Attenuation of Pseudomonas Aeruginosa Quorum Sensing by Natural Products: Virtual Screening, Evaluation and Biomolecular Interactions

International Journal of Molecular Sciences

Article Attenuation of Pseudomonas aeruginosa Quorum Sensing by Natural Products: Virtual Screening, Evaluation and Biomolecular Interactions

Lin Zhong, Vinothkannan Ravichandran, Na Zhang, Hailong Wang , Xiaoying Bian * , Youming Zhang * and Aiying Li * Helmholtz International Laboratory for Anti-Infectives, Shandong University-Helmholtz Institute of Biotechnology, State Key Laboratory of Microbial Technology, Shandong University, Qingdao 266237, China; [email protected] (L.Z.); [email protected] (V.R.); [email protected] (N.Z.); [email protected] (H.W.) * Correspondence: [email protected] (X.B.); [email protected] (Y.Z.); [email protected] (A.L.)

Received: 7 January 2020; Accepted: 17 March 2020; Published: 22 March 2020

Abstract: Natural products play vital roles against infectious diseases since ancient times and most drugs in use today are derived from natural sources. Worldwide, multi-drug resistance becomes a massive threat to the society with increasing mortality. Hence, it is very crucial to identify alternate strategies to control these ‘super bugs’. Pseudomonas aeruginosa is an opportunistic pathogen reported to be resistant to a large number of critically important antibiotics. Quorum sensing (QS) is a cell–cell communication mechanism, regulates the biofilm formation and virulence factors that endow pathogenesis in various bacteria including P. aeruginosa. In this study, we identified and evaluated quorum sensing inhibitors (QSIs) from plant-based natural products against P. aeruginosa. In silico studies revealed that catechin-7-xyloside (C7X), sappanol and butein were capable of interacting with LasR, a LuxR-type quorum sensing regulator of P. aeruginosa. In vitro assays suggested that these QSIs significantly reduced the biofilm formation, pyocyanin, elastase, and rhamnolipid without influencing the growth. Especially, butein reduced the biofilm formation up to 72.45% at 100 µM concentration while C7X and sappanol inhibited the biofilm up to 66% and 54.26% respectively. Microscale thermophoresis analysis revealed that C7X had potential interaction with LasR (KD = 933 369 nM) and thermal shift assay further confirmed the biomolecular interactions. These ± results suggested that QSIs are able to substantially obstruct the P. aeruginosa QS. Since LuxR-type transcriptional regulator homologues are present in numerous bacterial species, these QSIs may be developed as broad spectrum anti-infectives in the future.

Keywords: Pseudomonas aeruginosa; LuxR-type quorum sensing regulator; quorum sensing inhibitors; molecular docking; microscale thermophoresis; thermal shift assay

1. Introduction Globally, multi-drug resistant infections kill almost 700,000 people per year and it is predicted that it would increase up to 10 million deaths per year by 2050 [1]. This is due to the misuse and overuse of existing antimicrobials in humans, animals and plants, resulting in the development of multi-drug resistant (MDR) pathogenic bacteria [2]. Conventional antibiotics target the fundamental processes of bacteria leading to growth arrest which in turn creates selective pressure and further multi-drug resistant variants [3]. World Bank estimated that the antimicrobial resistance (AMR) will not only be a health burden but also cause reduction in gross domestic production in 2050 that would be comparable to the 2008–2009 global ﬁnancial crisis [4]. Pseudomonas aeruginosa is a Gram negative bacterium, common cause of pneumonia and infections especially in bloodstream, urinary tract and

Int. J. Mol. Sci. 2020, 21, 2190; doi:10.3390/ijms21062190 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 2190 2 of 22 surgical-sites and burn wounds and plays vital roles in cystic fibrosis. P. aeruginosa was reported to be resistant to many antibiotics, including aminoglycosides, cephalosporins, fluoroquinolones, and carbapenems [5,6]. Hence, P. aeruginosa was placed on the top of priority pathogen list (critical) by World Health Organization for prioritizing the new antibiotic development against MDR pathogens [7]. Quorum sensing (QS) is a cell–cell communication mechanism controlled by releasing, sensing and responding to signal molecules called autoinducers (AIs) to regulate the biofilm formation, virulence, bioluminescence, and antibiotic production based on population density in both Gram-negative and Gram-positive bacteria [8]. Gram-negative bacteria use LuxI/R type QS systems that contain LuxI-type auto inducer synthases that lead to the production of acyl homoserine lactone (AHL) molecules as AIs and cognate LuxR-type receptor proteins. LuxR-type receptors are a class of transcriptional regulatory proteins that mediate QS pathways by interacting with AIs. LuxR-type protein such as LuxR, LasR, and TraR are highly unstable without AIs and the concentration of AIs will be proportionally low at low cell density [9]. Once the cell density increases, AI concentration also respectively increases. And these accumulated AHLs interact with LuxR receptor, leading to the LuxR-AHL complex stabilization. Subsequently, the LuxR-AHL complex binds to respective promoter region, and leads to the expression of downstream genes [10]. In P. aeruginosa, there are three well-established quorum sensing pathways reported so far, such as two LuxI/R-type systems (LasI/R and RhlI/R), a PQS (Pseudomonas quinolone signal) [11]. These systems are well-organized with LasI/R at the top of the hierarchy in the cascade. LasR consists of two independently folded domains: A larger ligand binding domain (LBD) at the amino-terminal and a smaller DNA-binding domain (DBD) at the carboxy-terminal [12]. When N-(3-oxododecanoyl)-l-homoserine lactone (3OC12-HSL), an AI of LasI/R system, interacts with LasR, it activates many downstream genes including the lasI synthase, and leads to the production of 3OC12-HSL. The LasR-3OC12-HSL complex also initiates the expression of QS systems by positively regulating rhlR rhlI, pqsR, and pqsABCDE genes. Once RhlR interacts with N-butanoyl-l-homoserine lactone (C4-HSL), it activates many genes, including those are essential for virulence factor production and biofilm formation [13]. The comprehensive understanding that P. aeruginosa and many other pathogens, control much of their virulence by QS, offered a new direction to a novel and robust “drug target” [14–16]. Many studies revealed that curbing QS would effectively reduce the biofilm formation and virulence [17–19]. Unlike conventional antibiotics, quorum sensing inhibitors (QSIs) will not kill the bacteria, rather they disarm the bacterial pathogenicity alone and hence they seem to have only slighter chances to get resistance [20]. Hence interfering with quorum sensing is considered to be a practicable and potential alternate approach especially in handling multi-drug resistant P. aeruginosa [21]. Consequently, drugs capable of interfering with QS are prone to escalate the susceptibility of the pathogenic bacteria towards host defense mechanisms and further clearance [22]. Natural products from plants have been playing vital roles against infectious diseases since ancient times and many drugs in use today are derived from plant natural sources. Here, in this study, we have screened LasR-specific QSIs from plant-derived natural products and evaluated their efficacy in suppressing QS-related phenotypes, including biofilm formation and virulence factors. Further we also studied bimolecular interactions of these QSIs with LasR protein using microscale thermophoresis and thermal shift assays.

