pharmaceutics Article In Vivo Bactericidal Efficacy of GWH1 Antimicrobial Peptide Displayed on Protein Nanoparticles, a Potential Alternative to Antibiotics Jose V. Carratalá 1,2,3 , Eric Brouillette 4,5 , Naroa Serna 1,2,3 , Alejandro Sánchez-Chardi 6,7 , Julieta M. Sánchez 1,2,† , Antonio Villaverde 1,2,3 , Anna Arís 8 , Elena Garcia-Fruitós 8 , Neus Ferrer-Miralles 1,2,3, * and François Malouin 4,5, * 1 Institute for Biotechnology and Biomedicine, Autonomous University of Barcelona, Bellaterra, 08193 Barcelona, Spain; JoseVicente.Carratala@uab.cat (J.V.C.); naroa.serna@uab.cat (N.S.); jsanchezqa@gmail.com (J.M.S.); antonio.villaverde@uab.cat (A.V.) 2 Department of Genetics and Microbiology, Autonomous University of Barcelona, Bellaterra, 08193 Barcelona, Spain 3 Bioengineering, Biomaterials and Nanomedicine Networking Biomedical Research Centre (CIBER-BBN), C/Monforte de Lemos 3-5, 28029 Madrid, Spain 4 Centre d’Étude et de Valorisation de la Diversité Microbienne (CEVDM), Département de Biologie, Université de Sherbrooke, 2500 Boul. Université, Sherbrooke, QC J1K 2R1, Canada; eric.brouillette@usherbrooke.ca 5 Mastitis Network and Regroupement de Recherche Pour un Lait de Qualité Optimale (Op+Lait), Université de Montréal, 2900 Edouard Montpetit Blvd, Montréal, QC H3T 1J4, Canada 6 Microscopy Service, Autonomous University of Barcelona, Bellaterra, 08193 Barcelona, Spain; Alejandro.Sanchez.Chardi@uab.cat 7 Departament of Evolutionary Biology, Ecology and Environmental Sciences, University of Barcelona, Avda Diagonal 643, 08028 Barcelona, Spain 8 Department of Ruminant Production, Institute of Agriculture and Agrifood Research and Technology (IRTA), Caldes de Montbui, 08140 Barcelona, Spain; anna.aris@irta.cat (A.A.); elena.garcia@irta.cat (E.G.-F.) * Correspondence: neus.ferrer@uab.cat (N.F.-M.); francois.malouin@usherbrooke.ca (F.M.) † Permanent address: Faculty of Exact, Physical and Natural Sciences, ICTA and Chemistry Department, CONICET Institute of Biological and Technological Research (IIByT), National University of Cordoba, Velez Sarsfield Avenue 1611, X 5016GCA Córdoba, Argentina.  Received: 25 November 2020; Accepted: 14 December 2020; Published: 17 December 2020  Abstract: Oligomerization of antimicrobial peptides into nanosized supramolecular complexes produced in biological systems (inclusion bodies and self-assembling nanoparticles) seems an appealing alternative to conventional antibiotics. In this work, the antimicrobial peptide, GWH1, was N-terminally fused to two different scaffold proteins, namely, GFP and IFN-γ for its bacterial production in the form of such recombinant protein complexes. Protein self-assembling as regular soluble protein nanoparticles was achieved in the case of GWH1-GFP, while oligomerization into bacterial inclusion bodies was reached in both constructions. Among all these types of therapeutic proteins, protein nanoparticles of GWH1-GFP showed the highest bactericidal effect in an in vitro assay against Escherichia coli, whereas non-oligomerized GWH1-GFP and GWH1-IFN-γ only displayed a moderate bactericidal activity. These results indicate that the biological activity of GWH1 is specifically enhanced in the form of regular multi-display configurations. Those in vitro observations were fully validated against a bacterial infection using a mouse mastitis model, in which the GWH1-GFP soluble nanoparticles were able to effectively reduce bacterial loads. Keywords: mouse mastitis model; antimicrobial peptide; protein nanoparticle; inclusion body; recombinant protein; Escherichia coli; Staphylococcus aureus; therapeutic protein Pharmaceutics 2020, 12, 1217; doi:10.3390/pharmaceutics12121217 www.mdpi.com/journal/pharmaceutics Pharmaceutics 2020, 12, 1217 2 of 16 1. Introduction Novel antimicrobial and engineering approaches are urgently needed to cope with antibiotic resistance. Antimicrobial peptides (AMPs), a class of naturally occurring small (generally less than 50 amino acids) and positively charged peptides [1], have attracted attention in clinical research because of their broad-spectrum activity against a diverse group of microorganisms, including antibiotic-resistant pathogens [2]. On the other hand, cytokines are a group of small immunomodulatory proteins that play a central role in host defense by orchestrating the antimicrobial functions and conferring greater protection against different infectious agents [3]. One of the most studied cytokines is interferon-gamma (IFN-γ), which has proven to be a potent immunoprophylactic agent [4,5]. However, under physiological conditions, AMPs are subjected to proteolytic degradation and peptide inactivation by nonspecific interactions with anionic substances, which result in the low bioavailability and poor in vivo stability of these small molecules [6]. Furthermore, most cytokines, including IFN-γ, have very short half-lives, so their immunological function is limited [3]. Therefore, the accumulation of these proteins in naturally occurring bacterial inclusion bodies (IBs) and soluble self-assembling protein-only nanoparticles (PNPs) seems an appealing alternative to overcome these limitations. In 2017, Serna et al. described for the first time the use of PNPs as antibacterial agents [7]. These soluble PNPs were obtained following a modular protein design based on the fusion of a cationic peptide to a C-terminal his-tagged scaffold protein [8]. This modular configuration was presented as a transversal platform which has been replicated with several scaffold proteins [9,10]. The cationic α-helical GWH1 antimicrobial peptide [11], once fused to the amino terminus of green fluorescent protein (GFP), promoted the oligomerization in PNPs of around 50 nm size which showed a wide bactericidal effect against different pathogenic bacteria in cell cultures [7]. Due to the functional and structural versatility of this system, we wondered whether the fusion design between GWH1 and IFN-γ could also lead to the formation of these highly stable nanosized oligomers (PNPs). Besides, bacterial IBs, once considered as waste by-products derived from recombinant protein production, now provide a useful source of ready-to-use active protein. Inside these structures, therapeutic proteins are stored in native and native-like conformations and are released under physiological conditions [12–14]. The benefits of this system lie in the protective effect against degradation and the sustained release of the protein, which in both cases can significantly increase their half-lives. The aim of the present study is to characterize and to evaluate, in an in vivo mastitis mice model, the direct and non-direct antibacterial effects of two different protein designs (GWH1-GFP and GWH1-IFN-γ) assembled in two protein formats, namely, PNPs and IBs. Furthermore, we wanted to test the synergy of both activities, immunomodulation and bactericidal effect, in a single polypeptide with the potential to form PNPs. The results obtained here could throw some light on the development of new protein-based mastitis therapies but with transversal applicability in any clinical problem needing unconventional antimicrobial therapies. 