Investigating the Modes of Action of the Antimicrobial Chalcones BC1 and T9A

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Article Investigating the Modes of Action of the Antimicrobial Chalcones BC1 and T9A

1, 2, 2 3 Luana G. Morão †, André S. G. Lorenzoni †, Parichita Chakraborty , Gabriela M. Ayusso , Lucia B. Cavalca 2, Mariana B. Santos 3, Beatriz C. Marques 3, Guilherme Dilarri 1 , Caio Zamuner 1, Luis O. Regasini 3, Henrique Ferreira 1,* and Dirk-Jan Scheffers 2,* 1 Departamento de Bioquímica e Microbiologia, Instituto de Biociências, Universidade Estadual Paulista, 130506-900 SP Rio Claro, Brazil; [email protected] (L.G.M.); [email protected] (G.D.); [email protected] (C.Z.) 2 Department of Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, 9747 AG Groningen, The Netherlands; [email protected] (A.S.G.L.); [email protected] (P.C.); [email protected] (L.B.C.) 3 Departamento de Química e Ciências Ambientais, Instituto de Biociências, Letras e Ciências Exatas, Universidade Estadual Paulista, 15054-000 SP São José do Rio Preto, Brazil; [email protected] (G.M.A.); [email protected] (M.B.S.); [email protected] (B.C.M.); [email protected] (L.O.R.) * Correspondence: [email protected] (H.F.); d.j.scheff[email protected] (D.-J.S.); Tel.: +31-50-3632319 (D.-J.S.) These authors contributed equally to this work. † Academic Editors: Paola Di Donato and Brigida Silvestri Received: 7 September 2020; Accepted: 6 October 2020; Published: 9 October 2020 Abstract: Xanthomonas citri subsp. citri (X. citri) is an important phytopathogen and causes Asiatic Citrus Canker (ACC). To control ACC, copper sprays are commonly used. As copper is an environmentally damaging heavy metal, new antimicrobials are needed to combat citrus canker. Here, we explored the antimicrobial activity of chalcones, specifically the methoxychalcone BC1 and the hydroxychalcone T9A, against X. citri and the model organism Bacillus subtilis. BC1 and T9A prevented growth of X. citri and B. subtilis in concentrations varying from 20 µg/mL to 40 µg/mL. BC1 and T9A decreased incorporation of radiolabeled precursors of DNA, RNA, protein, and peptidoglycan in X. citri and B. subtilis. Both compounds mildly affected respiratory activity in X. citri, but T9A strongly decreased respiratory activity in B. subtilis. In line with that finding, intracellular ATP decreased strongly in B. subtilis upon T9A treatment, whereas BC1 increased intracellular ATP. In X. citri, both compounds resulted in a decrease in intracellular ATP. Cell division seems not to be affected in X. citri, and, although in B. subtilis the formation of FtsZ-rings is affected, a FtsZ GTPase activity assay suggests that this is an indirect effect. The chalcones studied here represent a sustainable alternative to copper for the control of ACC, and further studies are ongoing to elucidate their precise modes of action.

Keywords: chalcone; Asiatic citrus canker; Bacillus subtilis; Xanthomonas citri

1. Introduction Brazil is the world leader in sweet orange production, exporting more than 80% of concentrated orange juice consumed in the world. Xanthomonas citri subsp. citri, the causative agent of Asiatic Citrus Canker (ACC), is a bacterial phytopathogen that can infect all the commercially important species of sweet orange, and is a major threat to the orange juice industry [1]. The most eﬀective way to control the disease is to eradicate infected trees. However, in the current Brazilian legislation, eradication has been substituted by a set of combined measures known as risk mitigation systems, which include periodic

Molecules 2020, 25, 4596; doi:10.3390/molecules25204596 www.mdpi.com/journal/molecules Molecules 2020, 25, 4596 2 of 14 sprays of copper compounds to prevent the spread of X. citri in the orchards [2]. Spraying trees with copper-containing bactericides has led to the emergence of resistant strains [3]. In addition, copper is stable and accumulates in the soil, which has a negative environmental impact [4]. New antimicrobials are needed to tackle the problem of phytopathogens infecting citrus plants. In this study, we focus on the antimicrobial action of chalcones. Chalcones are plant-derived compounds, consisting of two aromatic rings linked by an α, β-unsaturated ketone (enone bridge), and are precursors of flavonoids and isoflavonoids that belong to plant defence mechanisms. Chalcones prevent damage by microorganisms, insects, and animals, and counteract reactive oxygen species, preventing molecular damage [5]. Many chalcones have been studied for their antimicrobial activity [5,6]. Licochalcone A, naturally produced by the roots of Glycyrrhiza inflata has activity against Bacillus subtilis and Staphylococcus aureus, and is able to prevent their growth at 3 µg/mL. However, this compound shows no activity against gram-negative bacteria [7,8]. Licochalcone A was shown to inhibit oxidation of NADH and oxygen consumption in Micrococcus luteus [8], and induce membrane permeability in S. aureus [6]. Despite being inactive against gram-negative bacteria, Licochalcone A is able to inhibit NADH oxidases in membranes isolated from Escherichia coli and Pseudomonas aeruginosa, suggesting that the compound cannot penetrate the outer membrane [7]. Accordingly, some chemically modified chalcones are effective against gram-negative bacteria [5,6], and Mycobacterium tuberculosis [9]. The antitubercular activity is associated with inhibition of the protein tyrosine phosphatase B [9]. Phloretin, a chalcone present in apple and kumquat, is effective only against gram-positive bacteria [10]. This compound inhibits acetate dehydrogenase, isocitrate dehydrogenase, and catalase from S. aureus [10]. DNA gyrase was reported to be a target for synthetic chalcones active against both gram-positive and gram-negative bacteria [11]. Finally, chalcones have antioxidant, anti-inflammatory, antitumoral, antimitotic, cytotoxic, and anti-infection activities, as reviewed in [5,12]. Due to their relatively low redox potential, they have a great probability of undergoing electron transfer reactions. In our continuing search for new antibacterial phenolic compounds, we have synthesized and evaluated hundreds of compounds using antibacterial phenotypic assays against gram-positive and gram-negative species [13–16]. The structural design of our library of curcumin analogs has been based on phenolic functionality, because most of bacterial division inhibitors display at least one phenol ring [17,18]. Among the tested phenolic compounds, the most potent compounds were BC1 and T9A, which were tested against human pathogens and found to be active against gram-positive and gram-negative species (to be published elsewhere). The most active compound against S. aureus, T9A (Figure1, hydroxychalcone 5, Table S1, Supplementary Material), inhibited growth of both bacteria albeit with a 16-fold higher MIC50 for the gram-negative bacterium. Plating of treated cells revealed that the cells were still alive at all concentrations of T9A tested, thus a minimal bactericidal concentration (MBC) for this compound could not be determined, and T9A seems bacteriostatic but not bactericidal for S. aureus. As both BC1 and T9A display antimicrobial activity against the gram-negative phytopathogen X. citri (see below), we decided to further characterize their modes of action. Here, we tested the influence of T9A and BC1 on macromolecular synthesis (DNA, RNA, protein, and peptidoglycan), ATP concentration, and membrane permeability using X. citri and B. subtilis. We chose B. subtilis as a gram-positive model organism, as this is commonly used as a model for mode of action studies, and several methods are established in our labs, including FtsZ purification and FT-IR spectrophotometry [14]. Molecules 2020, 25, 4596 3 of 14 Molecules 2020, 25, x FOR PEER REVIEW 3 of 14

Figure 1. Structures of the compounds used in this study.