2. Results Plants and plant-based natural products have always been used as important sources for novel drug development against various diseases. Hence, we intended to identify and evaluate QSIs from plant-based natural products. Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 3 of 22

Plants and plant-based natural products have always been used as important sources for novel drug development against various diseases. Hence, we intended to identify and evaluate QSIs from Int.plant J. Mol.-based Sci. 2020natural, 21, 2190 products. 3 of 22

2.1. Molecular Docking Studies 2.1. Molecular Docking Studies To screen QSIs against LasR, a transcriptional regulator of P. aeruginosa QS, virtual screening To screen QSIs against LasR, a transcriptional regulator of P. aeruginosa QS, virtual screening was performed using a natural product database of Chemfaces, China (http://www.chemfaces.cn) was performed using a natural product database of Chemfaces, China (http://www.chemfaces.cn) containing 4687 plant based natural products. containing 4687 plant based natural products. Docking results showed that the GScore forfor thethe cognatecognate ligandligand (3OC12-HSL)(3OC12-HSL) was -6.456-6.456 and it waswas ableable toto formform 55 H-bondsH-bonds withwith Tyr 56,56, TrpTrp 60,60, AspAsp 73,73, andand SerSer 129129 (2)(2) (Figure(Figure1 a,b).1a,b). Pose Pose analysis analysis revealedrevealed thatthat threethree compoundscompounds werewere ableable toto interact with LasR.LasR. ConsideringConsidering thethe pattern of interaction of 3OC12-HSL, 3OC12-HSL, only compounds compounds with similar pattern of interaction interaction with signiﬁcant significant GScore were were chosenchosen forfor furtherfurther investigations.investigations.

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Figure 1. The 2D (a) and 3D (b) interaction map of 3OC12-HSL and LasR. Tyr 56, Trp 60, Asp 73 and Figure 1. The 2D (a) and 3D (b) interaction map of 3OC12-HSL and LasR. Tyr 56, Trp 60, Asp 73 and Ser 129 (2). Ser 129 (2).

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 4 of 22 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 4 of 22 Int.Int. J.Int. Mol. J. Mol.J. Mol. Sci. Sci. 2020Sci.2020 2020, 21, 21, ,x 21, FOR 2190, x FOR PEER PEER REVIEW REVIEW 4 of4 4 of22 of 22 22 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 4 of 22 The identified compounds C7X, sappanol, butein, and C30 ((Z-)-4-Bromo-5-(bromomethylene)- TheTheThe identifiedidentified identified compoundscompounds compounds C7X,C7X, C7X, sappanol,sappanol, sappanol, bbuteinutein butein,, andand, and C30C30 C30 (((Z(Z- -())(Z--44--Bromo)Bromo-4-Bromo--55--(bromomethylene)(bromomethylene)-5-(bromomethylene)-- - 2(5H)The -identifiedfuranone) compounds[16] were C7X, having sappanol, the GScore butein ,– and13.508, C30 –(10.964,(Z-)-4-Bromo –9.245-5 - and(bromomethylene) –4.244 (Table- 1) 2(5H)2(5H)2(5H)--furanoneThefuranone-furanone identiﬁed)) [16][16]) compounds[16] were were were having having having C7X, the the sappanol, the GScore GScore GScore butein, ––13.508,13.508, –13.508, and ––10.964, C3010.964, –10.964, ((Z-)-4-Bromo-5-(bromomethylene)- ––9.2459.245 –9.245 and and and ––4.2444.244 –4.244 (Table (Table (Table 1) 1) 1) 2(5H)respectively.-furanone) C7X[16] was were able having to form the4 H - GScorebonds at– Tyr13.508, 56, Tyr–10.964, 64, Tyr – 9.24593, Leu and 125 –and4.244 a pi (Table–pi stalking 1) respectively.respectively.2(5H)-furanone)respectively. C7XC7X C7X was [was16 ]was wereableable able toto having formform to form the44 HH GScore-4-bondsbonds H-bonds atat13.508, TyrTyr at Tyr 56,56, 10.964, Tyr56,Tyr Tyr 64,64, Tyr64,Tyr9.245 Tyr 93,93, and Leu93,Leu Leu 4.244125125 125 andand (Table and aa pipi1 a–)– pi respectively.pi –stalkingstalkingpi stalking respectively.with Tyr 64 C7X (Figure was able2a,b ).to Sappanol form 4 H was-bonds able− at to Tyr form 56,− 4 Tyr H-bonds 64,− Tyr at 93, amino Leu− acids125 and Ty ra 64, pi –Thrpi stalking 75 (2), Leu withwithC7Xwith TyrTyr was Tyr 6464 able (Figure(Figure 64 (Figure to form 22aa,,bb 2).). 4a Sappanol,Sappanol H-bondsb). Sappanol was atwas Tyr was ableable 56, able toto Tyr formform to 64, form 44 Tyr HH -4-bondsbonds 93,H-bonds Leu atat 125 aminoamino at andamino acidsacids a pi–piacids TyTyrr stalking Ty 64,64,r ThrThr64, Thr 7575 with (2),(2), 75 Tyr (2),LeuLeu 64Leu with125 Tyr and 64 a (Figure pi–pi stalking 2a,b). Sappanol with Tyr was 47 (Figure able to 3forma,b). 4Whereas, H-bonds b atutein amino was acids able Tyto rform 64, Thr 3 H 75-bonds (2), Leu at Tyr 125125(Figure 125 andand and aa2 pipia,b). –a–pi pi Sappanolstalking–stalkingpi stalking withwith was with TyrTyr able Tyr 4747 to (Figure(Figure 47 form (Figure 4 33 H-bondsaa,,bb 3).).a Whereas,,Whereas,b). Whereas, at amino bbuteinutein b acidsutein waswas Tyr was ableable 64, able toto Thr formform to 75 form 3 (2),3 HH -3- Leubondsbonds H-bonds 125 atat and TyrTyr at aTyr 12547, and Thr a pi 75,–pi Ser stalking 129 and with a pi Tyr–pi 47 interaction (Figure 3a with,b). Whereas, Tyr 56 (Figure butein 4 wasa,b). able In contrast, to form 3C30 H- bondshas only at Tyrone H 47,47,pi–pi Thr47,Thr Thr stalking75,75, Ser75,Ser Ser129129 with 129 andand Tyr and aa 47pipi a–– (Figure pipi –interactioninteractionpi interaction3a,b). Whereas, withwith with TyrTyr butein Tyr 5656 (Figure (Figure56 was (Figure able 44aa,,b b4 to).).a ,InIn formb). contrast,contrast, In 3contrast, H-bonds C30C30 C30 ha athas Tyrs haonlyonlys 47, only oneone Thr one HH 75, H 47, bondThr 75, at Ser 129 and(Figure a pi 5–api.b interaction). with Tyr 56 (Figure 4a,b). In contrast, C30 has only one H bondbondSerbond 129atat SerSer at and Ser129129 a 129 pi–pi(Figure(Figure (Figure interaction 55aa..b b5)).a. .b). with Tyr 56 (Figure4a,b). In contrast, C30 has only one H bond at Ser bond at Ser 129 (Figure 5a.b). 129 (FigureTable5 a.b). 1. Docking results of quorum-sensing inhibitors against LasR, the quorum regulator of P. TableTableTable 1. 1. DockingDocking 1. Docking results results results of of quorum quorum of quorum--sensingsensing-sensing inhibitors inhibitors inhibitors against against against LasR, LasR, LasR, the the quorumthe quorum quorum regulator regulator regulator of of P. P. of P. Tableaeruginosa. 1. Docking results of quorum-sensing inhibitors against LasR, the quorum regulator of P. aeruginosa.aeruginosa.Tableaeruginosa. 1. Docking results of quorum-sensing inhibitors against LasR, the quorum regulator of aeruginosa. P. aeruginosa. H Bond forming Name Structure GScore HH H BondBondBond formingforming forming NameNameName StructureStructureStructure GScoreGScoreGScore HBonds Bondamino forming acids BondsBonds aminoBondamino acids Forming acids NameName Structure Structure GScore GScore Bonds H Bonds aminoTyr acids 56 Bonds aminoAmino acids Acids TyrTyr Tyr 5656 56 TyrTrp 56 60 3OC12-HSL –6.456 5 TrpTyr 60 56 3OC12-HSL –6.456 5 TrpAspTrp 60 7360 3OC123OC12-HSL-HSL –6.456–6.456 5 5 AspTrpTrp 7360 60 3OC123OC12-HSL-HSL –6.4566.456 5 5 AspSerAsp 73129 73 (2) − SerAsp 129Asp 73 (2) 73 SerSer Ser129 129 129 (2) (2) (2) Ser 129Tyr (2) 56 Tyr 56 TyrTyrTyr 56 6456 Catechin 7- TyrTyr 6456 TyrTyrTyr 64 56 64 CatechinCatechinCatechin 77-- 7- –13.508 4 TyrTyr 64 93 CatechinxylosideCatechin 7 (C7X)- ––13.50813.508–13.508 44 4 TyrTyrTyr Tyr 9393 64 93 xylosidexylosidexyloside (C7X)(C7X) (C7X) –13.50813.508 4 4 TyrLeu 93 125 xyloside7-xyloside (C7X) (C7X) − LeuLeuLeu Tyr 125125 93 125 LeuLeu 125125