2. Materials and Methods 2.1. Protein Design and Production All DNA sequences (GWH1-GFP, GWH1-IFN-γ, IFN-γ and GFP) were codon optimized (Escherichia coli), synthetized and cloned in pET22b plasmids by GeneArt (Waltham, MA, USA). IFN-γ from mouse origin was selected in sequence designs due to the species-specific nature of this protein. E. coli BL21 (DE3) (Novagen, Madison, WI, USA) was chosen as expression host. Protein production was performed in 2 L volume flasks containing 500 mL of LB medium with 100 µg/mL ampicillin (plasmid resistance). Cells were incubated at 37 ◦ C until the culture reached an optical density of around 0.5, at this point 0.1 mmol/L of isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to each culture and expression temperature was set at 20 ◦ C for 5 h. For IBs, production times were established for 3 h at 37 ◦ C. Cells were harvested by centrifugation (20 min, 5000 rpm) and stored at −80 ◦ C. Pharmaceutics 2020, 12, x 3 of 16 Pharmaceutics 2020, 12, 1217 3 of 16 2.2. Protein Purification ForProtein 2.2. soluble protein, purification procedures were performed as described before [9], with some Purification modifications. The cell pellet was resuspended in wash buffer (Tris 20 mmol/L, pH 8.0, NaCl 500 For soluble protein, purification procedures were performed as described before [9], with some mmol/L, imidazole 10 mmol/L) with DNase (1 mg/mL) (Thermo-Scientific, Waltham, MA, USA) and modifications. The cell pellet was resuspended in wash buffer (Tris 20 mmol/L, pH 8.0, NaCl 500 mmol/L, ethylenediamine tetra-acetic acid-free protease-inhibitor (complete EDTA-Free, F. Hoffmann-La imidazole 10 mmol/L) with DNase (1 mg/mL) (Thermo-Scientific, Waltham, MA, USA) and Roche AG, Basel, Switzerland). ethylenediamine French tetra-acetic acid-free press was selected protease-inhibitor as disruption (complete EDTA-Free, F.method for cell Hoffmann-La lysis (3 Roche rounds at 1200 psi). The protein amount was quantified by Bradford, aliquoted and AG, Basel, Switzerland). French press was selected as disruption method for cell lysis (3 rounds stored at −80 °C for further experiments. at 1200 psi). The proteinA amount schematic wasrepresentation of the different quantified by Bradford, aliquotedtypes of bioactive and stored at −80 ◦ C materials for obtained furtherafter protein purification experiments. is outlined inofScheme A schematic representation 1. Purified the different types of recombinant protein bioactive materials from the obtained soluble aftercell fraction protein by affinity purification chromatography is outlined in Scheme generated 1. Purifiedtwo types of protein recombinant samples protein from depending the soluble cell fraction by affinity on macromolecular chromatography organization generated (unassembled two types ofproteins and assembled protein (PNPs)). samples depending on Purified proteins macromolecular organization (unassembled and assembled proteins (PNPs)). were stored in sodium bicarbonate buffer containing 166 mmol/L NaHCO3 and 333 mmol/L NaCl. Purified proteins were storedIBs Bacterial in were sodium bicarbonate purified buffer containing and quantified 166 mmol/L as described NaHCO earlier andadditional [15].3An 333 mmol/Lstep NaCl. BacterialPress of French IBs were purified and quantified as described earlier [15]. An additional step of French Press disruption disruption (3 rounds at 1200 psi) was included at the beginning of the process. Sterile conditions were (3 rounds at 1200 psi) was included at the beginning of the process. Sterile conditions were maintained maintained throughout the process. Pelleted IBs were resuspended in sodium bicarbonate buffer throughout the process. Pelleted IBs were resuspended in sodium bicarbonate buffer prior to its use. priorPurification to its use.productivity Purification productivity of analyzed of analyzed recombinant proteinsrecombinant proteins as well as protein as iswell solubility as protein detailed in solubility is detailed Supplementary in Supplementary Table S1. Table S1. E. coli cell culture Cell lysis Soluble Insoluble GWH1-GFP Purification IBs cell cell GWH1-IFN-γ fraction fraction IFN-γ GFP FPLC purification Solubilization Unassembled Assembled 0.2 % N-lauroylsarcosine Disassembled Protein protein protein UPs PNPs GWH1-IFN-γ GWH1-GFP GWH1-GFP IFN-γ GFP Scheme Scheme 1. Schematic 1. Schematic representationofofthe representation the protein protein formats formatsusedusedthrough through thethe study. During study. protein During protein production, recombinant protein was accumulated inside E. coli cells as insoluble production, recombinant protein was accumulated inside E. coli cells as insoluble aggregates aggregates (inclusion bodies—IBs) or in a soluble state. After cell lysis and centrifugation, IBs were recovered from the cell (inclusion bodies—IBs) or in a soluble state. After cell lysis and centrifugation, IBs were recovered debris. Supernatant was used for soluble protein purification purposes. Positive protein peaks were from the cell debris. Supernatant was used for soluble protein purification purposes. Positive protein analyzed by dynamic light scattering (DLS) and protein samples were categorized as unassembled peaks were analyzed by dynamic light scattering (DLS) and protein samples were categorized as protein versions or assembled protein versions. Assembled protein GWH1-GFP was transformed into unassembled protein disassembled versions protein or assembled by incubation with mild protein versions. detergent. The names Assembled protein of the protein GWH1-GFP samples used in the was transformed mastitis mice model are labeled beside or below the protein versions enclosed in a light blueofbox. into disassembled protein by incubation with mild detergent. The names the protein samples used in the mastitis mice model are labeled beside or below the protein versions enclosed in 2.3. Nanoparticle Size Characterization a light blue box. Size distribution of protein samples was determined by dynamic light scattering (DLS). Average 