2. Results 2.1. Chalcones Inhibit Growth of X. citri and B. subtilis 2.1. Chalcones Inhibit Growth of X. citri and B. subtilis The minimum inhibitory concentrations (MIC) of BC1 and T9A for X. citri and B. subtilis were The minimum inhibitory concentrations (MIC) of BC1 and T9A for X. citri and B. subtilis were determined using the broth microdilution method (Table1). Overall, X. citri (MICs of BC1 = 90 µg/mL, determined using the broth microdilution method (Table 1). Overall, X. citri (MICs of BC1 = 90 μg/mL, and T9A = 50 µg/mL) was slightly more resistant to the compounds when compared to B. subtilis and T9A = 50 μg/mL) was slightly more resistant to the compounds when compared to B. subtilis (BC1 = 50 µg/mL, and T9A = 40 µg/mL). Nevertheless, both compounds inhibited X. citri and B. subtilis (BC1 = 50 μg/mL, and T9A = 40 μg/mL). Nevertheless, both compounds inhibited X. citri and B. with values very close and within the same order of magnitude as the positive control kanamycin subtilis with values very close and within the same order of magnitude as the positive control (~20 µg/mL). Moreover, both BC1 and T9A were bactericidal for X. citri, and only bacteriostatic for kanamycin (~20 μg/mL). Moreover, both BC1 and T9A were bactericidal for X. citri, and only B. subtilis at their respective MICs. Compared to our earlier results (Table S1, supplementary material), bacteriostatic for B. subtilis at their respective MICs. Compared to our earlier results (Table S1, we note that B. subtilis seemed less resistant to T9A compared to the gram-positive model S. aureus, supplementary material), we note that B. subtilis seemed less resistant to T9A compared to the gram‐ whereas X. citri was nearly as resistant to this compound as the gram-negative bacterium P. aeruginosa. positive model S. aureus, whereas X. citri was nearly as resistant to this compound as the gram‐ Though the MIC50 values in Table S1 were assessed by determining the metabolic activity after negative bacterium P. aeruginosa. Though the MIC50 values in Table S1 were assessed by determining incubation for 24 h using the Resazurin Microtiter Assay (REMA), the results cannot be directly the metabolic activity after incubation for 24 h using the Resazurin Microtiter Assay (REMA), the compared, since short incubations already strongly aﬀect the metabolic activity of the cells (see below). results cannot be directly compared, since short incubations already strongly affect the metabolic The MIC values determined here were used throughout to guide our investigations about the modes activity of the cells (see below). The MIC values determined here were used throughout to guide our of action of the compounds. investigations about the modes of action of the compounds.

Table 1. Minimal inhibitory concentrations of the compounds (µg/mL). Table 1. Minimal inhibitory concentrations of the compounds (μg/mL). X. citri B. subtilis CompoundCompound X. citri B. subtilis # BC1BC1 90 *90 * 5050 # # T9AT9A 50 *50 * 4040 # # * *bactericidal; bactericidal; # bacteriostatic.bacteriostatic.

2.2. BC1 Permeabilizes the Membrane of B. subtilis The ability to disrupt the bacterial membrane was assessed by measuring PI permeability in the presence ofof the the compounds. compounds. T9A T9A had had no enoffect effect on the on membrane the membrane of both ofB. both subtilis B. andsubtilisX. citriand, whereasX. citri, BC1whereas did not BC1 aff didect X.not citri affectbut X. did citri permeabilize but did permeabilizeB. subtilis membranes B. subtilis (Tablemembranes2; Figure (Table S1). The2; Figure. di fference S1). inThe permeabilization difference in permeabilization combined with combined the fact that with BC1 the has fact a slightlythat BC1 lower has a MIC slightly for B. lower subtilis MICthan for for B. X.subtilis citri, suggeststhan for thatX. citri BC1, suggests mode of actionthat BC1 may mode be di ffoferent action between may thebe twodifferent organisms. between It hasthe to two be notedorganisms. that measured It has to be membrane noted that permeabilization measured membrane does notpermeabilization necessarily mean does permanent not necessarily damage mean to thepermanent membranes, damage as MICsto the for membranes, membrane as active MICs compounds for membrane that active we measure compounds are generally that we measure tenfold higherare generally than the tenfold concentration higher than at which the concentration 50% of cells have at which membrane 50% of damage cells have [19 ],membrane and B. subtilis damagestill displayed[19], and severalB. subtilis other still activities displayed (respiration, several synthesis other activities of ATP and(respiration, macromolecules, synthesis below) of underATP and the appliedmacromolecules, conditions. below) under the applied conditions.

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Table 2. Membrane permeabilization of X. citri and B. subtilis cells by chalcones.

Permeabilized X. citri Cells Permeabilized B. subtilis Cells Treatment (Mean SD) (Mean SD) Molecules 2020, 25, x FOR PEER REVIEW ± ± 4 of 14 Negative control 2.38 0.08% 2.86 1.78% ± ± DMSOTable 1% 2. Membrane permeabilization 2.95 of X.0.53% citri and B. subtilis cells by chalcones. 4.02 3.16% ± ± BC1 (15 min) 2.22 1.34% 66.23 0.62% Treatment Permeabilized X. citri Cells± (Mean ± SD) Permeabilized B. subtilis Cells± (Mean ± SD) BC1 (30 min) 6.98 1.67% 75.32 0.28% Negative control 2.38 ± 0.08% ± 2.86 ± 1.78% ± T9ADMSO (15 1% min) 2.95 ± 0.53% 2.91 0.89%4.02 ± 3.16% 3.67 0.63% ± ± T9ABC1 (30(15 min) min) 2.22 ± 1.34% 3.86 0.22%66.23 ± 0.62% 4.81 3.29% ± ± Heat shockBC1 (60(30 min)C; 20 min)6.98 ± 1.67% 97.16 0.76%75.32 ± 0.28% not done ◦ ± Nisin (5.0T9Aµ (15g/mL; min) 15 min)2.91 ± 0.89% not done3.67 ± 0.63% 97.22 2.72% T9A (30 min) 3.86 ± 0.22% 4.81 ± 3.29% ± ResultsHeat shock from (60 two °C; independent 20 min) replicates97.16 with ± 200 0.76% cells each. Concentrations used:not BC1 done 90 µg/mL (X. citri) or 50 µNising/mL (5.0 (B. μ subtilisg/mL; 15); T9Amin) 50 µg/mL (X. citri)not or done 40 µg /mL (B. subtilis). 97.22 ± 2.72% Results from two independent replicates with 200 cells each. Concentrations used: BC1 90 μg/mL (X. 2.3. Eﬀectscitri on) Macromolecularor 50 μg/mL (B. subtilis Synthesis); T9A 50 μg/mL (X. citri) or 40 μg/mL (B. subtilis).