Tyr 64 TyrTyr Tyr 6464 64 Sappanol –10.964 4 TyrThr 64 75 (2) SappanolSappanolSappanol ––10.96410.964–10.964 44 4 ThrThr Thr 7575Tyr (2) (2)75 64 (2) SappanolSappanol –10.96410.964 4 4 ThrThr Leu75 75(2) 125 (2) − LeuLeuLeu 125125 125 LeuLeu 125125

Tyr 47 TyrTyr Tyr 4747 47 TyrThr 47 75 Butein –9.245 3 ThrThrTyr Thr 7575 47 75 ButeinButeinButein ––9.2459.245–9.245 33 3 ThrSer 75 129 Butein 9.245 3 Ser ThrSer129 75129 Butein –9.245− 3 Ser 129 SerSer 129 129

C30 –4.244 1 Ser 129 C30C30C30C30 ––4.2444.2444.244–4.244 11 11 SerSer Ser Ser129129 129 129 C30 –4.244− 1 Ser 129

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Figure 2. The 2D (a) and 3D (b) interaction map of C7X and LasR. Tyr 56, Tyr 64, Tyr 93, and Leu 125 Figure 2. The 2D (a) and 3D (b) interaction map of C7X and LasR. Tyr 56, Tyr 64, Tyr 93, and Leu 125 are the interacting residues. are the interacting residues.

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Figure 3. The 2D (a) and 3D (b) interaction map of sappanol with LasR. Tyr 64, Thr 75 (2), and Leu 125 Figureare the 3. interacting The 2D (a residues) and 3D and (b) Thrinteraction 75 is a key map residue. of sappanol with LasR. Tyr 64, Thr 75 (2), and Leu 125 are the interacting residues and Thr 75 is a key residue.

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Figure 4. The 2D (a) and 3D (b) interaction map of butein and LasR. Tyr 47, Thr 75 and Ser 129 are the Figure 4. The 2D (a) and 3D (b) interaction map of butein and LasR. Tyr 47, Thr 75 and Ser 129 are the interacting residues. Tyr 47 was reported to be present in L3 loop crucial for the inhibitor binding. interacting residues. Tyr 47 was reported to be present in L3 loop crucial for the inhibitor binding.

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Figure 5. The 2D (a) and 3D (b) interaction map of C30 which interacting only with Ser 129. Figure 5. The 2D (a) and 3D (b) interaction map of C30 which interacting only with Ser 129. 2.2. In vitro studies 2.2. In vitro studies 2.2.1. Influence on Biofilm Formation and Growth 2.2.1. Influence on Biofilm Formation and Growth Biofilm formation is one of the key factors which is controlled by QS and plays a fundamental roleBiofilm in pathogenesis formation is [17 one,19 ].of Allthe three key factors compounds which screened is controlled in this by studyQS and were plays found a fundamental to reduce the rolebiofilm in pathogenesis formation significantly[17,19]. All three at all thecompounds tested concentrations screened in (1,this 10 study and 100 wereµM). found All QSIsto reduce reduced the the biofilmbiofilm formation more than significantly 50% at 100 atµ Mall concentrationthe tested concentrations (Figure6a). Especially,(1, 10 and 100 butein μM). significantly All QSIs reduced reduced the biofilm more than 50% at 100 μM concentration (Figure 6a). Especially, butein significantly

Int. J. Mol. Sci. 2020, 21, 2190 9 of 22 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 9 of 22 thereduced biofilm the formation biofilm formation about 72.45% about with 72.45 100%µ Mwith concentration. 100 μM concentration. While, C7X While, and sappanol C7X and reduced sappanol the biofilmreduced about the biofilm 66% and about 54.26% 66% respectively and 54.26% withrespectively 100 µM with concentration. 100 μM concentration. To differentiate To thedifferentiate quorum sensingthe quorum inhibition sensing activity inhibition of these activity QSIs of fromthese antibiotic QSIs from activity, antibiotic the activity, cell growth the cell was growth also analyzed. was also Itanalyzed. was found It was that found none ofthat QSIs none had of influence QSIs had on influence the growth on the of P.growth aeruginosa of P.(Figure aeruginosa6b). (Figure 6b).

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FigureFigure 6.6. EffectEffect of QSIs on on biofilm biofilm and and growth growth after after 24 24 h h (n (n = =3).3). (a) ( aQSIs) QSIs effect effect onon biofilm. biofilm. Butein Butein (B) (B)inhibited inhibited the thebiofilm biofilm formation formation significantly significantly when when compared compared to the to untreated the untreated control control (C). Data (C). shown Data shown as mean SD of triplicate wells ** p < 0.01 vs. control; (b). None of the QSIs have affected the as mean ± SD of± triplicate wells ** p < 0.01 vs. control; (b). None of the QSIs have affected the growth growthestimated estimated by medial by medial cloudiness cloudiness.. C is control, C is control, C7X, C7X,sappanol sappanol (S), b(S),utein butein (B) (B) were were QSIs QSIs and and C30 C30 is isfuranone, furanone, the the known known inhibitor. inhibitor. Data Data was was statistically statistically analyzed analyzed and and the the P P-values-values were estimated byby aa t student’sstudent’s t-test.-test. 2.2.2. Confocal and Scanning Electron Microscope Studies 2.2.2. Confocal and Scanning Electron Microscope Studies The biofilm inhibition was studied using the confocal laser scanning microscopy (CLSM). It was The biofilm inhibition was studied using the confocal laser scanning microscopy (CLSM). It was found that all the tested QSIs suppressed the biofilm formation when treated with 100 µM concentration found that all the tested QSIs suppressed the biofilm formation when treated with 100 μM (Figure7). To be specific, butein significantly reduced the biofilm formation when administered with concentration (Figure 7). To be specific, butein significantly reduced the biofilm formation when 100 µM. The scanning electron microscope (SEM) analysis showed a very similar pattern of inhibition administered with 100 μM. The scanning electron microscope (SEM) analysis showed a very similar which was confirmed by CLSM results. In control, we could see a lot of colonies in different size pattern of inhibition which was confirmed by CLSM results. In control, we could see a lot of colonies varying by compound to compound. All the QSIs were able to reduce the biofilm formation, especially in different size varying by compound to compound. All the QSIs were able to reduce the biofilm butein was able to reduce the biofilm significantly equivalent to C30 (Figure8). formation, especially butein was able to reduce the biofilm significantly equivalent to C30 (Figure 8).