2.3. Nanoparticle values Size Characterization were obtained after the independent measurement of protein samples in triplicates at 633 nm in a Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, UK). Nanoscale morphological characterization Size distribution of protein samples was determined by dynamic light scattering (DLS). Average of isolated IBs was performed with two high resolution electron microscopy techniques adapted values weretype to this obtained after the[16–23]. of biomaterial independent Sample measurement preparation for of protein the fieldsamples emissioninscanning triplicates at 633 nm electron in amicroscope Zetasizer (FESEM) Nano ZS (Malvern Instruments Ltd., Malvern, UK). Nanoscale observation was described previously [12]. Pellets of the same four isolated morphological characterization of isolated IBs was performed with two high resolution electron microscopy techniques adapted to this type of biomaterial [16–23]. Sample preparation for the field emission scanning electron microscope (FESEM) observation was described previously [12]. Pellets of the same four isolated IBs were processed following conventional transmission electron microscopy (TEM) procedures as previously described [16,17]. Pharmaceutics 2020, 12, 1217 4 of 16 IBs were processed following conventional transmission electron microscopy (TEM) procedures as previously described [16,17]. 2.4. Bactericidal Activity Assay The effect of the different antimicrobial candidates was evaluated against E. coli ATCC 25922 and Staphylococcus aureus ATCC 29737. The assay was performed using a broth micro-dilution method. In 96-well plates, after a two-fold dilution process in 50 µL, each well contained a specific amount of the corresponding protein, ranging from 6 to 0.4 µmol/L. After protein distribution, 50 µL of Mueller Hinton Broth Cation-adjusted medium (MHB-II, Sigma-Aldrich, Saint Louis, MO, USA) containing 106 CFU/mL (colony forming units per mL) of E. coli or S. aureus was inoculated in each well. Maximal growth was achieved in control wells with no protein. Each concentration was evaluated in duplicates. After microorganism inoculation, the 96-well plates were gently agitated and then incubated without agitation at 37 ◦ C for 18 h. Bacterial viability was evaluated using the commercially available BacTiter-Glo™ Microbial Cell Viability Assay (Promega, Madison, WI, USA) following the manufacturer’s instructions. Luminescence was measured using the Multilabel Plater Reader VICTOR3 (PerkinElmer, Waltham, MA, USA). 2.5. Disassembling of Soluble Nanoparticles GWH1-GFP PNPs were disassembled after the addition of the mild-solubilization agent N-lauroylsarcosine 0.2% (Sigma-Aldrich, Saint Louis, MO, USA) (see Scheme 1). The disassembling process was evaluated by DLS at 633 nm in a Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, UK). Bactericidal activity of assembled and disassembled GWH1-GFP proteins was evaluated as described in Section 2.4, apart from the protein concentration range, that in this case ranged from 0.5 to 8 µmol/L. 2.6. Protein Solubilization from IBs The different protein IBs (GWH1-GFP, GWH1-IFN-γ, IFN-γ and GFP) were resuspended in 250 µL of sodium bicarbonate buffer. The conditions for solubilization were established at 37 ◦ C for 18 h with agitation. After the incubation, the solubilized protein in the supernatant was recovered by centrifugation (15 min, 15,000× g). Size distribution of the solubilized protein was evaluated by DLS and the amount of protein released from the IBs was determined by Western blot, as detailed [12]. 2.7. Determination of Murine IFN-γ Biological Activity The procedure for the activity evaluation of IFN-γ was adapted from a nitric oxide (NO)-based bioassay [24]. Detailed modifications are described as follows. Serial dilutions of the different IFN-γ constructs (commercial E. coli-derived murine IFN-γ (R&D systems, Minneapolis, MN, USA), recombinant in-house murine IFN-γ and GWH1-IFN-γ) at quantities ranging from 6 to 120 nmol/L were incubated with murine microglial cells (BV-2) for 48 h at 37 ◦ C. No protein was added in control wells. To study the specificity of the NO response, a specific concentration (120 nmol/L) of a commercial recombinant mouse IFN-γ was evaluated in the absence and presence of 500 µmol/L L-NMA (Sigma-Aldrich, Sant Louis, MO, USA), an inhibitor of nitric oxide synthase (i-NOS). 2.8. Mouse Model of Infectious Mastitis The mouse mastitis model was previously described [25–27]. CD-1 lactating micewere used. Experiment specifications are briefly reported here. A total of 100 µL of bacteria (100 CFUs of either E. coli ATCC 25922 or S. aureus ATCC 29737) and 100 µL of the corresponding protein compound (assembled PNPs, unassembled soluble protein or IBs) were administered in each mammary gland pair. A minimum of three mice (6 mammary glands) were employed to test each protein compound. Protein concentrations in all cases were adjusted to 60 µmol/L after dilution (unassembled versions Pharmaceutics 2020, 12, 1217 5 of 16 and PNPs) or resuspension (IBs) with sodium bicarbonate buffer with salt. In experimental controls, 100 µL of sodium bicarbonate buffer with salt was administered instead of the protein-containing solutions. At relevant time points post-inoculation (6, 12 and 24 h for kinetics growth studies and 8 h for compound efficacy studies), mice were sedated and euthanized. Bacterial CFU contained in samples were evaluated by plating serial dilutions of gland homogenates on tryptic soy agar (TSA) plates and raw CFU counts converted into base 10 logarithm values. The animal experiments were performed in accordance with the guidelines of the Canadian Council on Animal Care and approved by the ethics committee on animal experimentation of the Faculté des Sciences of Université de Sherbrooke (protocol 2017–1966 FM2017-01B). 2.9. Statistical Analysis All statistical analyses were conducted in GraphPad Prism version 8.0.0 for Windows (GraphPad Software, San Diego, CA, USA) with at least three independent replicates unless otherwise indicated. Bars in figures are expressed as mean ± standard deviation. Quantitative data were tested for normality and equality of variances with Kolmogorov–Smirnov and Levene tests, respectively, prior to analyses. Depending on the experiment, statistical divergences were evaluated using one-way ANOVA with Tukey’s multiple comparisons test or two-tailed unpaired Student’s t test. Statistical significance in relevant results is indicated in figures as * p < 0.05, ** p < 0.01, and *** p < 0.001. 