To2.3. investigate Effects on Macromolecular whether BC1 Synthesis or T9A can inhibit the synthesis of macromolecules such as DNA, RNA, proteins, and peptidoglycan, we followed the incorporation of radiolabeled precursors of each To investigate whether BC1 or T9A can inhibit the synthesis of macromolecules such as DNA, of these macromolecules (Figure2). Both compounds inhibited most pathways to some extent in both RNA, proteins, and peptidoglycan, we followed the incorporation of radiolabeled precursors of each X. citriof (Figurethese macromolecules2A) and B. subtilis (Figure(Figure 2). Both2 compoundsB), although inhibited T9A did most not pathways a ffect DNA to some synthesis, extent in both and also had littleX. citri to (Figure no effect 2A) on and protein B. subtilis synthesis (Figure 2B) in X., although citri. In T9A all did cases, not BC1 affect is DNA more synthesis, effective and at blockingalso macromolecularhad little to no synthesis effect on thanprotein T9A. synthesis Initially, in X. we citri. established In all cases, MICs BC1 foris more all control effective antibiotics at blocking used in B.macromolecular subtilis and X. citrisynthesis, and than used T9A. MIC Initially, as this we was established the concentration MICs for all at control which antibiotics BC1 and used T9A in were testedB. [ subtilis20]. To and our X. surprise, citri, and used all control MIC as antibioticsthis was the wereconcentration quite e ffatective which inBC1 blocking and T9A their were respectivetested pathways[20]. To in ourB. subtilissurprise,, but all control not in antibioticsX. citri, where were quite only effective tetracycline in blocking blocked their protein respective synthesis pathways almost completely.in B. subtilis Increasing, but not the in X. concentration citri, where only of controltetracycline antibiotics blocked toprotein four timessynthesis the almost MIC did completely. not result in Increasing the concentration of control antibiotics to four times the MIC did not result in a stronger a stronger inhibition of incorporation of precursors [20]. As the inhibition observed in X. citri was inhibition of incorporation of precursors [20]. As the inhibition observed in X. citri was often similar often similar to the inhibition observed with the control, it is difficult to comment on the inhibition to the inhibition observed with the control, it is difficult to comment on the inhibition of pathways in of pathwaysX. citri. inHowever,X. citri .as However, in both bacteria as in both various bacteria pathways various were pathways more or were less moreinhibited or less by inhibitedboth by bothcompounds, compounds, it is obvious it is obvious that these that compounds these compounds do not target do one not specific target one macromolecular specific macromolecular synthesis synthesispathway. pathway.

Figure 2. Effects of BC1 and T9A on the four major macromolecular synthesis pathways of B. subtilis (A), andFigureX. 2. citri Effects(B), of in BC1 comparison and T9A on to the negative four major controls macromolecular ( ) and antibioticssynthesis pathways with specific of B. subtilis activities (A), and X. citri (B), in comparison to negative controls (−−) and antibiotics with specific activities targeting DNA, RNA, Protein, and Peptidoglycan syntheses, Ciprofloxacin (Cip), Rifampicin (Rif), targeting DNA, RNA, Protein, and Peptidoglycan syntheses, Ciprofloxacin (Cip), Rifampicin (Rif), Tetracycline (Tet), and (A) Benzyl penicillin (PenG), (B) Vancomycin (Van), respectively. Each Tetracycline (Tet), and (A) Benzyl penicillin (PenG), (B) Vancomycin (Van), respectively. Each circle circlerepresents represents an anexperimental experimental replicate; replicate; bars indicate bars indicate the median the relative median incorporation relative incorporation at each condition at each conditiontested. tested. 2.4. Effects on Metabolic Activity and ATP Levels 2.4. Effects on Metabolic Activity and ATP Levels To determineTo determine whether whether the inhibition the inhibition of macromolecular of macromolecular synthesis synthesis was was specific, specific, or causedor caused by by overall retardationoverall ofretardation growth, orof growth, cell death, or cell the death, metabolic the metabolic activity ofactivity the cells of the in cells the presencein the presence of BC1, of T9A,BC1, and pathwayT9A, specific and pathway antibiotics specific was antibiotics measured was using measured the REMA using assay.the REMA This assay. assay This is based assay on is thebased reduction on the reduction of resazurin to resorufin as a result of bacterial respiration. X. citri or B. subtilis cells

Molecules 2020, 25, 4596 5 of 14 of resazurin to resorufin as a result of bacterial respiration. X. citri or B. subtilis cells were exposed to Molecules 2020, 25, x FOR PEER REVIEW 5 of 14 the compounds/antibiotics and their metabolic activity was measured in 20 min intervals for up to 120 minwereMolecules (Figure exposed 20203, ,25 Figure ,to x FORthe PEER S2).compounds/antibiotics REVIEW For X. citri, even and though their there metabolic was some activity inhibition was measured of metabolic in 20 5min of activity, 14 this didintervals not go for below up to 75%120 min of uninhibited (Figure 3, Figure activity, S2). For nor X. displayed citri, even a though large decline there was over some the inhibition 2 h period in were exposed to the compounds/antibiotics and their metabolic activity was measured in 20 min of metabolic activity, this did not go below 75% of uninhibited activity, nor displayed a large decline whichintervals activity for was up followed. to 120 min Thus, (Figure inhibition 3, Figure S2). of synthesis For X. citri pathways, even though to levels there was below some 70% inhibition in Figure 3 over the 2 h period in which activity was followed. Thus, inhibition of synthesis pathways to levels is notof only metabolic caused activity, by the this inhibition did not go of below growth. 75% For of uninhibitedB. subtilis ,activity, the results nor displayed were diff aerent, large decline and rather below 70% in Figure 3 is not only caused by the inhibition of growth. For B. subtilis, the results were puzzling.over Thethe 2 compound h period in which T9A almost activity completely was followed. blocked Thus, metabolicinhibition of activity, synthesis which pathways is striking, to levels as the different, and rather puzzling. The compound T9A almost completely blocked metabolic activity, effectbelow of T9A 70% on in macromolecular Figure 3 is not only synthesis caused by in theB. inhibition subtilis was of growth. not very For pronounced B. subtilis, the (Figure results 2werea). BC1 which is striking, as the effect of T9A on macromolecular synthesis in B. subtilis was not very different, and rather puzzling. The compound T9A almost completely blocked metabolic activity, reducedpronounced the respiration (Figure 2a). to about BC1 reduced 60%. the respiration to about 60%. which is striking, as the effect of T9A on macromolecular synthesis in B. subtilis was not very pronounced (Figure 2a). BC1 reduced the respiration to about 60%.