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Figure 7. Biofilm inhibition by quorum sensing inhibitors (QSIs). Obvious biofilm inhibition was Figure 7. Biofilm inhibition by quorum sensing inhibitors (QSIs). Obvious biofilm inhibition was observedFigure 7. whenBiofilm treated inhibition with bybu teinquorum (B), C7X sensing, C30 inhibitors and sappanol (QSIs) (S). Obvious using FITC biofilm-CoA inhibition staining dye was, observed when treated with butein (B), C7X, C30 and sappanol (S) using FITC-CoA staining dye, comparedobserved whento the treatedcontrol with(C) where butein there (B), C7Xis abundance, C30 and of sappanol biofilm. (S)Butein using reduced FITC- CoAthe biofilm staining to dye the, compared to the control (C) where there is abundance of biofilm. Butein reduced the biofilm to the maximumcompared whento the comparedcontrol (C) to where the C 30.there is abundance of biofilm. Butein reduced the biofilm to the maximum when compared to the C30.C30.

FigureFigure 8. SEMSEM analysis analysis of of biofilm biofilm inhibition. inhibition. Butein Butein (B) (B) and and C30 C30 inhibited inhibited the the biofilm biofilm formation. formation. ObviousFigureObvious 8. biofilmbiofilm SEM analysisreduction reduction of was was biofilm observed inhibition. when Buteintreated (B)with and butein. C30 inhibited the biofilm formation. Obvious biofilm reduction was observed when treated with butein. 2.3. Influence on Virulence Factors 2.3. Influence on Virulence Factors 2.3. InfluenceProduction on Virulence of pyocyanin Factors and rhamnolipid and elastase expression are also controlled by Production of pyocyanin and rhamnolipid and elastase expression are also controlled by P. P. aeruginosa, and involved in its pathogenesis [17,19]. All the tested QSIs inhibited the pyocyanin aeruginosaProduction, and involved of pyocyanin in its andpathogenesis rhamnolipid [17 ,19and]. Allelastase the testedexpression QSIs are inhibited also controlled the pyocyanin by P. production and especially butein showed 72.36% inhibition when treated with 100 µM concentration productionaeruginosa, and especiallyinvolved inbutein its pathogenesis showed 72.36 [17% inhibition,19]. All thewhen tested treated QSIs with inhibited 100 μM theconcentration pyocyanin (Figure9a). C30 also inhibited the pyocyanin production, comparable to that of C7X. Butein inhibited (Figureproduction 9a). C3and0 alsoespecially inhibited butein the showedpyocyanin 72.36 production,% inhibition comparable when treated to that with of 100C7X. μM Butein concentration inhibited the elastase activity to about 33.98%, comparable to C30 (37.65%). C7X and sappanol also reduced the the(Figure elastase 9a). activityC30 also to inhibited about 33.98 the% pyocyanin, comparable production, to C30 (37.65%). comparable C7X toand that sappanol of C7X. alsoButein reduced inhibited the elastase activity significantly (Figure9b). All the tested QSIs decreased the rhamnolipid production, elastasethe elastase activity activity significantly to about 33.98(Figure%, comparable9b). All the testedto C30 QSIs(37.65%). decreased C7X and the s rhamnolipidappanol also production,reduced the especially butein which is comparable to that of C30 (Figure7C). especiallyelastase activity butein significantly which is comparable (Figure 9b to). that All theof C30 tested (Figure QSIs 7C).decreased the rhamnolipid production, especially butein which is comparable to that of C30 (Figure 7C).

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FigureFigure 9.9. EEffectsﬀects ofof QSIsQSIs onon virulencevirulence factorsfactors afterafter 2424 hh (n(n == 3). ( a) Pyocyanin production; (b) Elastase;Elastase; ((cc)) Rhamnolipid.Rhamnolipid. Reduction in the QS-regulatedQS-regulated virulence factors in thethe presencepresence ofof buteinbutein (B)(B) waswas observed.observed. B Buteinutein reduced the pyocyanin production more signiﬁcantlysignificantly than C30,C30, sappanolsappanol (S)(S) andand control (C). Data shown as mean SD of triplicate wells ** p < 0.01 vs. control. control (C). Data shown as mean ±± SD of triplicate wells ** p < 0.01 vs. control.

2.4. Inhibition on QS-Related Gene Expression To investigate the impact of QSIs (100 μM) on the genes related to the quorum sensing in P. aeruginosa, qRT-PCR studies were performed. The effect of QSIs on lasI, lasR, rhlI, rhlR, and proC genes

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2.4. Inhibition on QS-Related Gene Expression

Int. J. Mol.To investigateSci. 2020, 21, x theFOR impactPEER REVIEW of QSIs (100 µM) on the genes related to the quorum sensing12 of in22 P. aeruginosa, qRT-PCR studies were performed. The eﬀect of QSIs on lasI, lasR, rhlI, rhlR, and proC wasgenes evaluated. was evaluated. Data suggested Data suggested that the that butein the butein was wasable ableto significantly to signiﬁcantly suppress suppress the theexpression expression of lasof RlasR andand rhlrhlRR (Figure(Figure 10), 10 whereas), whereas laslasII andand rhlrhlII werewere found found to tobe becomparatively comparatively less less expressed expressed..

Figure 10. The influenceinfluence ofof QSIs QSIs on on the the quorum quorum sensing sensing (QS) (QS) genes. genes. qRT-PCR qRT-PCR studies studies revealed revealed that thatlasR lasRand rhlRand wererhlR were inhibited inhibited significantly significantly when comparedwhen compared to lasI andto lasI rhlI and(n = rhlI3). The(n = level3). The of QS-regulatedlevel of QS- gene expression was suppressed by QSIs especially by butein (B) (100 µM) when compared to the regulated gene expression was suppressed by QSIs especially by butein (B) (100 μM) when compared untreated control (C), sappanol (S) and C30. Data shown as mean SD of triplicates ** p < 0.01 to the untreated control (C), sappanol (S) and C30. Data shown as mean± ± SD of triplicates ** p < 0.01 vs. control. vs. control. 2.5. Microscale Thermophoresis (MST) Analysis 2.5. Microscale Thermophoresis (MST) Analysis For the LasR protein expression, we used RecET technology to clone the target directly. LasR-encodingFor the LasR gene proteinlasR fromexpression, genomic we DNA used ofRecETP. aeruginosa technologytransferred to clone into the thetarget expression directly. vectorLasR- encodingand expressed gene successfullylasR from genomic in E. coli DNA(Figure of P. S1). aeruginosa The purified transferred LasR was into used the forexpression MST measurement. vector and expressedMST experiment successfully was carried in E. coli out (Figure to investigate S1). The the purified bio-molecular LasR was interaction used for MST between measurement. LasR and MST QSIs. experimentVariations in was normalized carried fluorescence out to investigate of the bound the bio and-molecular unbound interaction states will provide between the LasR fraction and bound QSIs. Variationsand thus the in dissociation normalized constant fluorescence is calculated. of the bound All values and areunbound multiplied states by will a factor provide of 1000 the to fraction get the boundrelative and fluorescence thus the dissociation change in per constant thousand. is calculated. All values are multiplied by a factor of 1000 to getMST the relative results revealed fluorescence that allchange the QSIs in per have thousand. significant binding ability with LasR. It is noteworthy to mention that the dissociation constant (K ) of C7X is 933 369 nM which is comparatively lesser MST results revealed that all the QSIs haveD significant binding± ability with LasR. It is noteworthy than other QSIs tested whereas the K of 3OC12-HSL is 271 58 nM (Figure 11a,e). Surprisingly, to mention that the dissociation constantD (KD) of C7X is 933 ± 369± nM which is comparatively lesser the Fnorm value of sappanol and butein are in same downward trend with the fitted K of 5730 than other QSIs tested whereas the KD of 3OC12-HSL is 271 ± 58 nM (Figure 11a,e). Surprisingly,D the± 2870 nM and 3481 1631 nM respectively and the dose dependent downward trend is similar to Fnorm value of sappanol± and butein are in same downward trend with the fitted KD of 5730 ± 2870 that of 3OC12-HSL (Figure 11b,c) whereas C30 (K = 20425 16003 nM) has a different upward nM and 3481 ± 1631 nM respectively and the dose dependentD ±downward trend is similar to that of 3OC12trend in-HSL the normalized(Figure 11b,c fluorescence.) whereas C30 In (K upwardD = 20425 trend ± 16003 the fluorescentnM) has a different molecules upward diffuse trend away in from the normalized(positive thermophoresis) fluorescence. In and upward in downward trend the trend fluorescent toward (negative molecules thermophoresis) diffuse away from the heat (positive focus. Wethermophoresis) speculate that and upward in downward trend of C7X trend and towardC30 is possibly (negative caused thermophoresis) by an irreversible the heat binding focus. mode. We speculate that upward trend of C7X and C30 is possibly caused by an irreversible binding mode.

