Molecular Modifications of the Pseudomonas Quinolone Signal In
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Journal of Fungi Article Molecular Modifications of the Pseudomonas Quinolone Signal in the Intermicrobial Competition with Aspergillus Hasan Nazik 1,†,‡, Gabriele Sass 1,†, Paul Williams 2 , Eric Déziel 3 and David A. Stevens 1,4,* 1 Infectious Diseases Research Laboratory, California Institute for Medical Research, San Jose, CA 95128, USA; [email protected] (H.N.); [email protected] (G.S.) 2 Biodiscovery Institute and School of Life Sciences, University of Nottingham, Nottingham NG7 2RD, UK; [email protected] 3 Centre Armand-Frappier Santé Biotechnologie, Institut National de la Recherche Scientifique (INRS), Laval, QC H7V 1B7, Canada; [email protected] 4 Division of Infectious Diseases and Geographic Medicine, Stanford University Medical School, Stanford, CA 94305, USA * Correspondence: [email protected]; Tel.: +1-408-998-4554 † These authors contributed equally to this work. ‡ Current address: National University of Health Sciences, Pinellas Park, FL 33781, USA. Abstract: The Pseudomonas quinolone signal (PQS) is an important quorum-sensing molecule for Pseudomonas aeruginosa that regulates virulence factors, chelates iron, and is an important factor in interactions with eukaryotes, including fungi and mammalian hosts. It was previously shown to inhibit or boost Aspergillus, depending on the milieu iron concentration. We studied several molecular modifications of the PQS molecule, and their effects on Aspergillus biofilm metabolism and growth in vitro, and the effects of iron supplementation. We found that most molecules inhibited Aspergillus at concentrations similar to that of PQS, but with relatively flat dose-responses, and Citation: Nazik, H.; Sass, G.; all were less potent than PQS. The inhibition was reversible by iron, suggesting interference with Williams, P.; Déziel, E.; Stevens, D.A. fungal iron metabolism. Stimulation of Aspergillus was not noted. We conclude that the critical Molecular Modifications of the Aspergillus-inhibiting moeities of the PQS molecule were partially, but not completely, interfered Pseudomonas Quinolone Signal in with by molecular modifications at several sites on the PQS molecule. The mechanism, as with PQS, the Intermicrobial Competition with appears to relate to fungal iron metabolism. Aspergillus. J. Fungi 2021, 7, 343. https://doi.org/10.3390/jof7050343 Keywords: Aspergillus; Pseudomonas; Pseudomonas quinolone signal; intermicrobial competition Academic Editor: David S. Perlin Received: 16 March 2021 1. Introduction Accepted: 21 April 2021 Published: 28 April 2021 Pseudomonas aeruginosa (Pa) and Aspergillus fumigatus (Af) encounter each other in nature in soil and water, and are, in most studies, the most frequently found bacterium Publisher’s Note: MDPI stays neutral and fungus in the airways of immunocompromised hosts, including persons with cystic with regard to jurisdictional claims in fibrosis (CF) [1,2]. Thus, it is expected that these two microbes likely compete for their published maps and institutional affil- establishment and growth in various milieu. This has been studied extensively in vitro, iations. and to a lesser extent in animal models, a subject reviewed in detail elsewhere [3–9]. Among the molecules identified in the intermicrobial competition is 2-heptyl-3- hydroxy-4(1H)-quinolone, the Pseudomonas Quinolone Signal (PQS). PQS is a quorum- sensing molecule, coordinating many Pa functions [8,10]. It chelates iron (Fe) and delivers Copyright: © 2021 by the authors. it to the bacterial cell membrane in conjunction with Pa siderophores [10–13]. It upregulates Licensee MDPI, Basel, Switzerland. genes involved in Pa oxidative stress responses, but can also be a pro-oxidant and induce This article is an open access article oxidative stress; it regulates Pa virulence factors, and in intermicrobial interactions it can distributed under the terms and be used as a quorum sensing signal by other bacterial species, inhibit the respiratory chain, conditions of the Creative Commons or induce membrane vesicle formation in other bacteria; and it can be inactivated by other Attribution (CC BY) license (https:// bacteria [14–16]. Relevant to eukaryotes, PQS production can be inhibited by fungi, and in creativecommons.org/licenses/by/ contrast, PQS can inhibit fungal biofilm formation [14,15,17,18]. In eukaryotic hosts, PQS 4.0/). J. Fungi 2021, 7, 343. https://doi.org/10.3390/jof7050343 https://www.mdpi.com/journal/jof J. Fungi 2021, 7, x FOR PEER REVIEW 2 of 17 J. Fungi 2021, 7, 343 2 of 16 fungi, and in contrast, PQS can inhibit fungal biofilm formation [14,15,17,18]. In eukary- oticcan hosts, be toxicPQS tocan host be cells,toxic andto host impair cells, host and defenses impair host [12,14 defenses,19]. Its probable[12,14,19] relevance. Its probablein vivo relevanceis heightened in vivo byis heightened reports of its by presence reports of in its body presence fluids in of body patients flui [ds12 of,14 patients]. [12,14]. In aIn