3. Results 3.1. Characterization of Self-Assembling Nanoparticles In the modular protein designs, GWH1 was N-terminally fused to two different his-tagged protein scaffolds (Figure 1A), namely, the previously reported GWH1-GFP [7] and the new design, GWH1-IFN-γ. After purification of recombinant proteins accumulated in the soluble cell fraction, high purity was reached with all protein designs (Figure 1(B1)). Self-assembling of monomers into soluble protein nanoparticles (PNPs) is driven by the fusion of a cationic peptide (GWH1) with His6-tagged scaffolds [10]. However, under the tested experimental conditions, protein oligomerization in soluble PNPs was only achieved with GWH1-GFP (Figure 1D) as previously described [7]. In DLS analysis, the peak size for this oligomer was around 35 nm. The high polydispersity showed by the sample could indicate the presence of a diverse mixture of PNPs with different oligomeric states (Figure 1D). Modular protein GWH1-IFN-γ showed an average size of around 10 nm (Figure 1D). When compared to IFN-γ, only a slight difference in size was observed, suggesting that protein oligomerization was not achieved with this fusion protein design. As expected, GFP and IFN-γ did not form PNPs (Figure 1D). 3.2. In Vitro Evaluation of Antibacterial Activity The antimicrobial potential of soluble GWH1-GFP (PNPs) and soluble GWH1-IFN-γ (unassembled protein (UP) forms) was evaluated in an in vitro assay against two of the most common pathogens involved in mastitis, S. aureus and E. coli. GFP and IFN-γ were used as controls for their antimicrobial peptide carrier counterparts. GWH1-GFP showed a dose-dependent antibacterial activity against E. coli reducing cell viability by up to 90% (Figure 2A). On the other hand, GWH1-IFN-γ UPs did not follow the same pattern, only showing antibacterial activity at the highest concentration, reducing bacterial viability by 40%. As expected, GFP and IFN-γ had no significant effect on cell viability (Figure 2A). On S. aureus, a dose-dependent pattern was observed again with GWH1-GFP PNPs (Figure 2B). However, its effect on cell viability was not as pronounced as that observed with E. coli. GWH1-IFN-γ UPs had a similar effect, reducing cell viability by 40% at the highest concentration. Control proteins, GFP and IFN-γ, showed an unspecific effect on cell viability, reducing bacterial loads to the same extent as that observed for the GWH1 carrier versions (Figure 2B). sample could indicate the presence of a diverse mixture of PNPs with different oligomeric states (Figure 1D). Modular protein GWH1-IFN-γ showed an average size of around 10 nm (Figure 1D). When compared to IFN-γ, only a slight difference in size was observed, suggesting that protein oligomerization was not achieved with this fusion protein design. As expected, GFP and IFN-γ did Pharmaceutics 2020, 12, 1217 6 of 16 not form PNPs (Figure 1D). Pharmaceutics 2020, 12, x 6 of 16 and facilitate self-assembling process. (B) TGX Stain-freeTM analysis showing the purity of the different protein designs in both, soluble proteins (B1) and IBs (B2). (C) Western blot showing the qualitative amount of protein released from the different IBs. (D) Evaluation of self-assembling capabilities of purified soluble proteins by size determination using DLS. PNPs: protein nanoparticles; UPs: unassembled proteins. (E) Size distribution of the protein samples obtained after solubilization from different IBs analyzed by DLS. 3.2. In Vitro Evaluation of Antibacterial Activity The antimicrobial potential of soluble GWH1-GFP (PNPs) and soluble GWH1-IFN-γ (unassembled protein (UP) forms) was evaluated in an in vitro assay against two of the most common pathogens involved in mastitis, Characterization theS.different of the aureus and different E. coli.formats protein GFP and IFN-γ were used study. as controls for their Figure Figure 1.1. Characterization of protein formats employed employed in in this this study. (A) (A)Schematic Schematic antimicrobial peptide representationofofmultimeric carrier multimericfusion counterparts. fusionproteins. GWH1-GFP proteins.The The showed presence a dose-dependent antibacterial representation presence ofof a cationic a cationic peptide peptide (GWH1) (GWH1) in in thethe N- activity against N-terminus, plus E. thecoli reducing His6 tag in cell viability may C-terminus, by upinduce to 90 % (Figure the 2A). On formation of the other hand, protein-only GWH1- nanoparticles terminus, plus the His6 tag in C-terminus, may induce the formation of protein-only IFN-γ UPs did not follow the same pattern, only showing antibacterial activity at the highest nanoparticles (PNPs). A linker between GWH1 and the scaffold protein was added in order to increase flexibility and (PNPs). A linker reducing concentration, between bacterial GWH1 and the scaffold viability by 40 %.protein was added As expected, GFP andin order IFN-γ tohad increase flexibility no significant facilitate self-assembling process. (B) TGX Stain-freeTM analysis showing the purity of the different effect on cell viability (Figure 2A). On S. aureus, a dose-dependent pattern was observed again with protein designsPNPs GWH1-GFP in both, soluble (Figure 2B). proteins However,(B1) and IBs its effect on(B2). (C) Western cell viability was blot not asshowing the qualitative pronounced as that amount observed with E. coli. GWH1-IFN-γ UPs had a similar effect, reducing cell viability by 40capabilities of protein released from the different IBs. (D) Evaluation of self-assembling % at the of highest purified soluble proteins concentration. Controlbyproteins, size determination GFP and IFN-γ, using DLS.an showed PNPs: protein unspecific nanoparticles; effect UPs: on cell viability, unassembled proteins. reducing bacterial (E)toSize loads thedistribution same extent of as the thatprotein observedsamples for theobtained after solubilization GWH1 carrier versions (Figurefrom 2B). IBs analyzed by DLS. different A B ** *** * *** C D ** Figure Figure 2. Antimicrobial 2. Antimicrobial activity activity of of assembled (PNPs) assembled (PNPs) and andunassembled unassembled soluble proteins soluble (UPs) in proteins vitro.in vitro. (UPs) (A) Effect on cell viability of increasing amounts of GWH1-GFP and GWH1-IFN-γ against E. coli and (A) Effect on cell viability of increasing amounts of GWH1-GFP and GWH1-IFN-γ against E. coli and (B) S. aureus (C). Dynamic light scattering (DLS) analysis showing the nanoparticle disassembling of (B) S. aureus (C). Dynamic light scattering (DLS) analysis showing the nanoparticle disassembling GWH1-GFP in presence of 0.2 % N-lauroylsarcosine. (D) Comparative effect on cell viability of of GWH1-GFP disassembled in presence (promoted of 0.2% N-lauroylsarcosine. in presence (D) Comparative of 0.2% N-lauroylsarcosine) and assembledeffect GWH1-GFPon cell viability of against disassembled E. coli in (promoted in presence vitro. IFN-γ and GFP wereof 0.2% used N-lauroylsarcosine) as controls. andare Significant results assembled shown as *GWH1-GFP p ≤ 0.05, ** p ≤ against E. coli in vitro. 