2.6. Thermal Shift Assay (TSA) We conducted the thermal unfolding studies in the presence and absence of the inhibitors with a temperature gradient of 0.05 °C/s from 25 to 99 °C. No shift in ΔT occurred upon the addition of the DMSO as negative control solvent. Boltzmann Tm (Tm B) is the melting temperature (°C) calculated by fitting data in the region of analysis to the Boltzmann equation.

A large shift in the Tm B (3.88 °C) (Figure 12a,b) occurred when 3OC12-HSL was added to LasR as positive control, indicating strong interaction between LasR and 3OC12-HSL. SYPRO orange

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 13 of 22 seems to be normal, whereas Protein Thermal Shift™ Dye produces comparatively lower florescence suggesting strong interaction between LasR and 3OC12-HSL that in turn forbids the exposure of the hydrophobic region of LasR protein where the assay dye was supposed to bind (Figure 12a). C7X and Int.sappanol J. Mol. Sci. has2020 Tm, 21 B, 2190 of 2.2°C and 3.1°C (Figure 12b) respectively suggesting a potential interaction13 of 22 with LasR.

C7X Sappanol

(a) (b)

Butein C30

3OC12-HSL

(e)

Figure 11. The molecular interaction of QSIs with LasR protein. (a) C7X binds to LasR with a K of Figure 11. The molecular interaction of QSIs with LasR protein. (a) C7X binds to LasR with a KDD of 933 369; (b) Sappanol binds to LasR with a KD of 5730 2870 nM; (c) Butein binds to LasR with a KD of 933±369± ; (b) Sappanol binds to LasR with a KD of 5730±2870± nM; (c) Butein binds to LasR with a KD of 3481 1631 nM; (d) C30 binds to LasR with a K of 20425 16003 nM; (e) 3OC12-HSL binds to LasR ± D ± with a K of 271 58 nM. D ±

2.6. Thermal Shift Assay (TSA) We conducted the thermal unfolding studies in the presence and absence of the inhibitors with a temperature gradient of 0.05 ◦C/s from 25 to 99 ◦C. No shift in ∆T occurred upon the addition of the Int. J. Mol. Sci. 2020, 21, 2190 14 of 22

DMSO as negative control solvent. Boltzmann Tm (Tm B) is the melting temperature (◦C) calculated by ﬁtting data in the region of analysis to the Boltzmann equation. A large shift in the Tm B (3.88 ◦C) (Figure 12a,b) occurred when 3OC12-HSL was added to LasR as positive control, indicating strong interaction between LasR and 3OC12-HSL. SYPRO orange seems to beInt. normal, J. Mol. Sci. whereas 2020, 21 Protein, x FOR PEER Thermal REVIEW Shift ™ Dye produces comparatively lower ﬂorescence suggesting14 of 22 strong interaction between LasR and 3OC12-HSL that in turn forbids the exposure of the hydrophobic region3481±1631 of LasR protein nM; (d) where C30 binds the to assay LasR dye with was a KD supposed of 20425±16003 to bind nM (Figure; (e) 3OC12 12a).-HSL C7X binds and to sappanol LasR with has Tm B ofa K 2.2D of◦ C271±58 and 3.1nM.◦ C (Figure 12b) respectively suggesting a potential interaction with LasR.

(a) (b)

Figure 12. Thermal-shift analyses of purified LasR with QSIs. (a) Boltzmann Tm (Tm B) of the tested compounds. Data show that the interaction of sappnol (S) and C7X with LasR is comparatively strong as 3OC12-HSL-LasR interaction. No fluorescence was observed when treated with butein (B); Figure 12. Thermal-shift analyses of purified LasR with QSIs. (a) Boltzmann Tm (Tm B) of the tested (b) normalized fluorescence data represent a strong interaction with 3OC12-HSL-LasR, whereas the compounds. Data show that the interaction of sappnol (S) and C7X with LasR is comparatively strong florescence revealed that the 3OC12-HSL-LasR interaction is strong enough to forbid the exposure of as 3OC12-HSL-LasR interaction. No fluorescence was observed when treated with butein (B); (b) hydrophobic binding pocket where the dye used on this study supposed to bind. normalized fluorescence data represent a strong interaction with 3OC12-HSL-LasR, whereas the 3. Discussionflorescence revealed that the 3OC12-HSL-LasR interaction is strong enough to forbid the exposure of hydrophobic binding pocket where the dye used on this study supposed to bind. Plants have always been a great source for novel drug compounds, as plant derived medicines have3. Discussion made huge aids to human health. In this study we identified and evaluated three QSIs against P. aeruginosa from natural products database. Catechin-7-Xyloside (C7X) is flavan-3-ols, from SpiraeaPlants hypericifolia have always. Sappanol been is a agreat 3, 4-dihydroxyhomoisoflavan, source for novel drug compounds, from Caesalpinia as plant sappanderived. Buteinmedicines is ahave chalcone, made from hugeToxicodendron aids to human vernicifluum, health. In thisand study C30 as we a knownidentified inhibitor and evaluated against P. three aeruginosa QSIs against[17], isP (Z-)-4-Bromo-5-(bromomethylene)-2(5H)-furanone.. aeruginosa from natural products database. Catechin-7-Xyloside (C7X) is flavan-3-ols, from Spiraea hypericifoliaTargeting. Sappanol QS is the is mosta 3, 4-dihydroxyhomoisoflavan, extensively studied alternative from Caesalpinia strategy and sappan demonstrated. Butein is a chalcone, against variousfrom Toxicodendron multi-drug resistant vernicifluum pathogens., and C30 Since as QSIs a known curb severalinhibitor virulence against factors P. aeruginosa and biofilm [17], withoutis (Z-)-4- affBromoecting- growth5-(bromomethylene) unlike antibiotics,-2(5H) it leads-furanone to the. attenuation of pathogenesis while preventing selective pressureTargeting and development QS is the of most antibiotic extensively resistance studied [18]. Hence, alternative QS interference strategy and seems demonstrated to be a promising against approachvarious multi to address-drug there resistant MDR pathogens pathogens. Since and canQSIs be curb developed several asvirulence standalone factors anti-infective and biofilm drugs without or inaffecting combination growt withh unlike conventional antibiotics, antibiotics. it leads to the attenuation of pathogenesis while preventing selectiveHere, pressure it is noteworthy and development to mention of that antibiotic docking resistance results revealed [18]. Hence, butein QS was interference able to form seems H-bond to be witha promising carbonyl oxygenapproach of Tyrto address 47. McCready there MDR et al., pathogens 2018, reported and thatcan Tyrbe developed 47 present inas loopstandalone L3 of LasR anti- isinfective able to adopt drugs multiple or in combination conformations with and conventional hence it can antibiotics. accommodate the different ligands [23]. When LasR Tyr 47 was mutated with other amino acids such as Ser and Arg, the sensitivity to towards AHLs was foundHere, to it be is decreased.noteworthy These to mention results that suggested docking that results the interactions revealed b ofutein the was acyl able side to chain form with H-bond the loopwith region carbonyl promotes oxygen increased of Tyr 47. protein McCready stability, et al., in turn2018, activates reported LasR. that Tyr It is 47 notable present that in theloop packing L3 of LasR of Tyris 47able against to adopt the acylmultiple chain conformations protects the LBD and from hence the it bulk can solvent accommodate and alterations the different at L3 loopligands region, [23]. leadingWhen LasR to protein Tyr 47 instability was mutated [12]. with Also, other LasR amino is agonized acids such by ligands as Ser and that Arg, were the able sensitivity to form to H-bonds towards withAHLs Trp60 was and found Asp73 to be residues, decreased. and These antagonized results ifsuggested they interacted that the with interactions Tyr 47. of the acyl side chain with the loop region promotes increased protein stability, in turn activates LasR. It is notable that the packing of Tyr 47 against the acyl chain protects the LBD from the bulk solvent and alterations at L3 loop region, leading to protein instability [12]. Also, LasR is agonized by ligands that were able to form H-bonds with Trp60 and Asp73 residues, and antagonized if they interacted with Tyr 47.