previous a previous study study [18], [18 we], wefocused focused on onPQS PQS inhibition inhibition of Af of Afbiofilm biofilm metabolism metabolism in a in a lowlow Fe Feenvironment environment (RPMI (RPMI-1640-1640 medium), medium), and and in in ancillary ancillary experiments, experiments, PQS PQS inhibited Af Af growth on on agar agar as as well. well. The The biofilm biofilm inhibition inhibition was was reversed reversed by by the the addition addition of ofFe. Fe. Most Most remarkably,remarkably, and and paradoxically, paradoxically, in in an an enriched enriched Fe Fe environment, environment, PQS PQS + + Fe Fe boosted boosted Af biofilmbio- filmmetabolism, metabolism, as as well well as as planktonic planktonic Af Af growth growth and and growth growth on on agar. agar. This This boosting boosting exceeded ex- ceededthe boosting the boosting effect effect of Fe of alone. Fe alone. Perhaps Perhaps uniquely uniquely biologically, biologically, the maximum the maximum boosting boost- effect ingrequired effect required Af siderophore Af siderophore involvement. involvement. ResearchResearch (with (with some some interest interest in inthe the possibility possibility of of generating generating inhibitors inhibitors of of Pa) Pa) in in sev- several erallaboratories, laboratories studying, studying molecular molecular modifications modifications of of the the PQS PQS molecule, molecule has, has indicated indicated a higha highdegree degree of of structural structural PQS PQS specificityspecificity forfor its functions functions in in Pa Pa physiology physiology [17,20,21] [17,20,21. ].We We thereforetherefore studied studied whether whether such such congeners congeners would would act actsimilarly similarly to PQS to PQS in the in theintermicrobial intermicrobial interactionsinteractions des describedcribed for forPQS. PQS. The The chemical chemical formulae formulae for forthe themolecules molecules studied studied are aregiven given in Figurein Figure 1 [20,221[20,22]. ]. Figure 1. Molecules in this study. In all, R1 is an H atom. In Pseudomonas quinolone signal (PQS), Figure 1. Molecules in this study. In all, R1 is an H atom. In Pseudomonas quinolone signal (PQS), 2-heptyl-4-hydroxyquinoline (HHQ), 2-heptyl-3-chloro-4(1H)-quinolone (3Cl-PQS) and 2-heptyl-3- 2-heptyl-4-hydroxyquinoline (HHQ), 2-heptyl2 -3-chloro-4(1H)-quinolone (3Cl-PQS) and 2-heptyl-3- amino-4(1H)-quinolone (3NH2-PQS)2 R is an alkyl side chain, C7H15. In C1-PQS, C3-PQS, C5-PQS, amino-4(1H)-quinolone (3NH2-PQS) R is an alkyl side chain, C7H15. In C1-PQS, C3-PQS, C5-PQS, 2 andand C9-PQS C9-PQS R2 is R anis alkyl an alkyl side sidechain chain of 1, of3, 5 1,, and 3, 5, 9 and carbon 9 carbon molecules, molecules, respectively. respectively. In PQS In and PQS and 3 3 HHQ,HHQ, R3 is R a ishydroxyl a hydroxyl group group or an or anH atom, H atom, respectively. respectively. In In3Cl 3Cl-PQS-PQS and and 3NH 3NH2-PQS,2-PQS, R3 R is isa chlo- a chlorine rineor or primary primary amine amine group, group, respectively. respectively. HHQHHQ (2-heptyl (2-heptyl-4-hydroxyquinoline)-4-hydroxyquinoline) is a is PQS a PQS precursor, precursor, and and it also it also activate activatess the thetran- tran- scriptionalscriptional regulator regulator PqsR PqsR (also (also known known as M asvfR) MvfR) [20] [ 20(2-]heptyl (2-heptyl-3-amino-4(1-3-amino-4(1H)-Hquinolone)-quinolone,, 3NH3NH2-PQS2-PQS retains retains this this capability capability).). PqsR PqsR is the is thePa protein Pa protein that that plays plays a central a central role role in the in the alkylalkyl quinolone quinolone-dependent-dependent quorum quorum-sensing-sensing cascade, cascade, and and is a isregulator a regulator of multiple of multiple viru- viru- lence factors (recently reviewed in [8]). The NH group is isosteric with OH, but would lence factors (recently reviewed in [8]). The NH2 group2 is isosteric with OH, but would havehave opposite opposite electronic electronic effects effects on onthe the bicyclic bicyclic ring. ring. 2-Heptyl 2-Heptyl-3-chloro-4(1-3-chloro-4(1H)-Hquinolone)-quinolone (3Cl(3Cl-PQS)-PQS) is unable is unable to toactivate activate PqsR, PqsR, and and the the 3- 3-ClCl substitution substitution will will have have different different molecularmolec- ulareffects effects than than the the 3-OH 3-OH on on the the bicyclic bicyclic ring. ring. ModificationsModifications ofof thethe 2-alkyl2-alkyl sideside chain,chain, ofof all all the substitutions, would be likely to retain Fe Fe-chelating-chelating properties of of PQS PQS ( (inin contrast contrast to 3NH2-PQS and 3Cl-PQS that lack the adjacent oxygen molecules at the 3 and 4 positions to 3NH2-PQS and 3Cl-PQS that lack the adjacent oxygen molecules at the 3 and 4 positions on the molecule), but result in different hydrophobicities that could impact uptake into on the molecule), but result in different hydrophobicities that could impact uptake into and/or export from cells and the interaction targets. and/or export from cells and the interaction targets. 2. Methods 2. Methods 2.1. Reagents 2.1.