ORIGINAL RESEARCH published: 19 June 2019 doi: 10.3389/fmicb.2019.01357 Testing a Human Antimicrobial RNase Chimera Against Bacterial Resistance Guillem Prats-Ejarque, Jiarui Li † , Fatima Ait-Ichou † , Helena Lorente and Ester Boix* Faculty of Biosciences, Department of Biochemistry and Molecular Biology, Universitat Autònoma de Barcelona, Barcelona, Spain The emergence of bacterial resistance to the most commonly used antibiotics encourages the design of novel antimicrobial drugs. Antimicrobial proteins and peptides (AMPs) are the key players in host innate immunity. They exert a rapid and multifaceted action that reduces the development of bacterial adaptation mechanisms. Human antimicrobial RNases belonging to the vertebrate specific RNase A superfamily participate in the maintenance of tissue and body fluid sterility. Among the eight human canonical RNases, RNase 3 stands out as the most cationic and effective bactericidal protein against Gram-negative species. Its enhanced ability to disrupt the bacterial Edited by: cell wall has evolved in detriment of its catalytic activity. Based on structure-functional Alessandra Polissi, University of Milan, Italy studies we have designed an RNase 3/1 hybrid construct that combines the high Reviewed by: catalytic activity of RNase 1 with RNase 3 bactericidal properties. Next, we have Gabriela Olmedo-Alvarez, explored the ability of this hybrid RNase to target the development of bacterial Unidad Irapuato (CINVESTAV), Mexico resistance on an Acinetobacter baumannii cell culture. Synergy assays were performed Jürgen Harder, in combination with colistin, a standard antimicrobial peptide used as an antibiotic University of Kiel, Germany to treat severe infections. Positive synergism was observed between colistin and the *Correspondence: RNase 3/1 hybrid protein. Subsequently, using an in vitro experimental evolution assay, Ester Boix Ester.Boix@uab.es by exposure of a bacterial culture to colistin at incremental doses, we demonstrated the † These authors have contributed ability of the RNase 3/1 construct to reduce the emergence of bacterial antimicrobial equally to this work resistance. The results advance the potential applicability of RNase-based drugs as antibiotic adjuvants. Specialty section: This article was submitted to Keywords: RNase, antimicrobial resistance, antibiotic adjuvant, gram-negative bacteria, antimicrobial peptides Antimicrobials, Resistance and Chemotherapy, a section of the journal Frontiers in Microbiology INTRODUCTION Received: 29 November 2018 The emergence of bacterial resistance to conventional antibiotics is becoming a serious sanitary Accepted: 31 May 2019 and economical threat. There is an urgent need to develop alternative drugs that address Published: 19 June 2019 this global health issue and overcome the spread of infectious diseases (Lohrasbi et al., 2018; Citation: Mustazzolu et al., 2018). In particular, novel therapeutic approaches will be required to fight Prats-Ejarque G, Li J, Ait-Ichou F, the dissemination of multi-drug resistant bacterial strains that threaten our public health system Lorente H and Boix E (2019) Testing a Human Antimicrobial RNase to return to the “pre-antibiotic era” (Wright, 2016). Currently, the majority of antimicrobial Chimera Against Bacterial Resistance. agents that are available in the pharmaceutical market rely on the direct elimination of the Front. Microbiol. 10:1357. microorganisms. Notwithstanding, recent literature highlights the potentiality of drug combination doi: 10.3389/fmicb.2019.01357 and simultaneous targeting of diverse cellular processes (Brochado et al., 2018; Lázár et al., 2018) to Frontiers in Microbiology | www.frontiersin.org 1 June 2019 | Volume 10 | Article 1357 Prats-Ejarque et al. An Antimicrobial RNase Targeting Bacterial Resistance overcome the bacterial development of resistance mechanisms. RNase enzymatic activity as a self-defense mechanism. Indeed, Being aware that full eradication of antibiotic resistance might most of our current antibiotics are derived from natural be unattainable, we should gather all our available means to compounds produced by microorganisms against competing minimize its dissemination and impact. One of the favorite species (Blair et al., 2015). Among them, bacteriocins, expressed strategies to overcome the so called “antibiotic resistance by commensal species and endowed with enzymatic activities, era” relies on the discovery of unconventional drugs and are being considered as appealing alternative antibiotics to fight combinatorial approaches (Brown and Wright, 2016). pathogenic strains (Mathur et al., 2018). In the search of novel antibiotic candidates, our own Another alternative approach proposed to fight resistance innate immunity system represents one of the best reservoirs. mechanisms against antimicrobial agents is the targeting of Upon infection, our innate immune cells secrete a variety of the bacterial community integrity. Recent studies reveal the antimicrobial proteins and peptides (AMPs) that protect the host previously underestimated complexity of bacterial communities biological fluids against pathogen invasion. AMPs are usually and identify collective resistance mechanisms (Vega and Gore, small polypeptides that present a non-specific wide-spectrum 2014). Novel strategies can be engineered to target the targeting of microorganisms (Hancock and Diamond, 2000). bacterial community cohesion and thereby weaken collective A growing clinical interest for AMPs is derived from their resistance. Collective resistance ensures the survival of the low toxicity to mammalian cells, together with the fact that microbial community upon exposure to antibiotic conditions bacterial resistance to these molecules seems inherently more that otherwise would be lethal to individual bacterial cells (Vega difficult to acquire in comparison to conventional antibiotics and Gore, 2014). One of the main mechanisms that ensures (Lai and Gallo, 2009; Nakatsuji and Gallo, 2012; Casciaro et al., bacterial survival relies on the intercellular horizontal transfer 2018). Within the AMPs, the largest group corresponds to of information that facilitates a rapid response to any external cationic peptides (Hancock and Diamond, 2000). The mode of injury and ensures the community’s adaptation to a hostile action of cationic antimicrobial peptides is different from that environment (Papenfort and Bassler, 2016). Recent discoveries of conventional antibiotics and is often related to interaction have identified signaling molecules that contribute to community with bacterial walls through electrostatic forces and subsequent quorum sensing, such as regulatory small RNAs (Papenfort cell lysis (Yeaman, 2003; Bahar and Ren, 2013). AMPs are and Vogel, 2010). Therefore, an antimicrobial agent endowed mostly amphiphilic in nature and they are comprised of with RNase activity that can target bacterial quorum-sensing hydrophobic and hydrophilic residues aligned on opposite sides signaling might work as an antibiotic adjuvant. Indeed, the use of of the peptides, facilitating their easy penetration through cell antibiotic adjuvants is one of the selected strategies to minimize membranes (Torrent et al., 2011a,b; Brandenburg et al., 2012). the emergence and impact of resistance phenomena (Wright, Fortunately, although several cases of bacterial resistance to 2016). Addition of adjuvants can lower the needed antibiotic AMPs have been observed (Perron et al., 2006; Moffatt et al., dose to reach a therapeutic effect (Brown and Wright, 2016). 