FigureFigure 3. Assessment 3. Assessment of the of the overall overall metabolic metabolic activity activity of ofB. B. subtilis subtilisand andX. X. citri citriexposed exposed toto BC1BC1 andand T9A. T9A. Absolute fluorescence of resorufin measured over time in 20 min intervals in B. subtilis (A), and AbsoluteFigure fluorescence 3. Assessment of resorufin of the overall measured metabolic over activity time in of 20 B. min subtilis intervals and X. incitriB. exposed subtilis (toA ),BC1 and andX. citri X. citri (B) cells. Fluorescence relative to the control is depicted in supplementary Figure S2. Control (B) cells.T9A. Fluorescence Absolute fluorescence relative to of the resorufin control measured is depicted over in time supplementary in 20 min intervals Figure in S2. B. Controlsubtilis (A compound), and compound 7b‐BF4 is a compound that completely blocks B. subtilis respiration [20]. 7b-BFX.4 is citri a compound (B) cells. Fluorescence that completely relative blocks to the B.control subtilis is depictedrespiration in supplementary [20]. Figure S2. Control Thecompound relative 7b intracellular‐BF4 is a compound ATP levels that completely were determined blocks B. subtilisin X. citri respiration and B. subtilis[20]. after 80 or 30 min The relative intracellular ATP levels were determined in X. citri and B. subtilis after 80 or 30 min treatment with the compounds, respectively (Figure 4). At 1x MIC, T9A and BC1 cause a decrease in treatmentThe with relative the compounds, intracellular ATP respectively levels were (Figure determined4). At in 1x X. MIC, citri and T9A B. andsubtilis BC1 after cause 80 or a 30 decrease min X. citri ATP levels compared to the control, to a similar extent as their effect on overall respiratory treatment with the compounds, respectively (Figure 4). At 1x MIC, T9A and BC1 cause a decrease in in X.activity. citri ATP BC1, levels at 2x compared the MIC led to theto an control, almost to complete a similar loss extent of ATP, as theirwhen e comparedffect on overall to the control respiratory X. citri ATP levels compared to the control, to a similar extent as their effect on overall respiratory activity.CCCP BC1, (Figure at 2x the4B). MIC Strikingly, led to in an B. almost subtilis complete, the ATP loss levels of ATP,increased when for compared BC1 and toT9A the at control 1x MIC. CCCP activity. BC1, at 2x the MIC led to an almost complete loss of ATP, when compared to the control (FigureHowever,4B). Strikingly, T9A at 2x in MICB. subtilisled to an, the almost ATP complete levels increased loss of ATP, for which BC1 andis in T9Aline with at 1x the MIC. complete However, CCCP (Figure 4B). Strikingly, in B. subtilis, the ATP levels increased for BC1 and T9A at 1x MIC. T9A atblock 2x of MIC respiration led to an observed almost (Figure complete 4A). lossThis suggests of ATP, whichthat, in the is in presence line with of either the complete compound, block B. of However, T9A at 2x MIC led to an almost complete loss of ATP, which is in line with the complete subtilis cells may first switch to anaerobic metabolism, together with an inhibition of ATP‐consumingB. subtilis respirationblock of observed respiration (Figure observed4A). This(Figure suggests 4A). This that, suggests in the that, presence in the of presence either compound, of either compound, B. cells pathways, after which, at higher concentrations of T9A, metabolic activity stops completely. may firstsubtilis switch cells may to anaerobic first switch metabolism, to anaerobic togethermetabolism, with together an inhibition with an inhibition of ATP-consuming of ATP‐consuming pathways, after which,pathways, at higher after which, concentrations at higher concentrations of T9A, metabolic of T9A, activity metabolic stops activity completely. stops completely.

Figure 4. ATP concentration (relative light units): (A) after 30 min treatment of B. subtilis cells and (B) after 80 min treatment of X. citri cells, with 1x and 2x the MIC of BC1 or T9A or 40 μg/mL of CCCP. FigureFigure 4. ATP 4. ATP concentration concentration (relative (relative light light units): units): ( (AA)) after 30 30 min min treatment treatment of ofB. subtilisB. subtilis cellscells and and(B) (B) 100% is defined as the median luminescence of the control sample (without any compound added); after 80after min 80 treatmentmin treatment of X.of citriX. citricells, cells, with with 1x 1x and and 2x the MIC MIC of of BC1 BC1 or or T9A T9A or 40 or μ 40g/mLµg/ mLof CCCP. of CCCP. each circle represents a replicate, and bars indicate the median of each condition tested. 100%100% is deﬁned is defined as the as medianthe median luminescence luminescence of of the the controlcontrol sample (without (without any any compound compound added); added); each circleeach circle represents represents a replicate, a replicate, and and bars bars indicate indicate the the median median of each each condition condition tested. tested.

Molecules 2020, 25, 4596 6 of 14

2.5. FT-IR Spectrophotometry Molecules 2020, 25, x FOR PEER REVIEW 6 of 14 FT-IR of B. subtilis cells treated with BC1 and T9A was measured, together with a negative control 2.5. FT‐IR Spectrophotometry and a positive control using nisin (Figure5). FT-IR is a technique that can provide a fingerprint of differentFT‐ componentsIR of B. subtilis of cells the bacterialtreated with cell, BC1 and and thus T9A can was also measured, report ontogether the disruption with a negative of cellular structurescontrol [14 and]. The a positive peaks shown control in using the spectrum nisin (Figure represent 5). FT perturbations‐IR is a technique of diverse that chemicalcan provide structures a fingerprint of different components of the bacterial cell, and thus can1 also report on the disruption of from compounds present in B. subtilis [21]. The peaks at 1083 cm− are due to stretching of C-O bonds cellular structures [14]. The peaks shown in the spectrum represent perturbations of diverse chemical present in glycogen. The peaks at 1231 cm 1 are due to PO asymmetric stretching, mainly from − 2− −1 structures from compounds present in B. subtilis [21]. The peaks at 1083 cm are due to 1stretching of nucleic acids with little contribution from phospholipids. The peaks at 1404 cm− corresponds to C‐O bonds present in glycogen. The peaks at 1231 cm−1 are due to PO2− asymmetric stretching, mainly 1 COOfrom− symmetric nucleic acids stretching with little from contribution amino acid from side phospholipids. chains and fatty The peaks acids. at The 1404 peaks cm−1 atcorresponds 1450 cm− are 1 due toto CHCOO2 −bending symmetric from stretching lipids from present amino in theacid cytoplasmic side chains and membrane. fatty acids. The The peaks peaks atat 15401450 cm −−1 are fromare N-H due bending to CH2 bending and C-N from stretching lipids present from in amidethe cytoplasmic II present membrane. in α helixes The peaks of proteins. at 1540 cm Finally,−1 are the 1 peaksfrom at 1646 N‐H cm bending− are and from C C‐N= Ostretching stretching from from amide amide II present I α helixes. in α helixes The peaks of proteins. of the spectrumFinally, the of the negativepeaks control at 1646 (Figure cm−1 are5A) from were C=O similar stretching to the from spectrum amide I of α thehelixes. cells The treated peaks with of the T9A spectrum (Figure of5 D),the and the peaksnegative of the control spectrum (Figure of 5A) cells were treated similar with to the nisin spectrum (Figure of5 B)the were cells similartreated with to peaks T9A (Figure in the spectrum 5D), of theand cells the treated peaks of with the BC1spectrum (Figure of 5cellsC). Wetreated could with not nisin identify (Figure any 5B) obvious were similar di fferences to peaks between in the the spectrum of the cells treated with BC1 (Figure 5C). We could not identify any obvious differences spectra regarding a particular peak. However, the similar intensities between the spectrum of the between the spectra regarding a particular peak. However, the similar intensities between the negative control and T9A and between BC1 and nisin, corroborates with the results of our membrane spectrum of the negative control and T9A and between BC1 and nisin, corroborates with the results permeabilityof our membrane assay (Table permeability2). There assay we (Table can see2). There that we T9A can does see that not T9A cause does membrane not cause membrane disruption in B. subtilisdisruption, whereas in B. BC1subtilis is, ablewhereas to disrupt BC1 is able the to cellular disrupt membrane the cellular similarmembrane to nisin.similar We to nisin. attempted We to applyattempted the FT-IR to techniqueapply the FT on‐IRX. technique citri cells on asX. citri well. cells However, as well. However,X. citri produces X. citri produces xanthan xanthan gum and biofilmsgum [ 22and], biofilms generating [22], noise generating in the noise spectra, in the which spectra, prevents which prevents the detection the detection of the peaksof the peaks that report that on cellularreport structures. on cellular structures.