0.01 and *** IFN-γ andmultiple p ≤ 0.001, GFP were t test.used as controls. All experiments wereSignificant performed inresults are shown duplicates. as * p ≤ 0.05, Bars represent the and ** p ≤ 0.01 the mean *** p ±≤standard deviation. 0.001, multiple t test. All experiments were performed in duplicates. Bars represent the the mean ± standard deviation. Pharmaceutics 2020, 12, 1217 7 of 16 Pharmaceutics 2020, 12, x 7 of 16 3.3. 3.3. Importance Importance of of Nanoparticle Nanoparticle Format Format on on Antibacterial Antibacterial Activity Activity Given Given that thatthe thenanoparticulated nanoparticulatedformatformat seems seems to properly to properly display the the display antimicrobial activity, antimicrobial the activity, PNPs formed the PNPs by GWH1-GFP formed by GWH1-GFP were wereincubated incubated with a mild with a mildsolubilization solubilizationagent agent(N-lauroylsarcosine) (N-lauroylsarcosine) in in order to disassemble the oligomeric format while preserving the native-like proteinstructure order to disassemble the oligomeric format while preserving the native-like protein structure[28]. [28]. The presence presenceofof monomers monomers of 5size of 5 nm nm size after after achieving achieving the final concentration the final concentration of 0.2 % N- of 0.2% N-lauroylsarcosine lauroylsarcosine was observed bywas DLSobserved (Figureby DLS 2C). In(Figure E. coli, 2C). the In E. coli, the version monomeric monomeric version of GWH1-GFP of GWH1-GFP lacked the lacked the dose-dependent antimicrobial behavior showed by the PNP version, dose-dependent antimicrobial behavior showed by the PNP version, only acting at the highest only acting at the highest concentration concentration by reducing by reducing cell viability cell viability by 40%, bya40low%, antimicrobial a low antimicrobial effect effect compared compared with with its its fully fully activeactive version, version, whichwhich reduced reduced viability viability by more by more than(Figure than 90% 90 % (Figure 2D). 2D). 3.4. Bioactivity 3.4. Bioactivity Evaluation Evaluation of of IFN-γ-Based IFN-γ-Based Proteins Proteins The measurement The measurement of of nitric nitric oxide oxide (NO) (NO) produced produced byby the the murine murine microglial microglial cell cell line line BV-2, BV-2, in in the the presence of IFN-γ, provides presence provides a simple method for detection of bioactive mouse IFN-γ. The The use use of of aa selective inhibitor selective inhibitorofof iNOS, iNOS, NG-monomethyl-L-arginine NG-monomethyl-L-arginine (L-NMA) allowsallows (L-NMA) one to evaluate one to the specificity evaluate the of the response. specificity of the An inhibition response. in the NO in An inhibition production by BV-2 cells the NO production was observed by BV-2 cells was when L-NMA observed whenwasL- addedwas NMA in the presence added in theof presence commercial mouse IFN-γmouse of commercial (Figure 3A). (Figure IFN-γ The activity 3A). of Thetheactivity two mouse IFN-γ of the two designsIFN-γ mouse (IFN-γdesigns and GWH1-IFN-γ (IFN-γ and UPs) was compared GWH1-IFN-γ UPs) to a standard was compared calibrated mousecalibrated to a standard IFN-γ. BV-2 cells mouse produced IFN-γ. nitrite BV-2 cellsinproduced a dose-dependent nitrite inmanner in response tomanner a dose-dependent IFN-γ at inprotein responseconcentrations to IFN-γ at between protein 6 and 120 nmol/L. concentrations The biological between 6 and 120activity nmol/L.ofThe the biological standard mouse activityIFN-γ of thewas significantly standard mousehigher IFN-γ than was that of the recombinant significantly higher thanIFN-γ that ofproduced in this study. the recombinant IFN-γSurprisingly, produced in thisGWH1-IFN-γ UPs did not study. Surprisingly, show GWH1- any response IFN-γ UPs didinnot thatshow range of response any concentrations, in thatwhile rangerecombinant in-house of concentrations, INF-γ while was effective recombinant from in-house 12 nmol/L INF-γ was (Figure effective3B). from 12 nmol/L (Figure 3B). A B *** *** *** *** ** *** *** *** ** *** * ** Figure Figure 3.3. Nitric Nitricoxide oxide (NO)-based (NO)-based bioassay bioassay for mouse for mouse IFN-γIFN-γactivityactivity evaluation evaluation in the in the murine murine microglial microglial cell (A) cell line BV-2. lineInhibitory BV-2. (A) Inhibitory effect of effect of L-NMA L-NMA (NG-monomethyl-L-arginine) (NG-monomethyl-L-arginine) on nitric on nitric oxide oxide production by BV-2 in by production presence BV-2 inofpresence a commercial recombinant of a commercial mouse IFN-γ recombinant mouse(120 nmol/L). IFN-γ Significant (120 nmol/L). results Significant are shown results as *** pas≤*** are shown 0.001, unpaired p ≤ 0.001, Student’s unpaired t test.t test. Student’s (B) Nitric oxide (B) Nitric production oxide production(measured (measured by by its absorbance at 490 nm) for increasing amounts of the different IFN-γ-based constructs its absorbance at 490 nm) for increasing amounts of the different IFN-γ-based constructs using, as using, as standard, a commercial standard, recombinant a commercial mouse IFN-γ. recombinant mouse Significant results areresults IFN-γ. Significant shown * p ≤ 0.05, asshown are p ≤p 0.05, as * ** ≤ 0.01**and p≤ *** p and 0.01 ≤ 0.001, ≤ 0.001,t test. *** pmultiple All experiments multiple were performed t test. All experiments wereinperformed triplicates and data are represented in triplicates and data are as mean ± standard represented deviation. as mean ± standard deviation. 3.5. Growth Kinetics of E. coli in the Mastitis Mice Model 3.5. Growth Kinetics of E. coli in the Mastitis Mice Model The intramammary infection (IMI) was monitored in a time course experiment (Figure 4). The intramammary infection (IMI) was monitored in a time course experiment (Figure 4). The The medians of bacterial loads were 1.2 × 105 CFU, 3.8 × 107 CFU and 7.8 × 109 CFU per g of gland after medians of bacterial loads were 1.2 × 105 CFU, 3.8 × 107 CFU and 7.8 × 109 CFU per g of gland after 6, 6, 12 and 24 h, respectively. Results demonstrated a steady growth and since higher bacterial loads 12 and 24 h, respectively. Results demonstrated a steady growth and since higher bacterial loads affected the general health of the animals, an infection time of 8 h was selected as the experimental condition for therapeutic efficacy studies. Pharmaceutics 2020, 12, 1217 8 of 16 affected the general health of the animals, an infection time of 8 h was selected as the experimental condition for therapeutic Pharmaceutics 2020, 12, x efficacy studies. 