2010), the complex structure of bacterial envelopes hinder the Moreover, weakening the bacterial community cohesion would development of total resistance. Besides, AMPs are frequently reduce the emergence and dissemination of resistant pathogenic multifaceted molecules that combine a direct mechanical action strains (Starkey et al., 2014). to the bacterial envelope with a specific enzymatic activity In this study, we have committed ourselves to evaluating the that targets essential intracellular macromolecule components. potential contribution of RNase catalytic activity in reducing the A variety of multifunctional antimicrobial proteins, endowed emergence of bacterial resistance. Toward this end, we have used with protease, DNase or RNase activity, can participate in the host an experimental evolution assay by exposing an Acinetobacter defense system (Brogden, 2005; Hancock and Sahl, 2006; Boix baumannii bacterial culture to increasing concentrations of and Nogués, 2007; Arranz-Trullén et al., 2017). colistin. Colistin (also called polymyxin E) is a non-ribosomal In our laboratory, we are working on the structure-functional bacterial cyclic AMP only used in the clinics as a last resort relationship of human antimicrobial RNases that belong to to treat live-threatening infections, due to its reported toxicity. the vertebrate-specific RNase A superfamily and are secreted Here, we have tested the reduction of the antimicrobial resistance by epithelial and blood cells during infection (Gupta et al., against colistin upon treatment with an engineered RNase 2012; Koczera et al., 2016; Lu et al., 2018). Characterization construct that combines a high catalytic activity with specific of the mechanism of action of human antimicrobial RNases antimicrobial properties. suggests that a combination of activities are taking place (Salazar et al., 2016; Lu et al., 2018). In particular, the human RNase 3, mostly secreted by eosinophils upon infection, combines a high MATERIALS AND METHODS cationicity (pI ∼11), lipopolysaccharide (LPS) binding affinity, membrane destabilization and bacterial agglutination activities Materials (Boix et al., 2012; Torrent et al., 2012). The A. baumannii strain (CECT 452; ATCC 19606) and Interestingly, a striking structural homology between the Pseudomonas aeruginosa strains (CECT 4122; ATCC 15692) RNase A superfamily and bacterial RNases belonging to the are from the Spanish Type Culture Collection (CECT). The contact-dependent growth inhibition (CDI) toxins was recently Escherichia coli BL21(DE3) strain and the pET11c plasmid are reported (Batot et al., 2017; Cuthbert et al., 2018). CDI bacterial from Novagen. MRC-5 and HepG2 cells are from the American toxins work as inter-strain competition weapons and use the Type Cell Culture Collection (ATCCC). Mueller–Hinton broth, Frontiers in Microbiology | www.frontiersin.org 2 June 2019 | Volume 10 | Article 1357 Prats-Ejarque et al. An Antimicrobial RNase Targeting Bacterial Resistance LPS and RNase A (Type XII) are from Sigma-Aldrich. 0.2 cm path-length quartz cuvette. The percentage of secondary 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide structure was estimated with Spectra Manager II, as described (MTT), Isopropyl β-D-1-thiogalactopyranoside (IPTG) and (Yang et al., 1986). colistin are from Apollo Scientific. 1-aminonaphthalene-3,6,8- trisulfonate (ANTS), α,α0 -dipyridinium p-xylene dibromide Activity Staining Gel (DPX) and the fluorescent probe BODIPY TR cadaverine Zymograms were performed following the method previously are from Molecular Probes. Toludine blue is from Merck. described (Bravo et al., 1994). 15% polyacrylamide-SDS gels were RNase 3/1 gene was purchased from NZYTech. 1,2-dioleoyl-sn- cast with 0.3 mg/mL of poly(C) (Sigma Aldrich). Then, 20 ng glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero- of RNase 1, 3, and 3/1 were loaded, and the gel was run at a 3-phosphoglycerol (DOPG) were from Avanti Polar Lipids. E. coli constant current of 100 V for 1.5 h. Following, the SDS was lipid extract was obtained as described (Folch et al., 1957). removed from the gel with 10 mM Tris/HCl, pH 8, and 10% Human RNase 1 gene was a gift from Dr. Maria Vilanova, (v/v) isopropanol. The gel was then incubated during 1 h in Universitat de Girona, Spain, and human RNase 3 sequence was the activity buffer (100 mM Tris/HCl, pH 8) to allow enzymatic taken from a previously synthesized gene (Boix et al., 1999). digestion of the embedded substrate and then stained with 0.2% (w/v) toluidine blue in 10 mM Tris/HCl, pH 8, for 10 min. Protein Expression and Purification Positive bands appeared white against the blue background. The The RNase 1, 3, and 3/1 genes were subcloned into the plasmid loading buffer had no 2-mercaptoethanol to facilitate recovery pET11c for prokaryote high yield expression in the E. coli of active enzymes. BL21(DE3) strain. The recombinant protein was expressed and purified as previously described (Boix, 2001), with some Minimum Bactericidal Concentration modifications (Palmer and Wingfield, 2004). Briefly, bacteria were grown in terrific broth (TB), containing 400 µg/mL (MBC) Determination ampicillin. Recombinant protein was expressed after cell MBC100 was defined as the lowest protein/peptide concentration induction with 1 mM IPTG added when the culture showed that completely eradicated bacterial cells. RNase 3/1 was serially an OD600 of 0.6. The cell pellet was collected after 4 h of diluted in HBS (HEPES 20 mm pH 7.4, NaCl 100 mM) in 96-well culture at 37◦ C. Cells were resuspended in 10 mM Tris/HCl plate in a volume of 100 µL to achieve final concentrations from and 2 mM EDTA, pH 8 and 40 µg/mL of lysozyme, and 20 to 0.02 µM. Then, 2 µL of an exponential phase subculture sonicated after 30 min. The pellet was suspended in 25 mL of E. coli, A. baumannii or P. aeruginosa was added, previously of the same buffer with 1% triton X-100 and 1 M urea and adjusted to give a final concentration of approximately 5 × 105 was left stirring at room temperature for 30 min, before being colony-forming units (CFU)/mL in each well and the plate was centrifuged for 30 min at 22.000 × g. This procedure was repeated incubated for 4 h at 37◦ C and 100 rpm. Finally, samples were until the supernatant was completely transparent. In order to plated onto LB (Condalab) Petri dishes and incubated at 37◦ C remove the triton X-100, 200 mL of 10 mM Tris-HCl pH 8.5, overnight. All the assays were performed in triplicate. 