrate that anti-virulence interventions based on QS-blockade diminish natural selection towards reduced virulence and therefore may increase the prevalence of more virulent genotypes in the Hospital environment. More generally, the impact of intervention on the evolution of virulence of pathogenic bacteria should be assessed

Risk-Targeted Selection of Agricultural Holdings for Post-Epidemic Surveillance: Estimation of Efficiency Gains

Current post-epidemic sero-surveillance uses random selection of animal holdings. A better strategy may be to estimate the benefits gained by sampling each farm and use this to target selection. In this study we estimate the probability of undiscovered infection for sheep farms in Devon after the 2001 foot-and-mouth disease outbreak using the combination of a previously published model of daily infection risk and a simple model of probability of discovery of infection during the outbreak. This allows comparison of the system sensitivity (ability to detect infection in the area) of arbitrary, random sampling compared to risk-targeted selection across a full range of sampling budgets. We show that it is possible to achieve 95% system sensitivity by sampling, on average, 945 farms with random sampling and 184 farms with risk-targeted sampling. We also examine the effect of ordering samples by risk to expedite return to a disease-free status. Risk ordering the sampling process results in detection of positive farms, if present, 15.6 days sooner than with randomly ordered sampling, assuming 50 farms are tested per day

Gene expression in fungi

This contribution is based on the four presentations made at the Special Interest Group (SIG) meeting titled Gene Expression in Fungi held during IMC9 in Edinburgh. This overview is independent from other articles published or that will be published by each speaker. In the SIG meeting, basic principles of in vivo animal models for virulence studies were discussed. Infection associated genes of Candida albicans and fungal adaptation to the host was summarized. Azole susceptibility was evaluated as a combined result of several changes in expression of pertinent genes. Gene transfer in fungi, resulting in fungal evolution and gene adaptation to environmental factors, was reported

Pseudomonas aeruginosa Overrides the Virulence Inducing Effect of Opioids When It Senses an Abundance of Phosphate

The gut during critical illness represents a complex ecology dominated by the presence of healthcare associated pathogens, nutrient scarce conditions, and compensatory host stress signals. We have previously identified key environmental cues, opioids and phosphate depletion that independently activate the virulence of Pseudomonas aeruginosa. Opioids induce quinolone signal production (PQS), whereas phosphate depletion leads to a triangulated response between MvfR-PQS, pyoverdin, and phosphosensory/phosphoregulatory systems (PstS-PhoB). Yet how P. aeruginosa manages its response to opioids during nutrient scarce conditions when growth is limited and a quorum is unlikely to be achieved is important in the context of pathogenesis in gut during stress. To mimic this environment, we created nutrient poor conditions and exposed P. aeruginosa PAO1 to the specific k-opioid receptor agonist U-50,488. Bacterial cells exposed to the k-opioid expressed a striking increase in virulence- and multi-drug resistance-related genes that correlated to a lethal phenotype in C. elegans killing assays. Under these conditions, HHQ, a precursor of PQS, rather than PQS itself, became the main inducer for pqsABCDE operon expression. P. aeruginosa virulence expression in response to k-opioids required PqsE since ΔPqsE was attenuated in its ability to activate virulence- and efflux pumps-related genes. Extracellular inorganic phosphate completely changed the transcriptional response of PAO1 to the k- opioid preventing pqsABCDE expression, the activation of multiple virulence- and efflux pumps-related genes, and the ability of P. aeruginosa to kill C. elegans. These results indicate that when P. aeruginosa senses resource abundance in the form of phosphate, it overrides its response to compensatory host signals such as opioids to express a virulent and lethal phenotype. These studies confirm a central role for phosphate in P. aeruginosa virulence that might be exploited to design novel anti- virulence strategies

Co-evolutionary dynamics of collective action with signaling for a quorum

Collective signaling for a quorum is found in a wide range of organisms that face collective action problems whose successful solution requires the participation of some quorum of the individuals present. These range from humans, to social insects, to bacteria. The mechanisms involved, the quorum required, and the size of the group may vary. Here we address the general question of the evolution of collective signaling at a high level of abstraction. We investigate the evolutionary dynamics of a population engaging in a signaling N-person game theoretic model. Parameter settings allow for loners and cheaters, and for costly or costless signals. We find a rich dynamics, showing how natural selection, operating on a population of individuals endowed with the simplest strategies, is able to evolve a costly signaling system that allows individuals to respond appropriately to different states of Nature. Signaling robustly promotes cooperative collective action, in particular when coordinated action is most needed and difficult to achieve. Two different signaling systems may emerge depending on Nature's most prevalent states.Funding: This research was supported by FEDER through POFC - COMPETE, FCT-Portugal through grants SFRH/BD/86465/2012, PTDC/MAT/122897/2010, EXPL/EEI-SII/2556/2013, and by multi-annual funding of CMAF-UL, CBMA-UM and INESC-ID (under the projects PEst-OE/BIA/UI4050/2014 and UID/CEC/50021/2013) provided by FCT-Portugal, and by Fundacao Calouste Gulbenkian through the "Stimulus to Research" program for young researchers. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.info:eu-repo/semantics/publishedVersio

Microbial Communication, Cooperation and Cheating: Quorum Sensing Drives the Evolution of Cooperation in Bacteria

An increasing body of empirical evidence suggests that cooperation among clone-mates is common in bacteria. Bacterial cooperation may take the form of the excretion of “public goods”: exoproducts such as virulence factors, exoenzymes or components of the matrix in biofilms, to yield significant benefit for individuals joining in the common effort of producing them. Supposedly in order to spare unnecessary costs when the population is too sparse to supply the sufficient exoproduct level, many bacteria have evolved a simple chemical communication system called quorum sensing (QS), to “measure” the population density of clone-mates in their close neighborhood. Cooperation genes are expressed only above a threshold rate of QS signal molecule re-capture, i.e., above the local quorum of cooperators. The cooperative population is exposed to exploitation by cheaters, i.e., mutants who contribute less or nil to the effort but fully enjoy the benefits of cooperation. The communication system is also vulnerable to a different type of cheaters (“Liars”) who may produce the QS signal but not the exoproduct, thus ruining the reliability of the signal. Since there is no reason to assume that such cheaters cannot evolve and invade the populations of honestly signaling cooperators, the empirical fact of the existence of both bacterial cooperation and the associated QS communication system seems puzzling. Using a stochastic cellular automaton approach and allowing mutations in an initially non-cooperating, non-communicating strain we show that both cooperation and the associated communication system can evolve, spread and remain persistent. The QS genes help cooperative behavior to invade the population, and vice versa; cooperation and communication might have evolved synergistically in bacteria. Moreover, in good agreement with the empirical data recently available, this synergism opens up a remarkably rich repertoire of social interactions in which cheating and exploitation are commonplace