8 of 16 CFU/g of gland (log 10) Figure 4. Growth Figure 4. Growth of E. coli kineticskinetics of in E. the colimouse in the mammary gland at mouse mammary 6, 12atand gland 24 h 6, 12 after and 24 the administration h after the administration of 100 CFU through the teat channel. The bacterial loads for each time of 100 CFU through the teat channel. The bacterial loads for each time point were evaluated in 8 point were evaluated in 8 individual mammary glands (4 mice).Bars represent the mean ± standard deviation. individual mammary glands (4 mice).Bars represent the mean ± standard deviation. 3.6. Characterization 3.6. Characterization of IBsof IBs Recombinant protein production in bacteria induces the formation of discrete protein clusters Recombinant protein production in bacteria induces the formation of discrete protein clusters usually located at the cellular poles. These supramolecular complexes, known as IBs, contain usuallyfunctional located atrecombinant the cellularprotein. poles. Released These supramolecular recombinant proteinscomplexes, known as tend to co-purify IBs,other with contain functional cellular recombinant proteinsprotein. using IBsReleased purificationrecombinant methods. In anyproteins case, intend this to co-purify study, with other a representative cellular amount of theproteins produced using IBs recombinant purification protein methods. Inwas anyobserved case, ininthis all cases study, (Figure 1(B2)). IFN-γ IBs a representative showed amount ofthe thebest produced releasing efficiency, being the amount of protein observed qualitatively recombinant protein was observed in all cases (Figure 1(B2)). IFN-γ IBs showed the best releasingdifferent from the other candidates (Figure 1C). On the other hand, just a faint band was observed for GFP-based proteins, efficiency, being the amount of protein observed qualitatively different from the other candidates indicating a poor releasing process (Figure 1C). The size distribution of the solubilized protein was (Figurealso 1C).evaluated On the other (Figurehand, just a faint 1E). Although band soluble was observed aggregates for GFP-based were mainly released fromproteins, indicating all protein a poor designs, releasing onlyprocess GWH1-GFP (Figure included1C).a monomeric The size distribution, distributionwhich of the solubilized indicated protein the presence of two was also evaluated (Figure of populations 1E). Although different sizes.soluble aggregates characteristics The ultrastructural were mainly of released the IBs from were all protein analyzed bydesigns, electronic microscopy only GWH1-GFP included (EM) (Figure 5).distribution, a monomeric The results showed whichthe presence the indicated of round-shaped presence of IBstwowith a populations similar electrodensity in all samples but with marked differences between GWH1-based of different sizes. The ultrastructural characteristics of the IBs were analyzed by electronic microscopy IBs and the GWH1-free (IFN-γ and GFP) IBs (Figure 5). Interestingly, in E. coli, IFN-γ formed a high amount of (EM) (Figure 5). The results showed the presence of round-shaped IBs with a similar electrodensity in IBs with similar size and shape than GFP IBs. Conversely, GWH1-GFP and GWH1-IFN-γ IBs showed all samples but with a shape–size marked completely distribution differences between different from GWH1-based those observedIBs for and the GWH1-free the well-formed GFP IBs. (IFN-γ In and GFP) IBs (Figurea high addition, 5). Interestingly, in E. variability in size cancoli, IFN-γ formed be observed betweenathese highsamples, amountsuggesting of IBs withthesimilar presencesize and shape than of notGFP IBs. Conversely, completely GWH1-GFP formed IBs (Figure 5). and GWH1-IFN-γ IBs showed a shape–size distribution completely different from those observed for the well-formed GFP IBs. In addition, a high variability in size can be observed between these samples, suggesting the presence of not completely formed IBs (Figure 5). 3.7. Therapeutic Effect of PNPs and IBs in the Mouse Mastitis Model A single dose of 60 µmol/L of each different protein design, in both soluble (UPs or PNPs) and insoluble (IBs) formats, was administered just after the intramammary inoculation of E. coli. The soluble PNPs of GWH1-GFP and the soluble UP versions of IFN-γ and GWH1-IFN-γ provoked a significant decrease in bacterial loads in comparison to both the control (sodium bicarbonate buffer) or GFP treatments (Figure 6A). The presence of GFP had, however, some negative effect on E. coli growth (Figure 6A). IFN-γ and GWH1-IFN-γ UPs caused a decrease in bacterial loads by 2.8 and 2 times greater than that caused by GFP, respectively. Nevertheless, the strongest bactericidal effect was obtained by the nanoparticle-forming protein GWH1-GFP, which decreased the bacterial loads by 35 times in Figure 5. Ultrastructural characterization and analysis by electronic microscopy of purified inclusion comparisonbodies. to theRepresentative control treatment and byfields images of general 11.5and times compared nanoparticle with detail of the entire IBsGWH1-free of GFP, GWH1-GFP. On the other hand, the same protein designs produced as IBs showed a different outcome GFP, IFN-γ and GWH1-IFN-γ at two different magnifications, 11,000×/150,000× (Figure (FESEM) and6B). The GFP IBs had a similar effect on bacterial loads than that observed for GFP UPs. Surprisingly, IFN-γ IBs showed a significant effect, diminishing bacterial loads by almost 6 times when compared to GFP IBs and by 11 times compared to the control. The IB format for the GWH1 carrier proteins, GWH1-IFN-γ and GWH1-GFP, showed a significant effect over bacterial loads in control glands, causing a decrease designs, only GWH1-GFP included a monomeric distribution, which indicated the presence of two populations of different sizes. The ultrastructural characteristics of the IBs were analyzed by electronic microscopy (EM) (Figure 5). The results showed the presence of round-shaped IBs with a similar electrodensity in all samples but with marked differences between GWH1-based IBs and the GWH1-free (IFN-γ Pharmaceutics 2020, and GFP) IBs (Figure 5). Interestingly, in E. coli, IFN-γ formed a high amount 12, 1217 9 ofof 16 IBs with similar size and shape than GFP IBs. Conversely, GWH1-GFP and GWH1-IFN-γ IBs showed a shape–size distribution completely different from those observed for the well-formed GFP IBs. In of 6.4 and 5.2 times, respectively. However, for these two samples only a numerically decrease in addition, a high variability in size can be observed between these samples, suggesting the presence bacterial loads by 3.2 and 2.7 times in comparison to GFP IBs was observed, respectively. of not completely formed IBs (Figure 5). Figure Figure 5. 