2 mM EDTA was added to the pellet and centrifuged again for 30 min at 22.000 × g. The resulting pellet was suspended Cytotoxicity Assay in 25 mL of Tris-acetate 100 mM, pH 8.5, 2 mM EDTA, 6 Cytotoxicity was measured for the MRC-5 and HepG2 human M guanidine hydrochloride, and 80 mM of DTT. The protein cell lines using the MTT assay, as described previously (Pulido was then refolded for 72 h at 4◦ C by a rapid 100-fold dilution et al., 2018). Cells were grown in 5% CO2 at 37 ◦ C with minimal into 100 mM Tris/HCl, pH 8.5, 0.5 M of guanidinium chloride, essential medium supplemented with 10% fetal bovine serum and 0.5 M L-arginine, and oxidized glutathione (GSSG) was (FBS). Cells were plated at 5 × 104 cells/well in a 96-well plate and added to obtain a DTT/GSSG ratio of 4. The folded protein incubated overnight. Next, the medium was removed and serial was then concentrated, buffer-exchanged against 150 mM sodium dilutions of RNase 1, 3, and 3/1 were added at concentrations acetate, pH 5 and purified by cation-exchange chromatography ranging from 200 to 0.2 µM in 100 µL of medium without on a Resource S (GE Healthcare) column equilibrated with the serum. After 4 h of incubation, the medium was replaced with same buffer. The protein was eluted with a linear NaCl gradient fresh medium containing 0.5 mg/mL MTT solution and the from 0 to 2 M in 150 mM sodium acetate, pH 5. The protein mixture was incubated for 2 h in 5% CO2 at 37 ◦ C. The medium purity was checked by SDS-PAGE and reverse-phase HPLC. was then removed and formazan was dissolved by adding acidic Absence of unpaired Cys was confirmed by the Ellman’s reaction isopropanol. The optical density (OD) was recorded by using a (Ellman, 1959). Victor3 plate reader (PerkinElmer, Waltham, MA) set at 550 and 630 nm as references. Reference absorbance at 630 nm was used Circular Dichroism (CD) to correct for nonspecific background values. Each experiment Far-UV CD spectra were obtained from a Jasco-715 (Jasco), as was repeated at least three times. previously described (Torrent et al., 2009a). The spectra were registered from 195 to 240 nm at room temperature. Data from LPS Binding Assay four consecutive scans were averaged. Before reading, the sample The LPS-binding affinity was assessed using the fluorescent probe was centrifuged at 10.000 × g for 5 min. Protein spectra were BODIPY TR cadaverine (BC) as described (Torrent et al., 2008). obtained at 6 µM in 5 mM sodium phosphate, pH 7.5, with a RNase 1, 3, and 3/1were serially diluted in a 96-well fluorescence Frontiers in Microbiology | www.frontiersin.org 3 June 2019 | Volume 10 | Article 1357 Prats-Ejarque et al. An Antimicrobial RNase Targeting Bacterial Resistance plate from 20 to 0.02 µM in HEPES 10 mm pH 7.4. Then, next day, the 24-h culture (stationary phase) was diluted LPS (10 µg/mL) and BC (10 µM) were added in the same 1:150 times (approximately 2 × 107 CFU/mL) and placed buffer. Fluorescence measurements were performed on a Victor3 in a 96-well polypropylene plate. Before the inoculation of plate reader. The BC excitation wavelength was 580 nm, and the bacteria, 90 µL of colistin in Mueller–Hinton medium the emission wavelength was 620 nm. Occupancy factor was were added to 80 wells in order to achieve the desired calculated as described previously (Torrent et al., 2008). concentration in the final volume (100 µL). To 40 of these wells, 10 µL of 1 µM RNase 3/1 (to have a final concentration Liposome Preparation of 100 nM) in a cationic protein/peptide stabilizer solution Large unilamellar vesicles (LUVs) containing DOPC/DOPG (3:2 (acetic acid at 0.01%) was added (Wiegand et al., 2008). molar ratio, 1 mM stock concentration) or E. coli membrane To the other 40 wells, the same solution without protein lipids (5 mg/mL stock concentration) of a defined size were was added. Finally, Mueller–Hinton medium with 10% of the obtained from a vacuum drying lipid chloroform solution. After stabilizer solution was added to the other 16 wells, as a the chloroform evaporation, liposomes were suspended with negative control. 10 mM Tris/HCl, 20 mM NaCl, pH 7.4. The distribution and the In the first experiment (Assay 1), the plate was placed mean hydrodynamic range of the liposomes in suspension was overnight in an Infinite F Nano + (Tecan) plate reader determined by dynamic light scattering (DLS) with a Zetasizer at 37◦ C with orbital shaking, and the OD600 was recorded Nano ZS Malvern, and the data was analyzed by its built-in every 5 min. The day after, the cultures at the stationary software (Zetasizer 7.02). phase were diluted three times and 2 µL of the diluted bacteria were inoculated to a new plate prepared as explained Liposome Leakage above, in order to have a 1:150 final dilution and incubated The ANTS/DPX liposome leakage fluorescence assay was overnight to determine the bacterial growth profiles by DO600nm performed as previously described (Pulido et al., 2016a). Briefly, monitoring. The first plate was stored at −80◦ C with 15% a unique population of LUVs DOPC/DOPG (3:2) or E. coli of glycerol for later determination of colistin MIC value. The liposomes was prepared to encapsulate a solution containing dose of each day of colistin exposure was adjusted according 12.5 mM ANTS, 45 mM DPX, 20 mM NaCl, and 10 mM to the observed bacterial growth; when no reduction of Tris/HCl, pH 7.5 by applying three freeze-thaw cycles. The the bacterial growth was observed, the dose was increased; ANTS/DPX liposome stock suspension was diluted to 30 µM and, when bacteria growth reduction was observed, the and incubated at 37◦ C for 1 h with RNase 1, 3, and 3/1, serially dose was maintained. diluted from 20 to 0.015 µM in a microtiter fluorescence plate. In order to test if RNase 3/1 could also hinder the resistance Fluorescence measurements were performed on a Victor3 plate acquisition of the bacteria in a colistin-pre-exposed strain, a reader with an excitation wavelength of 386 nm and an emission second assay was performed, following a modification of a wavelength of 535 nm. ED50 values were calculated by fitting the previously described assay (Lázár et al., 2018) (named as Assay data to a dose–response curve with Origin 8.0. 2). The plate preparation was performed as previously explained with the following modifications. Before starting the treatment protocol, a fresh isolated colony of A. baumannii was preexposed Synergy Determination Assay for three 24-h cycles to a 0.3 µM initial dose of colistin at the The synergy test was carried out by determining the bacterial same conditions than the first exposure assay. Then, to each of minimum inhibitory concentration (MIC) in Müller Hinton 40 wells of the pre-exposed culture, the protein was added to medium, as follows. 