FigureFigure 5. FT-IR 5. FT spectra‐IR spectra of B. of subtilis B. subtiliscells cells after after 30 min30 min treatment treatment with with (A (A) no) no compounds compounds added; added; ((BB)) nisin (5 µgnisin/mL); (5 ( μC)g/mL); BC1 (50 (C) µBC1g/mL); (50 μ andg/mL); (D )and T9A (D (40) T9Aµg (40/mL). μg/mL).

Molecules 2020, 25, 4596 7 of 14

Molecules 2020, 25, x FOR PEER REVIEW 7 of 14 2.6. Cell Division Investigations 2.6. Cell Division Investigations To assess the ability of the compounds to disrupt cell division, reporter strains expressing a To assess the ability of the compounds to disrupt cell division, reporter strains expressing a functional fluorescent fusion to a cell division protein (GFP-ZapA in X. citri and FtsZ-GFP in B. subtilis) functional fluorescent fusion to a cell division protein (GFP‐ZapA in X. citri and FtsZ‐GFP in B. X. citri weresubtilis treated) were with treated T9A andwith BC1, T9A andand imagedBC1, and by imaged microscopy by microscopy (Figure6 ).(Figure 6).cells X. citri treated cells onlytreated with theonly vehicle with (1% the DMSO) vehicle showed(1% DMSO) a perpendicular showed a perpendicular fluorescent signal fluorescent at mid-cell, signal which at mid corresponds‐cell, which to thecorresponds division septum to the (Figure division6A). septum Exposure (Figure of X. 6A). citri Exposureto both compounds of X. citri to had both no apparentcompounds eff ecthad on no cell divisionapparent since effect we on could cell division still detect since localized we could GFP-ZapA still detect inlocalized the cells GFP (Figure‐ZapA6 B,C).in the Finally,cells (Figure exposure 6B, toC). hexyl-gallate, Finally, exposure our positive to hexyl control‐gallate, [23 our], ledpositive to a completecontrol [23], delocalization led to a complete of GFP-ZapA delocalization in X. of citri (FigureGFP‐6ZapAD). Conversely in X. citri (Figure with what 6D). wasConversely observed with for whatX. citri was, theobserved localization for X. citri of FtsZ-GFP,, the localization our marker of forFtsZ cell‐ divisionGFP, our in markerB. subtilis for cell(Figure division6E), was in B. a subtilisffected (Figure by both 6E), BC1 was and affected T9A (Figure by both6F,G, BC1 respectively). and T9A Here,(Figure the fluorescence6F,G, respectively). of FtsZ-GFP Here, the looks fluorescence dispersed of and FtsZ cytosolic,‐GFP looks similar dispersed to what and cancytosolic, be seen similar for the controlto what hexyl-gallate can be seen (compare for the control 6F and hexyl 6G with‐gallate 6H). (compare Since BC1 6F is and able 6G to disruptwith 6H). membrane Since BC1 permeability is able to in disruptB. subtilis membrane(disrupting permeability membrane in B. potential subtilis (disrupting as well), itmembrane is possible potential that FtsZ-GFP as well), it localization is possible is disruptedthat FtsZ due‐GFP to localization membrane is potential disrupted disruption due to membrane [24]. However, potential T9Adisruption did not [24]. disrupt However, membrane T9A permeabilitydid not disrupt in B. membrane subtilis, suggesting permeability that in the B. subtilis mode, ofsuggesting action of that T9A the could mode be of linked action toof cellT9A division. could Thus,be linked we tested to cell the division. effect of Thus, BC1 we and tested T9A the on theeffect GTPase of BC1 activity and T9A of on purified the GTPaseB. subtilis activityFtsZ of purified (Figure7 ). AsB. the subtilis GTPase FtsZ activity (Figure of7). FtsZ As the was GTPase not significantly activity of FtsZ affected was not by significantly BC1 or T9A affected in comparison by BC1 or with T9A the negativein comparison control DMSO,with the it isnegative unlikely control that FtsZ DMSO, is a directit is unlikely target of that the FtsZ chalcones. is a direct target of the chalcones.

FigureFigure 6. 6.E ﬀEffectsects of of BC1 BC1 and and T9A T9A on on bacterial bacterial cell division. Phase Phase contrast contrast (left) (left) and and GFP GFP (right) (right) images images of: X. citri expressing GFP‐ZapA treated for 30 min with (A) 1% DMSO, (B) BC1 (90 μg/mL), (C) T9A of: X. citri expressing GFP-ZapA treated for 30 min with (A) 1% DMSO, (B) BC1 (90 µg/mL), (C) T9A (50 μg/mL), (D) hexyl gallate (60 μg/mL), and B. subtilis expressing FtsZ‐GFP treated for 30 min with (50 µg/mL), (D) hexyl gallate (60 µg/mL), and B. subtilis expressing FtsZ-GFP treated for 30 min with (E) (E) 1% DMSO, (F) BC1 (50 μg/mL), (G) T9A (40 μg/mL), (H) hexyl gallate (60 μg/mL). Scale bar: 5 μm. 1% DMSO, (F) BC1 (50 µg/mL), (G) T9A (40 µg/mL), (H) hexyl gallate (60 µg/mL). Scale bar: 5 µm.