5. Ultrastructural Ultrastructural characterization characterization and and analysis analysis by by electronic electronic microscopy microscopy of of purified purified inclusion inclusion bodies. bodies. Representative images of Representative images ofgeneral generalfields fieldsand andnanoparticle nanoparticle detail detail of of entire entire IBsIBs of GFP, of GFP, GWH1- GWH1-GFP, GFP, IFN-γIFN-γ and GWH1-IFN-γ and GWH1-IFN-γ at twomagnifications, at two different different magnifications, 11,000×/150,000× 11,000×/150,000× (FESEM) and (FESEM) and 3000×/10,000× (TEM). Magnifications are equivalent in each micrograph to allow comparative visualization. The scale bars represent 500 nm. Figure 6. Bacterial loads (E. coli CFU/g of gland log10) measured after the administration of therapeutic and non-therapeutic protein formats into infected mammary glands. (A) Effect of unassembled protein forms (UPs) and assembled protein-only nanoparticles (PNPs) 8 h after the challenge with 100 CFU of E. coli. (B) Effect of the inclusion body (IB) format 8 h after the challenge with 100 CFU of E. coli. In all cases, 60 µM of the different protein formats were administered just after the infection with E. coli. Sodium bicarbonate buffer (166 mmol/L NaHCO3 , 333 mmol/L NaCl) and GFP were used as controls. The middle bars indicate median values for each group of glands (at least 6 glands) whereas the boxes specify quartiles Q1-Q3. Experiments were performed with at least three independent replicates. Significant results are shown as ** p ≤ 0.01 and *** p ≤ 0.001, one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test. The performance of the GWH1-GFP construct forming PNPs prompted us to expand our in vivo study in S. aureus-challenged mice. In that case, GWH1-GFP PNPs caused a significant decrease in bacterial loads, showing 3.3 times less bacteria within the glands than the control (Figure 7). Pharmaceutics 2020, 12, 1217 10 of 16 However, there was no difference when compared to the GFP UPs treatment, which also reduced S. aureus colonization. Pharmaceutics 2020, 12, x 10 of 16 * ** Figure 7.7. Antimicrobial Figure Antimicrobialeffect of of effect GFP GFPandand GWH1-GFP GWH1-GFP(PNPs) over S. (PNPs) aureus-infected over mammary S. aureus-infected glands mammary in vivo. in glands Mammary glands were vivo. Mammary harvested glands wereand processed harvested 8 h processed and after the simultaneous administration 8 h after the simultaneous of 100 CFU of S. aureus administration of 100and CFU60 of µmol/L of protein, S. aureus and 60 respectively. Sodium µmol/L of protein, bicarbonateSodium respectively. buffer (166 mmol/L bicarbonate NaHCO 3 , 333 buffer (166 mmol/L mmol/L NaCl) 3was NaHCO , 333used as control. mmol/L NaCl)The wasmiddle used asbars indicate control. Themedian middlevalues bars for each indicate group of glands (at least 6 glands) whereas the boxes specify quartiles Q1-Q3. The experiment median values for each group of glands (at least 6 glands) whereas the boxes specify quartiles Q1-Q3. was performed The experimentin triplicates. Significant was performed results areSignificant in triplicates. p ≤ 0.05, shown as *results are ** p ≤ 0.01, shown as *one-way p ≤ 0.05, analysis of ** p ≤ 0.01, variance one-way (ANOVA), analysis offollowed variance by Tukey’s multiple (ANOVA), followedcomparisons test. by Tukey’s multiple comparisons test. 4. Discussion 4. Discussion Novel therapies are urgently needed to tackle the reduced cure rates related to the development Novel therapies are urgently needed to tackle the reduced cure rates related to the development of bacterial resistance [29]. One alternative includes the use of alpha-helical AMPs [30]. However, of bacterial resistance [29]. One alternative includes the use of alpha-helical AMPs [30]. However, large-scale production of synthetic small peptides is challenging [31] and recombinant protein large-scale production of synthetic small peptides is challenging [31] and recombinant protein production in prokaryotic host is presented here as a promising alternative. Several experimental production in prokaryotic host is presented here as a promising alternative. Several experimental strategies have been explored to cope with the inherent toxicity of such peptides, when recombinantly strategies have been explored to cope with the inherent toxicity of such peptides, when produced, towards the prokaryotic host, including the fusion to partner proteins that seem to mask recombinantly produced, towards the prokaryotic host, including the fusion to partner proteins that the antimicrobial activity [32–34], as well as the accumulation in the insoluble cell fraction of AMPs seem to mask the antimicrobial activity [32–34], as well as the accumulation in the insoluble cell fused to scaffold proteins [35]. In any case, regardless of the mechanism of action of alpha-helical fraction of AMPs fused to scaffold proteins [35]. In any case, regardless of the mechanism of action AMPs against microorganisms, the recombinant production of these peptides might be achieved by the of alpha-helical AMPs against microorganisms, the recombinant production of these peptides might neutralization of their net charge by fusion to solubility-enhancing proteins [36–38] or by enhancing be achieved by the neutralization of their net charge by fusion to solubility-enhancing proteins [36– formation of IBs in producing cells by the fusion to aggregation-prone proteins [39]. 