8 × 8 well plates were used to check a concentration of 1 µM, while the colistin dose was increased the protein/colistin combinations. First, RNase 3/1 was serially at 1.2 × serial incremental doses, keeping constant the protein diluted from 12 to 0.1 µM, in eppendorf tubes at a 10-fold concentration. The plates were incubated in an incubator at the final concentration in 0.01% acetic acid (Wiegand et al., 37◦ C and 100 rpm. The OD600 was recorded after 24 h in a 2008). Then, colistin was serially diluted from 8 to 0.02 µM Victor3 plate reader. in Mueller–Hinton medium to a final volume of 90 µL, and 10 µL of the serially diluted protein aliquots were added in each corresponding well (except in the negative control, where only Minimum Inhibition Concentration (MIC) buffer was added). After that, 2 µL of a 1:3 diluted 24-h culture of Determination A. baumannii were added to each well to achieve a final 1:150 The MIC of each of the bacterial lineages generated during the bacterial dilution (approximately 2 × 105 CFU/mL), and the resistance evolution assay was determined. MIC was defined plate was incubated overnight at 37◦ C and 100 rpm. Positive or as the lowest protein/peptide concentration that completely negative bacterial growth was checked by optical density. All the inhibited bacterial growth. Colistin was serially diluted from 64 assays were performed in triplicate. to 0.5 µM in Mueller–Hinton. Next, 2 µL of a 1:3 diluted 24-h culture of each A. baumannii lineage were added to each well to Antimicrobial Resistance Evolution achieve a final 1:150 bacterial dilution (approximately 2 × 107 Assay by Colistin Exposure CFU/mL), and the plate was incubated overnight at 37◦ C and A fresh and isolated colony of A. baumannii was picked up 100 rpm. Presence or absence of bacterial growth were checked and left to grow overnight in Mueller–Hinton broth. The by visual inspection. Frontiers in Microbiology | www.frontiersin.org 4 June 2019 | Volume 10 | Article 1357 Prats-Ejarque et al. An Antimicrobial RNase Targeting Bacterial Resistance Statistical Analysis (Doucet et al., 2009; Gagné et al., 2015). In addition, the cationic Barlett’s test and Shapiro–Wilk’s method were used to study regions from Arg77 to Arg81 and Arg103 to Arg107, associated to the variance homogeneity and normal distribution of the RNase 3 bactericidal activity, were incorporated (Carreras et al., data, respectively. Due to unequal sample size among groups 2003). On the other hand, the residues that participate in the (unbalanced design), the Kruskall-Wallis non-parametric test binding of the main and secondary bases of the RNA substrate, followed by post hoc comparisons using Wilcoxon method were located at the 50 and 30 sides of the cleaved phosphodiester bond used for statistical comparison of continuous variable (time to (B1 and B2, respectively), were kept according to the RNase 1 reach exponential phase) for A. baumannii between and within sequence. The resulting final sequence and the structure model the three different groups (culture bacteria treated with colistin, are shown in Figure 1. colistin + RNase 3/1 or non-treated control). Sample size (n) was RNase 3/1 was successfully expressed in E. coli BL21(DE3) cells 40 for the two treatments and 16 for the control group. Statistical and purified from inclusion bodies at a final yield of 30 mg/L significance was set at p < 0.05, and all statistical tests were of bacterial culture. The proper 3D protein folding was checked 2-sided. Data are presented as means ± standard error. Statistical by circular dichroism (Supplementary Figure S1), giving the analysis and data visualization was performed using R Software characteristic secondary structure percentage distribution of the 3.4.4 (R Core Team, 2018). RNase A family (Fowler et al., 2011). Moreover, we confirmed by Ellman’s assay that all disulphide bonds were formed in the refolded protein and no free Cys residue was present RESULTS (A412 nm = 0.0003 and 0.001 for 4 and 8 µM of protein sample respect to control buffer). RNase 3/1 Rational Design Next, we assessed the protein catalytic activity using an activity RNase 3/1 has been designed in an effort to combine both staining gel. Results indicated that the RNase 3/1 hybrid displays the high bactericidal activity of human RNase 3 and the high a high catalytic efficiency toward a polynucleotide substrate. catalytic activity of human RNase 1, according to previous As shown in Figure 2, the catalytic activity of RNase 3/1 for structural-functional studies (Prats-Ejarque et al., unpublished polyuridine (poly(U)) is more than 30 times higher than that of results). Using the RNase 1 structure as a scaffold, antibacterial RNase 3, almost reaching half of the catalytic activity of RNase 1. regions of RNase 3 were added. Specifically, we incorporated the In view of these results, we wanted to check whether the N-terminal region of RNase 3, previously found to be related to engineered structural changes that achieved an increase in antimicrobial activity (Torrent et al., 2009a, 2010, 2013; Sánchez catalytic activity in relation to RNase 3 had any detrimental et al., 2011), but preserved the Asp17-Ser26 flexible loop of effect on the hybrid proteins antimicrobial activity. Therefore, RNase 1, considered essential for RNase 1 high catalytic activity we determined the MBC for the hybrid and parental RNases. FIGURE 1 | Design of RNase3/1 chimera. (A) Model of RNase 3/1 obtained by Modeler 9.12 (Webb and Sali, 2016). In green, B1 site. In pink, B2 site. In red, the active site. In beige, the RNase 1 skeleton. In light blue, the RNase 3 antimicrobial regions. (B) Alignment of RNase 1, 3 and 3/1 primary structures. The fully conserved amino acids are highlighted in red. The residues that are not conserved but have similar properties are marked with red letters. The secondary structure is indicated above the alignment. The green numbers indicate the disulphide bridge pairs. The alignment was done using Clustal Omega (Sievers and Higgins, 2018), and the image was obtained with ESPript7 (http://espript.ibcp.fr/ESPript/) (Robert and Gouet, 2014). Frontiers in Microbiology | www.frontiersin.org 5 June 2019 | Volume 10 | Article 1357 Prats-Ejarque et al. An Antimicrobial RNase Targeting Bacterial Resistance FIGURE 2 | Catalytic characterization of RNase3/1 by zymogram. (A) Activity staining gel showing the degradation of poly(U) with 20 ng of each protein. RNase 3 (lane 1), RNase 3/1 (lane 2), and RNase 1 (lane 3). (B) Densitometric analysis of the bands of the activity staining gel. Quantification was done from a scan image using Quantity One software (Bio-Rad R ). The results confirmed the acquisition of bactericidal activity of TABLE 2 | LPS-binding affinity and liposome leakage activity of RNases 1, 3 and 3/1. RNase 3/1 respect to the non-antimicrobial RNase 1; although a significant decrease of the hybrid bactericidal activity in LPS binding Liposome leakage comparison to RNase 3 is appreciated (Table 1). (LBC50 ) (µM) (LC20 ) (µM) Next, we evaluated whether our protein construct retained the high LPS-binding affinity observed for RNase 3 (Torrent DOPC:DOPG E. coli et al., 2008; Pulido et al., 2012, 2016b). LPS-binding affinity RNase 1 1.50 ± 0.28 N.D. N.D. was estimated by a displacement assay using the fluorescent RNase 3 0.38 ± 0.03 1.19 ± 0.19 1.87 ± 0.42 BODIPY-cadaverine (BC) probe as previously described (Torrent RNase 3/1 0.59 ± 0.02 1.18 ± 0.36 0.86 ± 0.18 et al., 2008). Results confirmed that RNase 3/1 retained The LPS binding concentration at the 50% displacement (LBC50 ) values have a similar LPS-binding affinity to RNase 3 (Table 2 and been determined from a dose-response curve adjusted by OriginPro 8 statistical Supplementary Figure S2). software. Liposome leakage was measured as the protein concentration that In addition, the liposome leakage ability of the RNases was induces 20% of