MoleculesMolecules2020 2020, 25, 459625, x FOR PEER REVIEW 8 of8 14 of 14

Figure 7. GTP hydrolysis activity of B. subtilis FtsZ in the presence of DMSO, BC1, or T9A. Figure 7. GTP hydrolysis activity of B. subtilis FtsZ in the presence of DMSO, BC1, or T9A. 3. Discussion 3. X.Discussion citri causes Asiatic Citrus Canker, leading to economic losses to citriculture [1]. Here, we investigatedX. citri the causes antibacterial Asiatic modeCitrus of Canker, action of leading the methoxychalcone to economic losses BC1 to and citriculture the hydroxychalcone [1]. Here, we T9Ainvestigated on X. citri andthe antibacterialB. subtilis, using mode techniques of action previouslyof the methoxychalcone used to investigate BC1 and the the mode hydroxychalcone of action of drugsT9A in on model X. citri organisms and B. subtilis such, as usingB. subtilis techniquesitself, andpreviously also E. coliusedand to S.investigate aureus [25 the–28 mode]. of action of drugsIn X. in citri model, all the organisms metabolic such pathways as B. subtilis are aff ecteditself, byand the also compounds E. coli and within S. aureus the 80[25–28]. min monitoring, which isIn a strongX. citri indication, all the ofmetabolic an aspecific pathways mechanism. are affected It has to beby noted the compounds that most positive within controls the 80 for min pathwaymonitoring, inhibition which in X.is a citri strongwere indication not very e ffofective an aspecific in blocking mechanism. incorporation It has of to metabolites—even be noted that most thoughpositive all the controls compounds for pathway used were inhibition confirmed in X. as citri bona were fide not antibiotics very effective [20]. Possibly, in blocking the slow incorporation growth of X.of citrimetabolites—evenmakes it difficult though to see di allfferences the compounds in incorporation; used were however, confirmed increasing as bona control fide concentrations antibiotics [20]. wasPossibly, not effective the slow within growth the timeframe of X. citri of makes the experiment. it difficult Longer to see differences exposure or in higher incorporation; concentrations however, of antibioticincreasing would control most concentrations likely lead to secondary was not effective effects, such within as overallthe timeframe growth reduction,of the experiment. which makes Longer it impossibleexposure or to higher specify concentrations whether reduced of antibiotic incorporation would is most due likely to pathway lead to inhibition. secondary Inhibitioneffects, such of as multipleoverall pathways growth reduction, is similar which to what makes was it described impossible by to Haraguchi specify whether et al. [7 reduced] on the modeincorporation of action is of due licochalconeto pathway A. inhibition. This chalcone Inhibition inhibited of multiple incorporation pathways of DNA, is similar RNA, to and what protein was described (peptidoglycan by Haraguchi was notet tested) al. [7] on in Micrococcusthe mode of luteusaction, of via licochalcone inhibition of A. oxygen This chalcone consumption inhibited and incorporation interference withof DNA, energy RNA, metabolismand protein [7]. Similarly,(peptidoglycan for B. subtilis was allnot the tested) metabolic in pathwaysMicrococcus are aluteusffected, via within inhibition the 60 min of tested.oxygen consumptionIntriguingly, and in ourinterference experiments, with energy we showed metabolism that both [7]. compounds Similarly, for (BC1 B. subtilis and T9A) all the caused metabolic an increasepathways in ATP are concentration affected within in theB. subtilis 60 min(Figure tested.4 ). This was surprising, as the observed membrane damageIntriguingly, with BC1, and in our the experiments, drop in respiration we showed for both that compounds, both compounds would (BC1 suggest and aT9A) decrease caused in an ATPincrease production, in ATP which concentration was only in observed B. subtilis at(Figure higher 4). concentrations This was surprising, of T9A, as whichthe observed collapsed membrane ATP. It coulddamage be with that both BC1, compounds and the drop cause in respiration a switch offorB. both subtilis compounds,to anaerobic would growth, suggest which a decrease allows ATPin ATP levelsproduction, to be maintained which was [29 only], but observed the increase at higher in ATP concentrations could also be of due T9A, to which processes collapsed such as ATP. cell It wall could andbe RNA that both synthesis compounds being inhibited cause a switch or blocked, of B. subtilis causing to anaerobic a difference growth, between which ATP allows production ATP levels and to consumption.be maintained The [29], latter but eff theect wasincrease previously in ATP observed could also for be the due protein to processes inhibitors such chloramphenicol as cell wall and and RNA streptomycinsynthesis being [30]. In inhibitedX. citri, a decreaseor blocked, in ATP causing levels wasa difference observed (Figurebetween4), especiallyATP production for BC1 atand higherconsumption. concentrations, The latter which effect is an was indication previously of indirect observed effect. for the protein inhibitors chloramphenicol andCell streptomycin division processes [30]. In X. were citri investigated, a decrease in by ATP observing levels was the localization observed (Figure of GFP 4), fusions especially with for cell BC1 divisionat higher proteins. concentrations, In X. citri whichneither is BC1an indication nor T9A causedof indirect delocalization effect. of our cell division marker (GFP-ZapA)Cell division which rulesprocesses out the were possibility investigated that by these observing compounds the localization cause a direct of GFP eff ectfusions on X. with citri cell celldivision division. proteins. In B. subtilis In X.both citri compounds neither BC1 caused nor T9A delocalization caused delocalization of our cell division of our marker cell division (FtsZ-GFP). marker BC1(GFP was‐ZapA) able to which permeabilize rules outB. the subtilis possibilitymembranes, that these therefore compounds this result cause could a direct be due effect to on membrane X. citri cell potentialdivision. disruption, In B. subtilis similar both compounds to that observed caused by delocalization Strahl, et al. [ 24of ].our However, cell division T9A marker was not (FtsZ able‐GFP). to permeabilizeBC1 was ableB. subtilisto permeabilizemembranes. B. subtilis Therefore, membranes, we tested therefore FtsZ GTPasethis result activity, could be and due since to membrane neither potential disruption, similar to that observed by Strahl, et al. [24]. However, T9A was not able to permeabilize B. subtilis membranes. Therefore, we tested FtsZ GTPase activity, and since neither

Molecules 2020, 25, 4596 9 of 14 chalcone caused an effect on GTPase activity, we rule out the possibility of FtsZ being directly affected by T9A or BC1. Summarizing, the chalcones affect both gram-negative and gram-positive cells but not in the same manner. In gram-positive cells, the cytoplasmic membrane is a target; in gram-negative cells the outer membrane may be a sink for these compounds that would prevent them from reaching the cytoplasmic membrane. It remains to be established however, how this would result in the overall decrease in ATP levels and inhibition of macromolecular synthesis observed upon exposure of a gram-negative bacterium to these compounds.