Int. J. Mol. Sci. 2020, 21, 2190 15 of 22

Paczkowski et al., 2019 revealed that Thr 75, Tyr 93, and Ala 127 were able to convert low-potency QSIs into high-potency compounds and inactive ligands into low-potency QSIs by mutational analysis [24]. Here, we found that sappanol and butein have potential interaction with Thr 75. The acyl side chain has van der Waals interactions with the following hydrophobic pocket residues such as Leu 36, Leu 40, Tyr 47, Ile 52, Val 76, and Leu 125 at the end of the LasR LBD. These interactions seem to be crucial since they are involved in stabilization of the LasR–3OC12-HSL complex [25]. Our results also revealed that C7X and sappanol were able to interact with Leu 125. Overall docking results revealed that these compounds were able to interact with Tyr 47, Thr 75 and Leu 125, especially sappanol which interacts with both Thr 75 and Leu 125 whereas the butein is able to form H bonds with Tyr 47 and Thr 75. Thus our findings are in consistent with the earlier reports [25,26] suggesting that these residues which are mostly in hydrophobic region upon interactions with these QSIs may destabilize the LasR protein. Many natural products of plant origin were reported to be QSIs but their specificity towards LasR or any other quorum regulators has been poorly investigated. Ouyang et al., 2015 reported that the pyocyanin production was decreased by quercetin (16 µg/mL) by 58% [27]. Curcumin decreased the biofilm formation and decreased the elastase by 2 fold when administered with 1–3 µg/mL [28]. Vandeputte et al., 2010 reported that naringenin (4 mM) reduced the QS-regulated virulence factors especially pyocyanin and elastase [29]. Kim et al., 2015 found that the rhamnolipid production was suppressed by almost 36%–60% when they administered 6-Gingerol (0.1–100 µM) [30]. Paczkowski et al., 2017 identified that flavonoids were able to down regulate many virulence related traits, and especially flavonoids possessing dihydroxyl moieties such as baicalein and quercetin were able to inhibit QS by interacting with LasR [26]. It is noteworthy to mention that C7X and Sappanol are flavonoids, whereas butein is chalcone. Our study revealed that all the tested QSIs were able to reduce the biofilm, pyocyanin, elastase, and rhamnolipid production at the minimal concentration when compared to earlier studies. Numerous studies have been carried out in search of potential QSIs over phytochemicals sources, in which all the virulence related traits shown to be decreased [16,31,32]. Natural products including furonones, quercetin, naringenin, curcumin, catechin, were reported to be QSIs with varying activity range. Most of the studies were focused on measuring the biofilm and virulence phenotypes but no further efforts have been taken to evaluate mode of action and understand the biomolecular interactions of these QSIs the respective molecular targets. CLSM and SEM results revealed that these QSIs are able to reduce the biofilm which confirms our in vitro studies. Kim et al., 2015 also observed similar kind of inhibition using 6-gingerol [30]. Packiavathy et al., 2012 showed that eugenol inhibited the biofilm [33] and Sarabhai et al., 2013 showed that ellagic acid reduced the biofilm formation [32]. It is important to note that our study showed significant QSI effect at lower concentrations of phytochemicals when compared to aforesaid reports. Kim et al., 2015 showed that lasR and rhlR expression was drastically reduced by 6-gingerol (100 µM) [30]. It is found that quercetin (16 µg/mL) significantly reduced lasI, lasR, rhlI, and rhlR expression [23]. O’Loughlin et al., 2013 showed that mBTL decreased the QS-related genes [19]. In our study, it is observed that all the tested QSIs were able to influence the QS-regulated genes, especially butein significantly down-regulated lasI, lasR, rhlI, and rhlR genes. In order to affirm the specificity of these QSIs on LasR protein, we have performed MST and TSA. MST revealed that the tested QSIs were potential interaction with LasR protein. The strong interaction of C7X is coherent with our docking results since it has the better GScore with 4 H-bonds with Tyr 56, Tyr 64, Tyr 93, and Leu 125. Overall results of MST revealed the strong interaction of these QSIs with LasR. It is worthy to mention that sappanol and butein have a similar downward trend and they both were able to form H-bond Thr 75. Though C30 was able to form only one H-bond (Ser 123), it significantly curbed the QS pathway. We assume that C30 occupies the AHL binding pocket by interacting with Ser 129 which forbids 3OC12-HSL’s entry into the binding pocket. Paczkowski et al., 2017, reported the importance of Thr 75 in suppressing quorum sensing through interacting with LasR [26]. To our knowledge, this is the first report to use MST to study interaction Int. J. Mol. Sci. 2020, 21, 2190 16 of 22 between QSIs with LasR. The movement of molecules in a temperature gradient can be studied using MST, and the movement could be tracked by a fluorophore which can covalently interact with the protein. The interaction of proteins and ligands modifies this movement and thus MST can be used to detect binding ability of the molecules. The thermophoresis of a protein significantly differs from the thermophoresis of a protein-ligand complex as the binding induces fluctuations in size, charge and energy. Even if the interaction is not able to change the size or charge of a protein significantly, MST can still detect the binding due to binding-induced changes in the solvation entropy of the molecules. MST requires relatively low amounts of materials and that is advantageous over conventional instruments such as ITC and SPR [34]. TSA clearly indicates the biomolecular interaction of LasR and QSIs. Unfortunately, we found no fluorescence when treated with butein and it may be due to the interaction of butein with Tyr 47, a very crucial amino acid present in L3 loop. Since Tyr-47 is positioned in the L3 loop region which covers the LBD, the Tyr-47 interaction in the absence of the autoinducer would result in loop flexibility, and subsequent exposure of the hidden hydrophobic residues in LBD resulting in protein aggregation [35]. Compounds that induce inward moment of L3 loop with the bond length of 3.5–3.9Å found to be potential LasR inhibitors [12]. In MST we could not find any misfolding of LasR since the fluorescent dye, its interaction with protein and buffer system are entirely different from Thermal shift assay system. Hence docking results revealed the interaction between butein and Tyr 47 and the bond length of hydroxyl group of butein with carbonyl oxygen of Tyr 47 is 2.21Å (Figure S2), we assume that butein induce an inward moment of L3 loop of LasR. We also speculate that butein induces the movement of L3 loop leading to misfolding of LasR, in turn inducing protein aggregation. In our previous work [36], we have reported that C7X, sappanol and butein inhibited the QS mechanism in Chromobacterium violaceum. Though they might have different mode of interaction with CviR in C. violaceum from LasR in P. aeruginosa, their QS inhibition against both pathogenic bacteria implied that they might represent a broad spectrum anti-infective activity. Structurally, Catechin-7-Xyloside (C7X) is flavan-3-ols, Sappanol is a 3, 4-dihydroxyhomoisoflavan, and butein is a chalcone. Chinese herbal products have been studied for many medical problems. The plants from which the QSIs have been identified are being used in traditional Chinese medicine (TCM) especially Toxicodendron vernicifluum used for the treatment of gastritis, stomach cancer, and atherosclerosis [37]. Paczkowski et al., 2017 revealed that flavonoids can potentially interact with LasR and suppress the quorum sensing in P. aeruginosa [26]. Among these QSIs, C7X, and sappanol are flavonoids whereas butein is a chalcone which showed comparatively significant quorum sensing inhibitory activity, implying structural diversity of potential QS-inhibitors.