liposome leakage (LC20 ). Experimental data plots are shown in Supplementary Figures S2, S3, respectively. N.D. indicates that no leakage was evaluated. As shown in Table 2, RNase 3/1 retains similar detected at the highest tested concentration (20 µM). liposome leakage activity to RNase 3, while RNase 1 does not present any membrane disruption ability. TABLE 3 | Cytotoxic activity characterization of RNase3/1. Finally, the potential cytotoxicity against host tissues was evaluated in vitro using two human cell lines: immortalized lung IC50 (µM) fibroblasts (MRC-5) and tumor hepatic cells (HepG2). The results indicated that RNase 3/1 shows no toxicity at the maximum RNase 1 RNase 3 RNase 3/1 concentration tested (200 µM) as observed for RNase 1 (Table 3). Lung fibroblasts (MRC-5) N.D. >100 N.D. Hepatocarcinoma epithelial N.D. 134.66 ± 0.95 N.D. RNase 3/1 Shows Synergy Activity With cells (HepG2) Colistin Against A. baumannii IC50 was determined as the concentration of protein where 50% of cells were still Next, we decided to test the potential synergistic activity of alive. Cytotoxicity was determined by the MTT assay. Each assay was performed in triplicate. N.D. means that, at the highest tested concentration (200 µM), no RNase 3/1 in combination with a common antimicrobial peptide reduction of cell viability was detected. TABLE 1 | Bactericidal activity characterization of RNase3/1. used in the clinics, colistin. The analysis of the combinatorial effect of RNase 3/1 with colistin showed that the combination MBC100 (µM) of colistin with RNase 3/1 improves significantly the MIC100 of colistin against A. baumannii (Figure 3). The MIC100 value for E. coli A. baumannii P. aeruginosa colistin alone in the assayed conditions was 0.9 ± 0.05 µM. RNase 1 >20 >20 18.33 ± 2.89 The calculated RNase 3/1 concentration where the MIC for RNase 3 1.88 ± 0.88 0.6 ± 0.07 0.6 ± 0.07 colistin is reduced by half was 1.35 ± 0.15 µM. Interestingly, RNase 3/1 6.25 ± 2.17 6.25 ± 2.17 3.13 ± 1.08 we observe a biphasic dose-response profile, where the MIC for MBC100 was determined as the lowest concentration of peptide where no cells colistin reaches a first plateau in the presence of 0.3 to 5 µM of were detected by Colony Forming Units counting in HBS solution. RNase 3/1 (∼1.5 × reduction of the original MIC value) and a Frontiers in Microbiology | www.frontiersin.org 6 June 2019 | Volume 10 | Article 1357 Prats-Ejarque et al. An Antimicrobial RNase Targeting Bacterial Resistance Moreover, addition of RNase 3/1 not only displaced the bacterial growth curve, producing a delay in the overall growth of the surviving bacterial lineages, but also resulted in increased mortality rate (see Table 4 and Figure 6A). Noteworthy, the cycles corresponding to the highest mortality rates (Day 9 and Day 11 in case of colistin alone; Day 4 and Day 11 in case of colistin combined with RNase 3/1) correlate with an increase in the time needed to reach the exponential phase. To note, at the 4th day of exposure, when the colistin dose was increased from 0.1 to 0.25 µM, we observed a marked decrease in growth in both colistin and colistin + RNase 3/1 conditions. In particular, the response to the increase of the colistin dose was most prominent in the RNase 3/1 supplemented condition, achieving, at 0.25 µM (Day 4), a 20% of mortality versus only a 2.5% in the cultures exposed only to colistin (Figure 6A). Likewise, we observed a pronounced shift of the growth curves (Figures 5, 6B). FIGURE 3 | Biphasic dose-response fit of the variation of the MIC100 of colistin against A. baumannii as a function of the RNase 3/1 concentration. A second peak of mortality was visualized between days The non-linear fitting was done using OriginLab 8. 8 and 9 (corresponding to 0.35 µM to 0.7 µM of colistin dose increase). At this point, only 35% of the original strains exposed to colistin + RNase 3/1 survived, versus a second plateau at concentrations higher than 10 µM of RNase 65% of survival for colistin alone. Interestingly, although the 3/1 (∼5 × reduction of the MIC value). condition exposed to colistin alone experimented a significant delay of growth (Figure 6B), this was not the case for the RNase 3/1 supplemented condition, suggesting that the Exposure of Bacterial Culture to RNase surviving strains had already acquired resistance to that 3/1 Reduces the Acquisition of Bacterial dose of colistin. Resistance to Colistin Eventually, a shock dose of colistin (10 × the dose of the Positive synergy results encouraged us to test our hybrid RNase in previous day) was applied at day 11, triggering a high mortality an antimicrobial resistance evolution assay. Toward this end, we and a strong delay of the growth of the surviving strains. performed two different exposure protocols, as described in the Following this shock dose, the surviving rate of the lineages methodology section, which we will refer to as Assays 1 and 2 (see exposed to colistin and RNase 3/1 was less than half the observed Figure 4). In Assay 1, we exposed non-treated A. baumannii cells in the cultures exposed to colistin alone (35–15% of survival, to either colistin alone or colistin supplemented with RNase 3/1 respectively), as shown in Figure 6A and Table 4. at 0.1 µM. RNase 3/1 final concentration was selected well below On the other hand, results highlighted an overall reduction by the minimum value required to display any potential synergy half in the calculated average MIC mean value for colistin in the effect in combination with colistin (see Figure 3). The MIC bacterial cultures supplemented with RNase 3/1 (Figure 7). value for RNase 3/1 in the assay conditions is ≥10 µM. During Next, we wanted to explore whether RNase 3/1 addition to the experiment, in order to increase the selection pressure, colistin could also hinder the bacterial resistance acquisition we gradually increased the dose of colistin in accordance to to a strain that had previously acquired resistance to colistin. the bacterial growth, while keeping the same initial RNase 3/1 With this goal in mind, we incubated an A. baumannii concentration at 0.1 µM. The cultures of 40 parallel lineages culture for 3 days in the presence of 0.3 µM of colistin, and were serially diluted and transferred for eleven consecutive cycles then, we performed a second experimental evolution assay of 24-h each, corresponding to a total of about 330 generations using this strain, with acquired resistance to colistin, as a as described (Moffatt et al., 2010). Colistin dose was adjusted starting seed (see Assay 2 in Figure 4). Then, the cultures according to the observed bacterial growth curves: the dose was were exposed to either colistin alone or colistin supplemented increased when no effect on the bacterial growth was observed, with RNase 3/1 at 1 µM final protein concentration. In this and kept constant when a significant reduction of bacterial experiment, RNase 3/1 concentration was added at 1 µM growth was registered. The growth of the bacteria following 1/150 to ensure a positive response when tested in combination dilution was monitored every 5 min and results were compared with colistin (see Figure 3). The 40 parallel