4. Materials and Methods

4.1. Chemical Synthesis Synthesis of BC1 was achieved by the reaction between acetovanillone (5 mmol) and benzaldehyde (5 mmol) at room temperature using ethanol as the solvent and ethanolic solution of NaOH (1.0 mol/L, 5 mL), using a protocol reported by Santos and collaborators with minor modifications [31]. The reaction mixture was stirred and monitored by successive thin-layer chromatography (TLC) analyses. When the reaction was finished, the residue was poured into crushed ice. The resulting precipitate was removed by filtration, and product obtained was purified by recrystallization from ethanol at 76 ◦C, with yield of 89%. In order to synthesize T9A, acetophenone (5 mmol) and 2-hydroxybenzaldehyde (5 mmol) were solubilized in a NaOH ethanolic solution (1.0 mol/L, 20 mL) at room temperature, using protocols reported by Kobelnik and collaborators with minor modifications [32]. The reaction was monitored by TLC analyses. When reagents were converted to the product, the reaction medium was poured into crushed ice and acidified with HCl solution (1.0 mol/L) at pH 2. The precipitate was filtered and the product obtained was recrystallized in ethanol, with yield of 54%. All reagents were purchased from Merck (Kenilworth, NJ, USA).

4.2. Minimal Inhibitory Concentration Assays The minimal inhibitory concentration (MIC) of the compounds against X. citri and B. subtilis was determined by broth microdilution. Exponentially growing bacteria were diluted to OD600 of 0.005 in 96 well plates containing twofold serial dilutions of the compounds, in a final volume of 200 µL of growth medium. Davis Minimal Medium (DMM) (casaminoacids 2.0 g/L; K2HPO4 7.0 g/L; KH2PO4 3.0 g/L; MgSO 7(H O) 0.1 g/L; (NH ) SO 1.0 g/L; Na C H O 2(H O) 0.5 g/L; glucose 7.0 g/L and 4· 2 4 2 4 3 6 5 7· 2 tryptophan 10 mg/L), modified from [33,34], was used for B. subtilis. Xam1 medium (glycerol 2.46 g/L; MgSO 7H O 0.247 g/L; (NH ) SO 1.0 g/L; K HPO 10.5 g/L; KH PO 4.5 g/L; Na C H O 2H O 4· 2 4 2 4 2 4 2 4 3 6 5 7· 2 0.5 g/L; casaminoacids 0.3 g/L; BSA 1 g/L (pH 5.4 adjusted with HCl)) was used for X. citri. The MIC was defined as the lowest concentration of compound that prevented bacterial growth after incubating cultures for 24 h at 30 ◦C[35]. MICs were also determined using the Resazurin Microtiter Assay Plate (REMA) essentially as described by [14]. The antibiotic kanamycin at 20 µg/mL was used as positive control in both evaluations. Three independent experiments were used to estimate the MIC data. Statistics were done using Graphpad Prism version 6 (Graphpad Software, San Diego, CA, USA). The bacteriostatic or bactericidal action of compounds was assessed by plating samples from REMA on solid-media following incubation for 24 h (B. subtilis) or 48 h (X. citri) in order to check for cell viability.

4.3. Membrane Permeability Assay Membrane permeability was tested using the Live/Dead BacLight bacterial viability kit (Thermo-Scientiﬁc Scientiﬁc, Waltham, MA, USA). An overnight culture of B. subtilis grown in 6 LB, or X. citri grown in NYGB [15], at 29 ◦C, was diluted to 10 cells/mL. B. subtilis cells were incubated in the presence of BC1 (50 µg/mL), T9A (40 µg/mL), DMSO (1%) as a negative control, and nisin (5.0 µg/mL) as a positive control; X. citri cells were incubated in the presence of BC1 (90 µg/mL), Molecules 2020, 25, 4596 10 of 14

T9A (50 µg/mL), DMSO (1%) as a negative control, and 20 min at 55 ◦C as a positive control. Treatments were carried out for 15 min and 30 min at 23 ◦C. Propidium iodide (5.0 mM) and SYTO 9 (835 µM) were added to each test. After incubation, cells were mounted on agarose pads for microscopy analysis and imaged using an Olympus BX-61 microscope with an OrcaFlash-2.8 camera using DAPI/FITC and Texas Red fluorescence filter cubes. Propidium iodide (shown in Texas Red fluorescence filter) is membrane impermeable, and thus only stains cells with disrupted membrane, whereas SYTO 9 (shown in DAPI/FITC fluorescence filter) is membrane permeable and stains all the cells. The Olympus cellSens platform was used for image analysis. Experiments were performed in triplicates.

4.4. Macromolecular Synthesis A total of four macromolecular synthesis pathways were evaluated by monitoring the incorporation of radioactively labelled precursors. [5-3H] uridine, [methyl-3H] thymidine, L-[3,4,5-3H(N)] leucine and D-[6-3H(N)] glucosamine hydrochloride (all at 0.5 µCi/mL) were used to respectively monitor RNA, DNA, protein and peptidoglycan synthesis. During this experiment B. subtilis cells were grown to early exponential phase in DMM and X. citri cells were grown to early exponential phase in Xam1. 1 mM of non-labelled precursor was used for RNA and peptidoglycan and 10 µM was used for DNA and protein. For DNA incorporation, B. subtilis cells were grown in LB medium instead of DMM. Cells were incubated in the presence of labelled precursors for 80 min, with shaking at 30 ◦C. B. subtilis was tested in the presence of 40 µg/mL of BC1 or T9A, without compound (negative control), or with an antibiotic known to inhibit the synthesis pathway tested as a positive control (RNA: rifampicin 0.625 µg/mL, DNA: ciprofloxacin 0.625 µg/mL, protein: tetracycline 10 µg/mL, peptidoglycan: vancomycin 1.0 µg/mL). X. citri was tested in the presence of 30 µg/mL of BC1, 20 µg/mL of T9A, without compound (negative control), or with an antibiotic supposed to inhibit the synthesis pathway tested as positive control (RNA: rifampicin 0.625 µg/mL, DNA: ciprofloxacin 10.0 µg/mL, protein: tetracycline 10 µg/mL, peptidoglycan: penicillin G 800 µg/mL). For every precursor tested, at least two technical replicates were taken, from three biological replicates (minimum six datapoints). During incubation, aliquots were taken and precipitated with ice-cold 12% trichloroacetic acid for 35 min and then filtered through nitrocellulose membranes (pore size 0.45 µm). The filters were washed with ice cold 12% trichloroacetic acid, transferred to 2 mL scintillation fluid Ultima Gold MV (PerkinElmer, Waltham, MA, USA), and measured in a Tri-Carb 2000CA liquid scintillation analyzer (Packard Instruments, part of Thermo-Scientific Scientific, Waltham, MA, USA).