4. Materials and Methods

4.1. Bacterial Strains and Growth Conditions Pseudomonas aeruginosa (PAO1) was a wild type strain and used as test organism in this study and grown in Lysogeny Broth (LB) medium for 24 h at 37 ºC unless otherwise mentioned. Test compounds Catechin 7-xyloside (C7X), sappanol, and butein were purchased from Chemfaces, China (http://www.chemfaces.cn). N-(3-oxo-dodecanoyl)-l-homoserine lactone (3OC12-HSL), (Z-)-4-Bromo-5-(bromomethylene)-2(5H)-furanone (C30), and other chemicals were purchased from Sigma-Aldrich, (Shanghai, China) unless otherwise stated.

4.2. In Silico Studies Molecular docking was performed using the Schrodinger suite software (Glide module V 8.2, New York, NY, USA) [38]. LasR protein (PDB 2UV0) was docked against natural products database. The ligands were prepared using LigPrep and the protein was processed further with Protein Preparation wizard. The receptor grid was generated based on 3OC12-HSL position in the crystal structure with the following residues present in the active site Tyr 56, Trp 60, Asp 73, and Ser 129. HTVS (High Int. J. Mol. Sci. 2020, 21, 2190 17 of 22

Throughput Virtual Screening) was performed to identify the QSIs and the XP (Extra Precision) was performed to further predict the interactions between these QSIs and LasR protein.

4.3. In Vitro Assays

4.3.1. Biofilm Inhibition Assay Biofilm formation was measured using the microtitre plate assay to study the efficacy of QSIs [36,39]. Briefly, overnight cultures of P. aeruginosa PAO1 (OD600 nm = 0.4) were added into 1 mL of fresh LB medium with QSIs in different concentrations (1, 10, and 100 µM). Bacteria were grown up for 24 h at 37 ºC without any agitation. To remove the free-floating planktonic cells, the microtitre plate was washed with PBS (pH 7.4) after incubation. The biofilm was stained by adding 200 µL of crystal violet (0.1%) solution. Crystal violet solution was removed after 15 min and 200 µL of ethanol (95%) was added. The absorbance at OD470 nm was read using a microplate reader (Infinite M200, Tecan, Männedorf, Switzerland).

4.3.2. Pyocyanin Production From overnight cultures of P. aeruginosa PAO1 were grown with or without QSIs (1, 10, and 100 µM) in LB broth at 37 ◦C for 24 h. Then, the supernatants were collected, and the pyocyanin was extracted by using chloroform, followed by the addition of 0.2 M HCl. The crude extract obtained appears to be a pink solution, and the absorbance at OD520 nm was read [30].

4.3.3. Elastase Activity The elastolytic activity was measured using elastin congo red (ECR) as substrate. Briefly, the P. aeruginosa PAO1 were grown in LB broth at 37 ◦C for 24 h with or without QSIs (1, 10, and 100 µM). After the incubation period, centrifuged at 15,000 g at 4 ◦C for 10 min and then the resultant supernatant (0.5 mL) was added to 1 mL of assay buffer (30 mM Tris buffer, pH 7.2) which contains 10 mg of ECR. The reaction mixture was then incubated for 6 h at 37 ◦C with agitation. Centrifugation at 1200 g for 10 min was done to remove insoluble substrate and absorbance of the supernatant was measured at OD495 nm [40].

4.3.4. Rhamnolipid Assay Rhamnolipid was measured as previously reported with minor modiﬁcations [41]. The overnight culture of P. aeruginosa PAO1 was inoculated in LB medium with or without QSIs (1, 10 and 100 µM) and incubated at 37 ◦C for 24 h with agitation. After incubation, the cultures were centrifuged at 12,000 g at 4 ◦C for 5 min after 24 hrs. 500 µL of cell supernatants were added to 1 mL of diethyl ether (100%) and then the ether fraction was evaporated. The resultant fraction was eluted with 500 µL ddH2O, and then 100 µL of the subsequent eluting fraction was mixed with 900 µL orcinol solution (0.19% orcinal in 53% H2SO4). The reaction mixture was boiled for 30 min, and then cooled for 15 min at RT. Rhamnolipid production was estimated by measuring OD421nm in a spectrophotometer.

4.4. Confocal Laser Scanning Microscopy Analysis Confocal Laser Scanning Microscopy (CLSM) analysis of the P. aeruginosa PAO1 biofilms was performed as reported earlier [42]. Briefly, P. aeruginosa PAO1 was inoculated (1: 100) in LB broth on the cover slips to form static biofilms and incubated overnight at 37 ºC inside of the 6-well plates with or without QSIs (100 µM). The biofilms were washed twice before to remove loosely bound cells and stained with FITC-ConA for 15 min. Then the cells were washed twice with PBS to remove the excess stains and the biofilms were analyzed using CLSM (Zeiss L800, Zeiss, Tokyo, Japan) with the excitation and emission wavelength of 488 nm and 520 nm respectively. Int. J. Mol. Sci. 2020, 21, 2190 18 of 22

4.5. SEM Analysis The effect of QSIs on biofilm formation was visualized by scanning electron microscopy (SEM) as described by Singh et al., 2017 [43]. Biofilms of P. aeruginosa PAO1 were grown on glass coverslips in 6 well plates immersed in LB broths with or without QSIs (100 µM) for 24 h at 37 ºC. After incubation, the cover slips with biofilm were incubated with 2.5% glutaraldehyde for 20 min followed by 4% OsO4 in 0.1 M phosphate buffer for 30 min. Then the samples were dehydrated with a gradient ethanol series (10%–95%) for 10 min. The dried biofilms were coated with gold and visualized under SEM (VEGA 3, TESCAN USA, Inc., Kohoutovice, Czech Republic).