lineages were to parallel non-exposed control cultures. exposed to gradually increasing concentrations of colistin Evaluation of the growth curves confirmed that the (1.2-fold following each transfer cycle of 24 h, starting from experimental evolution assay has been successful. The an initial concentration of 0.3 µM, see corresponding daily monitoring of culture optical density indicated that the exposure concentrations in Supplementary Figure S6). The tested colistin to increasing concentrations of colistin delayed bacterial growth range (from 0.3 to 3.21 µM) ensured the induction of (see Figure 5). Interestingly, the growth curve displacement is bacterial resistance and avoided massive cell mortality. Results more pronounced by the addition of RNase 3/1. indicated that the average increase of the MIC for colistin Frontiers in Microbiology | www.frontiersin.org 7 June 2019 | Volume 10 | Article 1357 Prats-Ejarque et al. An Antimicrobial RNase Targeting Bacterial Resistance FIGURE 4 | Graphical depiction of the experimental evolution of resistance assay protocol by culture exposure to colistin. 40 parallel lineages of A. baumannii were exposed to colistin or colistin + RNase 3/1. Assay 1 was started from a single colony without colistin preexposure incubated in the presence of 0.1 µM of RNase 3/1. Assay 2 was started from a culture pre-exposed to 0.3 µM of colistin during 3 days and addition of RNase 3/1 at 1 µM. FIGURE 5 | A. baumannii growth curves at representative days of exposure. Bacterial culture growth was monitored following 1/150 dilution by OD600 reading. Points represent the mean of all the lineages for each day and condition. In red is shown the negative control, in blue the colistin-exposed cultures and in green, the colistin + RNase 3/1 conditions. Error bars represent the standard error of the mean. A statistical analysis was applied to further evaluate the significance of the observed differences between the behavior of the cultures exposed only to colistin and the cultures exposed to both colistin and RNase 3/1. Taking as a reference the time needed for the bacteria to reach the exponential phase (OD600 = 0.4), we performed a comparison between cultures belonging to the same cycle (Figure 6B) and cultures of consecutive cycles within each condition (Supplementary Figures S4, S5). following 14 cycles of 24 h (about 420 generations) was DISCUSSION significantly decreased for the condition supplemented with RNase 3/1 in comparison to the non-supplemented sample The design of novel strategies for the eradication of bacterial (Figure 8 and Supplementary Figure S6). resistance forms is one of the main concerns of the global health Frontiers in Microbiology | www.frontiersin.org 8 June 2019 | Volume 10 | Article 1357 Prats-Ejarque et al. An Antimicrobial RNase Targeting Bacterial Resistance TABLE 4 | Evolution of the resistance acquisition of A. baumannii to colistin, considering the number of replicates alive after each day of exposure. Alive replicates after n days of exposure (%) D1 D2 D3 D4 D5 D6 D7 D8 D9 D10 D11 Colistin 100 100 100 97.5 97.5 97.5 97.5 97.5 65 65 35 Colistin + RNase 3/1 100 100 100 80 80 80 75 75 35 35 15 Negative control 100 100 100 100 100 100 100 100 100 100 100 Colistin dose 0.1 µM 0.25 µM 0.3 µM 0.35 µM 0.7 µM 2 µM 20 µM The initial number of replicate lineages was 40. Colistin dose was gradually increased according to bacterial growth, as detailed in the methodology. The colistin dose used in each exposure is indicated in the bottom row. RNase 3/1, when used, was kept constant at 0.1 µM. FIGURE 6 | Evolution of the resistance acquisition of A. baumannii to colistin in the presence and absence of RNase 3/1. (A) Graphical representation of the surviving replicates after each day of exposure. (B) Graphical representation of the time needed for the surviving bacteria upon treatment to reach exponential phase (OD600 = 0.4). The remaining alive replicates are indicated following each day of exposure. Significance is calculated between the different conditions of the same day; n.s. means no significance between the compared samples, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001 are indicated. The bottom red line indicates the calculated MIC of the colistin alone. organization and is among the top priority lines of action of delivered by bacterial extracellular vesicles. Bacterial extracellular the current research policy. The extensive use and abuse of vesicles ensure the exchange of proteins and nucleic acids (Kulp conventional antibiotics during the last decades have generated and Kuehn, 2010; Rumbo et al., 2011; Schwechheimer and a population of resistant bacteria. This phenomenon has been Kuehn, 2015; Jan, 2017) and mediate the rapid adaption of the aggravated by the fact that most commonly used antibiotics community to hostile environments. Interestingly, the transfer nowadays are based on bacterial secreted natural products. of RNA molecules has been identified (Guerrero-Mandujano Indeed, the pharmaceutical industry has deeply exploited the et al., 2017) to participate in transcription regulation mechanisms naturally produced self-defense toxins that bacteria secrete to (Sjostrom et al., 2015; Koeppen et al., 2016). Overall, the search fight against competitor strains. Following billions of years of of novel drugs that target the bacterial community signaling co-evolution, a reservoir of resistance genes has been created that mechanisms, also referred as quorum sensing inhibitors, is can confer bacterial protection to most conventional antibiotics. gaining prominence in the pharmaceutical research field. In addition, we cannot underestimate the inherent ability of Within this context, a compound endowed with RNase bacterial cells to generate diversity. This process can constantly activity would be able to interfere in the horizontal exchange enrich the bacterial resistome pool, which is easily mobilized in of RNA and thereby attack the community cohesion. To note, case of emergency (Blair et al., 2015; Knoppel et al., 2017). Last the use of the enzymatic activities of bacteriocin toxins was but not least, cooperative mechanisms are taking place between proposed as a strategy to weaken the biofilm community the individual cells of the bacterial community and can provide a viability (Mathur et al., 2018). Bacteriocins are antimicrobial collective antimicrobial resistance profile (Vega and Gore, 2014). proteins secreted by bacteria that specifically target quorum The intercellular network organization can play a prominent sensing systems. Among these proteins we can find enzymes role under stressful conditions and can facilitate an appropriate with protease, DNase and RNase activities that facilitate biofilm response to external injuries. Therefore, the blockage of bacterial removal. Interestingly, recent literature reveals the structure fold signaling systems should significantly reduce the emergence of homology between vertebrate secretory RNases and bacterial resistance mechanisms. Noteworthy, the horizontal exchange of RNases that work as inter-strain competition toxins (Batot material between the integrands of the community is