4.5. Resazurin Assay The Resazurin assay (REMA) was performed in order to evaluate the impact of the compounds on the respiratory activity after a short period of time [23,36]. Cells of B. subtilis or X. citri were incubated in contact with resazurin in the same medium used for the MIC evaluations (DMM and Xam1 respectively) in 96-well plates. The compound BC1 was used at a final concentration of 40 µg/mL for B. subtilis and 30 µg/mL for X. citri; T9A was used at 40 µg/mL for B. subtilis and 20 µg/mL for X. citri. Rifampicin 0.625 µg/mL, ciprofloxacin 0.625 µg/mL, tetracycline 10 µg/mL, and vancomycin 1.0 µg/mL were used as controls for B. subtilis, and rifampicin 0.625 µg/mL, ciprofloxacin 10.0 µg/mL, tetracycline 10 µg/mL, and penicillin G 800 µg/mL were used as controls for X. citri, along with samples without added compound. Resazurin was added to a final concentration of 0.1 mg/mL. Fluorescence (excitation 530 nm, emission 560 nm, bandwidth 9 nm) was recorded every 20 min up to 120 min in a BioTek Synergy Mx 96-well plate reader (BioTek instruments, Winooski, VT, USA).

4.6. Intracellular ATP Concentration Assay ATP levels were measured in B. subtilis and X. citri using the BacTiter-Glo™ Microbial Cell Viability Assay (Promega). CCCP (Carbonyl cyanide m-chlorophenyl hydrazine, 40 µg/mL) was used as a positive control for ATP depletion. Cells were incubated in a 96 well plate (30 ◦C, 1000 rpm), 30 min for B. subtilis (in DMM) and 80 min for X. citri (in Xam1) after which 100 µL of cell culture was added Molecules 2020, 25, 4596 11 of 14

to 100 µL of BacTiter-Glo™ Reagent for 5 min in a white 96 well plate (26 ◦C, 1000 rpm). Luminescence was measured in a Tecan Inﬁnite F200 Pro luminometer. The amount of light emitted is a measure for the intracellular ATP concentration.

4.7. FT-IR Spectrophotometry Fourier-transform infrared (FT-IR) spectrophotometry was done on B. subtilis cells in a FT-IR spectrophotometer (Shimadzu, model 8300, Shimadzu, Kyoto, Japan), as described by [14]. Cells were 5 cultivated in LB medium (29 ◦C) up to the concentration of 10 cells/mL, then treated with BC1 (50 µg/mL), T9A (40 µg/mL), nisin (5 µg/mL), or a negative control without the addition of any compound for 30 min. Aliquots of 1.5 mL of cells were then centrifuged at 10.000 x g for 2 min, washed with water, and centrifuged two more times to remove traces of medium. After that, the samples were air-dried, and mixed with 150 mg of KBr. Next, samples were compressed at 40 kN for 5 min. 1 1 1 Absorbance was measured from 400 cm− to 4000 cm− , with 32 scans at a resolution of 4 cm− . Data treatment and analyses were performed using the software Origin 8.00 (OriginLab, Northampton, MA, USA).

4.8. Cell Division Interference The effect of chalcones on cell division was investigated using GFP fusions to cell division proteins, and by evaluating the GTPase activity of FtsZ from B. subtilis. X. citri labeled with GFP-ZapA (X. citri amy: gfp-zapA [37] was grown in NYGB and induced with Xylose (0.5%) for 90 min. Cells were treated with BC1 (90 µg/mL), T9A (50 µg/mL), DMSO (1%, as a negative control) or hexyl gallate (60 µg/mL, as a positive control) for 30 min and samples were prepared for visualization as described before [37]. B. subtilis labelled with FtsZ-GFP [14] was cultivated in the presence of 0.02 mM IPTG (isopropyl-β-d-1-thiogalactopyranoside) to induce the expression of the protein fusion from the Pspac promoter. Cells were treated with BC1 (50 µg/mL), T9A (40 µg/mL), DMSO (1%) as a negative control, and hexyl gallate (60 µg/mL) as a positive control for 15 and 30 min. FtsZ from B. subtilis was expressed and purified using the ammonium sulfate precipitation method as described by [38]. The FtsZ GTP hydrolysis rate was determined using the malachite green phosphate assay as described by [38,39], with a few modifications. Stocks of BC1 (50 µg/mL), T9A (40 µg/mL), and DMSO (1%) as a negative control, with FtsZ (24 µM), MgCl2 (20 mM) and Triton X-100 (0.02%) prepared in polymerization buffer (HEPES 50 mM, KCl 300 mM, pH 7.5) were stabilized at 30 ◦C for 5 min. Then, GTP dissolved in the same buffer was added (1 mM final concentration) at different time points (0, 2.5, 5, 10, 15, 20, and 30 min). The reactions were kept at 30 ◦C and developed in accordance with Malachite Green Phosphate Assay Kit (MAK307, Sigma-Aldrich, St. Louis, MO, USA).

5. Conclusions The chalcones described in this study aﬀect both gram-negative and gram-positive cells but not in the same manner. In gram-positive cells, the cytoplasmic membrane is a target; in gram-negative cells the ATP levels drop and macromolecular synthesis is inhibited.

Supplementary Materials: The following are available online, Figure S1: Membrane permeability assay, Figure S2, Relative inhibition by compounds in the REMA assay, Table S1: Structures and minimum inhibitory concentrations of hydroxychalcones synthesized and tested by Ayusso and Regasini (unpublished results). Author Contributions: L.G.M.: Investigation (lead), Visualization (supporting), Writing original draft (equal); A.S.G.L.: Funding acquisition (supporting), Investigation (lead), Writing original draft (equal), Visualization (supporting); P.C.: Investigation (supporting), Visualization (supporting); G.M.A.: Funding acquisition (supporting), Resources (lead); L.B.C.: Investigation (supporting), Visualization (supporting); M.B.S.: Resources (supporting); B.C.M.: Funding acquisition (supporting), Resources (supporting); G.D.: Investigation (supporting), Visualization (supporting); C.Z.: Investigation (supporting); L.O.R.: Conceptualization (supporting), Funding acquisition (supporting), Resources (equal), Supervision (supporting); H.F.: Conceptualization (lead), Funding acquisition (lead), Project administration (lead), Supervision (lead), Writing original draft (supporting); D.-J.S.: Conceptualization (lead), Funding acquisition (lead), Project administration (lead), Supervision (lead), Writing original draft (equal). All authors have read and agreed to the published version of the manuscript. Molecules 2020, 25, 4596 12 of 14

Funding: This work was supported by grants 729004005 from the Netherlands Organization for Scientific Research (DJS); FAPESP grants 2017/50216-0 (HF); 2014/18330-0 and 2018/15083-2 (LOR); and FAPESP scholarships 2014/18577-5 and 2016/08084-7 (BCM), and 2014/18719-4 and 2016/07108-0 (GMA); CNPq grants 246986/2013-1 (ASGL), and 471129/2013-5, 306251/2016-7 and 429322/2018-6 (LOR). Acknowledgments: We would like to thank Rieza Aprianto (University of Groningen, NL) for help in using the Tecan Infinite F200 Pro luminometer, and Mark Enright (Manchester Metropolitan University, UK) for comments and suggestions. Conflicts of Interest: The authors declare no conflict of interest.

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Sample Availability: Samples of the compounds BC1 and T9A are available from the authors.

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