4.6. qRT-PCR Studies P. aeruginosa PAO1 cells were grown in 1 mL LB medium with or without QSIs (100 µM) at 37 ºC for 24 h. Total RNA was extracted with the RNA isolation kit as per the manufacturer’s instructions (TIANGEN Biotech Co., Ltd., Beijing, China). Primers for lasI, lasR, rhlI, rhlR, and proC are listed in Table S1 and were synthesized by Sangon Biotech (Shanghai, China). The reverse transcription reaction was performed using a Prime Script RT Reagent Kit (TaKaRa, Tokyo, Japan) using total RNA as a template for at 37 ◦C for 15 min for three times (reverse transcription), and at 85 ◦C for 5 min (inactivation of reverse transcriptase), with a total volume of 20 µL. The qRT-PCR was performed with the SYBR® Premix Ex TaqTM II Kit (TaKaRa, Japan) as per the manufacturer’s protocol. The cDNA was used as a template for qRT-PCR, and the total reaction system (20 µL) was made up of the following: 10 µL SYBR® Premix Ex TaqTM II (2 ), 0.8 µL forward primer, × 0.8 µL reverse primer, 0.4 µL ROX Reference Dye (50 ), 2 µL cDNA template, and 6 µL double-distilled × H2O (ddH2O). The qRT-PCR was performed using Applied Bio systems QuantStudio™ 3 Real-Time PCR System (Applied Biosystems Inc., Waltham, MA, USA). The reaction conditions used were as follow: pre-denaturation at 95 ◦C for 30 s, followed by 40 cycles of denaturation at 95 ◦C for 5 s and annealing and extension at 60 ◦C for 30 s. proC was used as an internal reference [26]. The relative mRNA expression of all the genes was calculated using the 2 ∆∆CT method and the experiment was − independently conducted in triplicates.

4.7. LasR-LBD Expression and Purification All recombination procedures were performed based on LLHR (linear plus linear homologous recombination) of Red/ET recombineering established in our lab [44] (Figure S3), and related cloning primers were listed on Table S2. Proteins were purified based on the protocols by Lugo et al., 2017 [45] and the ÄKTA explorer system mount with Superdex 75 10/300 was used for further purification and buffer exchanging (Tm shift buffer: 50 mM K2HPO4/KH2PO4, 150 mM NaCl, 1mM DTT, pH 7.8, MST buffer: 50 mM Tris-Hcl 400 mM NaCl, 10 mM MgCl2 pH 7.8). After purification, SDS-PAGE analysis was done.

4.8. Microscale Thermophoresis The interactions between QSIs and LasR were analyzed with the concentration gradient of 50 µM of QSIs with 20 µM of LasR, which was labeled with Monolith NT™ Protein Labeling Kit RED-NHS before analysis. The optimized buffer for MST (50 mM Tris-HCl, 150 mM NaCl, 10 mM MgCl2, 0.05% Tween 20) and LED power (20%) was used. Analysis was performed on Monolith NanoTemper (NT) 115 (NanoTemper Technologies GmbH, Munich, Germany) and standard-treated 4 µL-volume glass capillaries were employed to measure the molecular interactions. The fluorescence intensity was obtained by the MST measurement fitted using NT 1.5.41 analysis software and the resultant Kd values were given together with an error estimation [46]. Int. J. Mol. Sci. 2020, 21, 2190 19 of 22

4.9. Thermal Shift Assay (TSA) The interaction of QSIs with LasR-LBD have been measured by Protein Thermal Shift Assay (Thermofisher Scientific, Waltham, MA, USA) as per manufacturers’ protocol. Using 20 µM the test compounds and 4 µM protein, TSA replicates were set and compared with the none-compound groups and none-protein groups. The thermal shift assay was conducted in Applied Bio systems QuantStudio™ 3 Real-Time PCR System (Applied Biosystems Inc., Waltham, MA, USA). The protein melt reaction mixture was added to the wells of the 96-well PCR plate and then heated from 25 to 99 ◦C with a heating rate of 0.05 ◦C/S. The fluorescence intensity was measured with Ex/Em: 580/623 nm. Tm data were generated using the Boltzmann method and ∆Tm was calculated by comparing the Tm values for LasR-LBD without ligand to those of LasR with ligand. Data were collected at 1 ◦C intervals from 25 ◦C through 99 ◦C on the Real Time PCR System and were analyzed using Protein Thermal ShiftTM Software v1.3 (Thermofisher Scientific (Applied Biosystems), Waltham, MA, USA).

4.10. Statistical Analysis Graph pad prism software (v6.01) (GraphPad, San Diego, CA, USA) was used for statistical analysis. Multiple comparisons and one way ANOVA were applied wherever required. P-values (<0.05 and <0.001) were considered as statistically significant. All assays were performed in triplicates and the results were expressed as mean SD. ± 5. Conclusions In view of the emergence of multi-drug resistant pathogens, it is necessary to develop novel strategies to find effective drugs against these multi-drug resistant pathogens. Since natural products continually play a major role in medicine and human health, virtual screening of natural products against the molecular drug targets will be a productive approach towards drug designing. Overall results suggested that C7X, sappanol and butein are potential LasR-mediated quorum sensing inhibitors against P. aeruginosa, based on our in silico and in vitro assays, and further confirmation by CLSM and SEM studies. MST and Thermal shift assay confirmed bimolecular interactions of QSIs with LasR protein. Though C7X and sappanol show similar trends with C30, a known inhibitor, we speculate that butein induce misfolding and followed by aggregation of LasR protein by interacting with Tyr 47 present in L3 loop to allow inward moment of L3 loop. It is very clear that C7X and sappanol were down regulating the QS by potentially interaction with LasR protein of P. aeruginosa whereas further investigations are much warranted to ensure the mode of action of these QSIs in vivo. Since homologues in LuxR-family are present in more than 100 g negative pathogens, these QSIs here may be developed as a broad spectrum anti-infective drug candidates. It is evident that starting with biological evaluation, gene expression studies and molecular interactions using MST and thermal shift assay will help us to get an in-depth understanding of these QSIs and their proposed mode of action. Considering the multi-drug resistance emergence in P. aeruginosa, it is essential to further evaluate these QSIs in animal models.

Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/21/6/2190/ s1. Author Contributions: Conceptualization, V.R., X.B., Y.Z., A.L.; Formal analysis, L.Z., V.R.; Funding acquisition, V.R.,X.B., Y.Z., A.L; Investigation, L.Z., V.R.,H.W. and Y.Z.; Methodology, L.Z., V.R.and N.Z.; Project administration, V.R. and A.L.; Resources, X.B., Y.Z., A.L.; Software, V.R.; Supervision, X.B., Y.Z., A.L. and H.W.; Writing—original draft, L.Z., V.R., X.B., Y.Z. and A.L., Writing—review and editing, V.R., H.W., X.B., Y.Z., A.L. All authors have read and agreed to the published version of the manuscript. Funding: This study was supported by grants from National Key Research and Development Program of China (2019YFA0905700, 2018YFA0900400) and China post-doctoral grant (2015M582103). This study also supported by the National Natural Science Foundation of China (31670098 and 31670097), the Shandong Province Natural Science Foundation (ZR2019JQ11 and ZR2017MC031). This work was also supported by the Recruitment Program of Global Experts (Y.Z.) and the Qilu Youth Scholar Startup Funding of Shandong University (X.B.). We also thank Int. J. Mol. Sci. 2020, 21, 2190 20 of 22 the financial support from the Open Project Program of State Key Laboratory of Bio-based Material and Green Papermaking (KF201825) and the 111 Project (B16030). Acknowledgments: We would like to thank Wei Qian (Institute of Microbiology, Chinese Academy of Sciences) for providing the MST facility. Conflicts of Interest: The authors declare no conflict of interest

Abbreviations

QS Quorum sensing QSIs Quorum sensing inhibitors C7X Catechin-7-xyloside MDR Multi-drug resistant AMR Antimicrobial resistance AIs Autoinducers AHL Acyl homoserine lactone LBD Ligand binding domain DBD DNA-binding domain 3OC12-HSL N-(3-oxododecanoyl)-L-homoserine lactone C4-HSL N-butanoyl-L-homoserine lactone CLSM Confocal laser scanning microscopy SEM Scanning electron microscope MST Microscale thermophoresis KD Dissociation constant TSA Thermal shift assay Tm B Boltzmann Tm ECR Elastin congo red

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