mainly et al., 2017; Cuthbert et al., 2018). A parallelism can be Frontiers in Microbiology | www.frontiersin.org 9 June 2019 | Volume 10 | Article 1357 Prats-Ejarque et al. An Antimicrobial RNase Targeting Bacterial Resistance of RNase 3 (Torrent et al., 2008) together with its membrane leakage activity (Torrent et al., 2007, 2009b) (Table 2). On the other hand, RNase 3/1 shares with RNase 1 its non-toxicity to the tested human cell lines (Table 3). Indeed, human secretory RNases’ harmlessness is ensured thanks to the presence in the cytosol of all human tissue cells of the RNase inhibitor (RI), a horseshoe-shaped protein that binds to the human secretory RNase members (Lomax et al., 2014) in a 1:1 stoichiometry with an unusually high affinity (at the fM-nM range). The RI is ubiquitous and protects the host cells from the potential toxicity of secretory RNases (Dickson et al., 2005). Therefore, our RNase chimera combines a high affinity for bacterial membranes together with an elevated catalytic efficiency and no toxicity to the host. Here, we have investigated the potency of our lead construct to work as an adjuvant molecule and reduce the emergence of bacterial resistance to colistin, an antimicrobial peptide, commonly used in the clinics. Colistin targets bacterial cells by a direct action at the cell membrane and bacterial wall, FIGURE 7 | Comparison of the calculated average mean MIC value following Assay 1 treatment for RNase 3/1-supplemented versus non-supplemented and is mostly used to treat difficult infections, such as the bacterial strains exposed to colistin. Error bars show the standard error of the ones caused by Gram-negative multidrug resistant strains (Stein mean. ∗ indicates that the p-value is < 0.05. and Raoult, 2002; Michalopoulos et al., 2005; Sabuda et al., 2008; Mendelson et al., 2018). However, colistin can accumulate in many body tissues, such as kidney and liver, and shows established between bacterial RNases secreted as virulence toxins considerable collateral toxicity to the host in the long term, so and secretory RNases expressed by innate cells in vertebrates it is only prescribed as a last resort in critically ill patients (Stein (Lu et al., 2018). An interesting hypothesis puts forward the and Raoult, 2002; Michalopoulos et al., 2005; Sabuda et al., 2008; scenario where the proteins secreted by granulocytes could Mendelson et al., 2018). provide a link between a simple local response and a systemic The use of drug combinations is currently attracting attention organized signaling process associated to the increase in size in the fight against antimicrobial resistance (Tabbene et al., 2015; and complexity of multicellular organisms. In this context, Brochado et al., 2018; Lázár et al., 2018). The co-administration the host innate cells would have retained similar self-defense of an adjuvant molecule can significantly lower the required mechanisms as displayed by unicellular organisms. The ancestral effective dose to treat an infection. In the same vein, our present role of bacterial virulence toxins would have derived to a data using A. baumannii cultures indicate that by combining more specialized role toward body fluid protection against the engineered RNase construct with colistin we can reduce infection (Lee and Lee, 2005). Understanding the similarities its effective dose (Figure 3). Interestingly, this effect shows a between unicellular self-defense and mammalian innate cell biphasic dose-response profile, with the colistin MIC reaching a mechanisms can provide novel strategies to take profit of our first plateau when it is supplemented with 0.3–5 µM of RNase immune response system. Undoubtedly, our own host defense 3/1 (∼1.5 × decrease of the MIC value) and a second plateau at system constitutes one of the most attractive sources of novel concentrations of RNase 3/1 higher than 10 µM (∼5 × decrease antimicrobial agents. of the MIC). We can hypothesize that this two-step reduction In this work, we have explored the potentiality of an of the MIC for colistin is due to a dual mechanism of action of engineered RNase construct derived from a human antimicrobial our construct, where a first event is dependent on the protein member of the vertebrate-specific RNase A superfamily. Based on enzymatic activity and a second process is triggered by a direct our previous structural-functional studies we have engineered a action against the bacterial cell wall. hybrid RNase that combines a high catalytic activity together with Encouraged by the results of the synergy assay we decided specific antimicrobial properties. The RNase 3/1 form conserves to analyze whether the RNase addition could target the the RNase 1 scaffold that provides the essential requirements emergence of bacterial resistance. Toward this end, we set up for an elevated catalytic activity (Nogués et al., 1998; Raines, an in vitro evolution assay where resistance against colistin 1998; Doucet et al., 2009; Gagné et al., 2015) and encompasses was generated following the culture exposure to increasing the key features of RNase 3 antimicrobial activity (Figure 1) concentrations of the antimicrobial peptide. Forty parallel (Carreras et al., 2003; Boix et al., 2008, 2012; Torrent et al., lineages of A. baumannii cultures were exposed to increasing 2009a, 2013; Pulido et al., 2013). Experimental results confirmed concentrations of colistin from 0.1 to 20 µM. After 330 that our new construct successfully combines a high catalytic generations of exposure, we observed a 1.8-fold reduction of activity (Figure 2) together with a high bactericidal activity the calculated MIC value for colistin by addition of RNase for all tested Gram-negative species (Table 1). In addition, the 3/1. Moreover, when the experimental evolution assay was RNase 3/1 variant retained the characteristic high affinity for LPS started using a colistin-resistant strain, the acquisition of Frontiers in Microbiology | www.frontiersin.org 10 June 2019 | Volume 10 | Article 1357 Prats-Ejarque et al. An Antimicrobial RNase Targeting Bacterial Resistance FIGURE 8 | Evolution of the mean MIC value for colistin of each assayed condition in Assay 2. Overall reduction of calculated MIC for colistin is observed for samples supplemented with RNase 3/1. Red corresponds to the negative control, blue to the colistin alone condition and green to the colistin + RNase 3/1 condition. Data from selected days is shown. The raw data used to produce the figure is shown in Supplementary Figure S6. Error bars show the standard error of the mean. further resistance to colistin was also significantly delayed by However, further testing with other RNase constructs are supplementation of the RNase 3/1 protein. mandatory to understand the whole process. Likewise, we Results highlight that the RNase 3/1 chimera can significantly are planning to analyze the potential combinatorial effect reduce the emergence of bacterial resistance. The construct’s of our RNase construct in the presence of other families of efficacy to inhibit the emergence of A. baumannii resistance conventional antibiotics, to discard any specific synergistic to colistin at a concentration below its effective bactericidal effect unique to the antimicrobial mechanism of colistin. activity indicates that the mechanism of action of RNase 3/1 In addition, to fully interpret our results, it is important to is probably due to a combination of the protein specific analyze whether the antibiotic resistome profile differs between properties and not to a direct bactericidal action. The the control exposed to colistin and the RNase 3/1 treated present experimental results suggest that the RNase may bacterial cultures. act as a double-edged sword, with both enzymatic activity Our results emphasize once more the applicability of and membrane disruption/LPS binding being responsible host defense proteins to design novel anti-infective therapies. for the observed phenomenon. Further work is currently One of the main sources of effective adjuvants is the ongoing to determine the specific mechanism underlying host defense arsenal of proteins and peptides. We find RNase 3/1 reduction of the emergence of bacterial resistance several examples of host defense peptides that are not and ideate additional strategies to enhance its potential antimicrobial by themselves but can boost the host’s innate adjuvant properties. immunity. Some of them are currently on clinical trials Although further studies will be necessary to confirm for the development of novel adjuvants (Hancock et al., this hypothesis, we speculate that the RNase activity might 2012). Bearing in mind that antibiotic resistance mechanisms interfere with the bacterial community quorum sensing. Since might be palliated but are probably impossible to fully bacterial resistance to cell-wall targeting antibiotics is often eradicate (Wright, 2016), alternative strategies to conventional associated to alterations of the outer membrane composition therapies are urgently needed. Drug combination is nowadays (Moffatt et al., 2010; Lázár et al., 2018), an antimicrobial one of the best choices to combat multidrug resistance agent able to block bacterial intercellular communication (Brochado et al., 2018). Antibiotic adjuvants can explore should reduce the cell survival adaptation stratagems. In synergy events and significantly reduce the effective antibiotic particular, our RNase 3/1 chimera, with a high binding affinity dose (Wright, 2016). Although it would be necessary to to LPS, a positive liposome leakage and high enzymatic fully characterize the mechanism through which RNase 3/1 activity, might block the inter-bacterial communication. hinders the bacterial resistance acquisition, RNases and their Frontiers in Microbiology | www.frontiersin.org 11 June 2019 | Volume 10 | Article 1357 Prats-Ejarque et al. An Antimicrobial RNase Targeting Bacterial Resistance derivatives appear as new lead compounds in the fight against manuscript. EB, FA-I, GP-E, and JL revised the final manuscript. antimicrobial resistance. All authors approved the final version of the manuscript. CONCLUSION FUNDING Targeting RNA to fight bacterial resistance is a promising This work was supported by Fundació La Marató de TV3 approach toward the development of alternative antibiotic and/or (Ref. 20180310), the Ministerio de Economía y Competitividad adjuvant drugs. Our results highlight the ability of an RNase (SAF2015-66007P) and by the AGAUR, Generalitat de Catalunya hybrid protein (RNase 3/1) that combines a high catalytic (2016PROD00060 and 2017SGR1010), co-financed by the activity with specific antimicrobial properties, to display synergic FEDER funds. GP-E was a recipient of a PIF (UAB) predoctoral activity when combined with colistin. Colistin is an antimicrobial fellowship. JL was a recipient of a CSC predoctoral fellowship. cyclic peptide that is frequently used as a last resort antibiotic to treat multidrug resistant bacteria in severe infections but has some toxic side effects. The addition of our RNase lead compound can significantly reduce the effective antimicrobial ACKNOWLEDGMENTS dose of colistin. Moreover, we have demonstrated that the RNase We thank Núria Crua, Gabriel Serra, Sandra Villegas, and Marc 3/1 construct can efficiently target the bacterial antimicrobial Torrent for providing the necessary infrastructure to perform the resistance evolved upon colistin exposure. After 330 generations assays, Lu Lu and Helena Carbó for laboratory technical support, of exposure to colistin, we observed about a twofold reduction Alejandro Ramos for statistical suggestions and Clara Villalba for of the calculated MIC value when supplementing the culture thoroughly checking the manuscript. Spectrofluorescence assays with the RNase 3/1 construct. The present data anticipate were performed at the Laboratori d’Anàlisi i Fotodocumentació, the potential development of RNase-based lead molecules as Universitat Autònoma de Barcelona. antibiotic adjuvant candidates. AUTHOR CONTRIBUTIONS SUPPLEMENTARY MATERIAL EB and GP-E conceived and designed the experiments. FA-I, The Supplementary Material for this article can be found GP-E, HL, and JL performed the experimental work. EB, FA-I, online at: https://www.frontiersin.org/articles/10.3389/fmicb. GP-E, and JL analyzed the data. EB and GP-E drafted the 2019.01357/full#supplementary-material REFERENCES protein antimicrobial activity. Biol. Chem. 393, 801–815. doi: 10.1515/hsz-2012- 0160 Arranz-Trullén, J., Lu, L., Pulido, D., Bhakta, S., and Boix, E. (2017). Boix, E., Torrent, M., Sánchez, D., and Nogués, M. V. (2008). The antipathogen Host antimicrobial peptides: the promise of new treatment strategies activities of eosinophil cationic protein. Curr. Pharm. Biotechnol. 9, 141–152. against tuberculosis. Front. Immunol. 8:1499. doi: 10.3389/fimmu.2017. doi: 10.2174/138920108784567353 01499 Brandenburg, L. O., Merres, J., Albrecht, L. J., Varoga, D., and Pufe, T. Bahar, A. A., and Ren, D. (2013). 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The conducted in the absence of any commercial or financial relationships that could generation of antimicrobial peptide activity: a trade-off between charge and be construed as a potential conflict of interest. aggregation? Angew. Chem. Int. Ed. Engl. 50, 10686–10689. doi: 10.1002/anie. 201103589 Copyright © 2019 Prats-Ejarque, Li, Ait-Ichou, Lorente and Boix. This is an open- Torrent, M., Cuyás, E., Carreras, E., Navarro, S., López, O., De La Maza, A., access article distributed under the terms of the Creative Commons Attribution et al. (2007). Topography studies on the membrane interaction mechanism of License (CC BY). The use, distribution or reproduction in other forums is permitted, the eosinophil cationic protein. Biochemistry 46, 720–733. doi: 10.1021/bi061 provided the original author(s) and the copyright owner(s) are credited and that the 190e original publication in this journal is cited, in accordance with accepted academic Torrent, M., de la Torre, B. G., Nogués, V. M., Andreu, D., and Boix, E. (2009a). practice. No use, distribution or reproduction is permitted which does not comply Bactericidal and membrane disruption activities of the eosinophil cationic with these terms. Frontiers in Microbiology | www.frontiersin.org 14 June 2019 | Volume 10 | Article 1357