38] or by enhancing formation of IBs in producing cells by the fusion to aggregation-prone proteins In the present work, we have designed GWH1-containing recombinant proteins, with the idea [39]. of building bifunctional molecules where each individual part of the recombinant fusion design In the present work, we have designed GWH1-containing recombinant proteins, with the idea could maintain its original function, and at the same time, act as a building block for protein self- of building bifunctional molecules where each individual part of the recombinant fusion design could assembling [8,10]. In that sense, it has been demonstrated that GFP, toxins and pro-apoptotic proteins are maintain its original function, and at the same time, act as a building block for protein self-assembling able to act as scaffold partners for protein self-assembling while retaining their biological activity [9,40]. [8,10]. In that sense, it has been demonstrated that GFP, toxins and pro-apoptotic proteins are able to Protein production was affected by the expression of these AMP carrying proteins. The addition act as scaffold partners for protein self-assembling while retaining their biological activity [9,40]. of GWH1 provoked a drastic decrease in protein yields in comparison to the non-AMP-containing Protein production was affected by the expression of these AMP carrying proteins. The addition of counterparts, see Supplementary Table S1. However, proteins were produced in enough amounts GWH1 provoked a drastic decrease in protein yields in comparison to the non-AMP-containing to allow an effective purification process. Still, in the tested conditions, GWH1-IFN-γ arrangement counterparts, see Supplementary Table S1. However, proteins were produced in enough amounts to was unable to promote the formation of soluble PNPs (Figure 1D), suggesting the inability of the allow an effective purification process. Still, in the tested conditions, GWH1-IFN-γ arrangement was mouse IFN-γ to act as a scaffold domain for protein oligomerization. Mature mouse IFN-γ forms unable to promote the formation of soluble PNPs (Figure 1D), suggesting the inability of the mouse noncovalently linked homodimers of 20–25 kDa [41]. The formation of dimers in both, IFN-γ and IFN-γ to act as a scaffold domain for protein oligomerization. Mature mouse IFN-γ forms GWH1-IFN-γ could explain the bigger sizes observed by DLS and the bigger size of GWH1-IFN-γ noncovalently linked homodimers of 20–25 kDa [41]. The formation of dimers in both, IFN-γ and over IFN-γ due to the presence of the GWH1 peptide. Moreover, the N-terminal addition of the GWH1-IFN-γ could explain the bigger sizes observed by DLS and the bigger size of GWH1-IFN-γ GWH1 antimicrobial peptide has somehow diminished or truncated the mouse IFN-γ functionality as over IFN-γ due to the presence of the GWH1 peptide. Moreover, the N-terminal addition of the observed when comparing the activity of the IFN-γ and GWH1-IFN-γ proteins (Figure 3B). This fact is GWH1 antimicrobial peptide has somehow diminished or truncated the mouse IFN-γ functionality not surprising, since there is evidence that the N-terminus region of mouse IFN-γ plays an important as observed when comparing the activity of the IFN-γ and GWH1-IFN-γ proteins (Figure 3B). This fact is not surprising, since there is evidence that the N-terminus region of mouse IFN-γ plays an important role in receptor binding. Therefore, the failure to bind prevents internalization and later events that result in the induction of the immune response [42,43]. Pharmaceutics 2020, 12, 1217 11 of 16 role in receptor binding. Therefore, the failure to bind prevents internalization and later events that result in the induction of the immune response [42,43]. Surprisingly, antimicrobial performance of GWH1-containing proteins in different hosts, showed an inhibitory activity against E. coli, whereas S. aureus was more resistant to the treatment (Figure 2A,B). The different membrane composition may influence the mechanism of action of α-helical AMPs, especially for Gram-negative and Gram-positive bacteria [44]. In this sense, the GWH1 constructs produced in this work, not only have displayed a preferred antimicrobial activity against the Gram-negative E. coli, but, most importantly, the antimicrobial performance in this microorganism was enhanced when the GWH1-fusion proteins formed nanoparticles (Figure 2A). The main mechanism of action of AMPs is membrane permeabilization and structural disruption. To achieve the antimicrobial effect, a minimal AMP concentration is required in the target surface. Such characteristic is described as the threshold concentration [45] and is experimentally expressed as the peptide-to-lipid ratio (P/L). The propensity of GWH1-GFP to self-assemble in multimeric complexes could enhance the proximity of effective monomeric units on cell surface, diminishing the local peptide concentration required to reach higher P/L values. As the P/L ratio increases, the peptides start to insert and traverse the membrane. AMP monomeric forms require higher concentrations to achieve threshold concentrations. This was demonstrated in the case of the monomeric GWH1-GFP, which lacked a dose-dependent antimicrobial activity, only showing antimicrobial effect at the highest concentration, suggesting the need to reach a critical concentration in order to display its activity (Figure 2D). A similar result was observed with the unassembled GWH1-IFN-γ (Figure 2A). The presented evidence validates the multimeric format as a more effective arrangement than the monomeric format, when it comes to antimicrobial activity. In order to explore the influence of the multiple display configuration of AMPs in the in vivo settings, we analyzed the performance of the engineered protein nanoparticles in a mouse mastitis model. Infectious mastitis is one of the most relevant diseases in dairy cattle, and antibiotic usage is the leading strategy for its treatment and prevention, since this disease is the costliest for dairy producers [46]. Despite the high antimicrobial activity showed by the multimeric format of GWH1-GFP, in vitro assays do not always correlate with in vivo efficacies inside the mammary gland [47,48]. Various aspects are influenced by the complex milk environment [25], and, therefore, a proof of concept in vivo approach is necessary. To date, different studies have used mouse models to assess the effect of antimicrobials [48–51]. However, most of these studies were dedicated to the Gram-positive pathogen S. aureus [25] and only few of them characterized the efficacy of antimicrobial agents on other relevant mastitis-causing pathogens such as E. coli [52,53]. To the best of our knowledge, this is the first study in which the antimicrobial capacity of AMP-containing nanoparticles has been determined in an in vivo mouse mastitis model of E. coli infection. Based on the in vitro assays for GWH1-GFP PNPs, a single dose of 60 µmol/L, i.e., ten times the MIC measured in vitro for GWH1-GFP in E. coli (Figure 2A), was selected for the intramammary administration after the bacterial challenge. As a result, the GWH1-GFP PNPs further reduced by 1-Log the bacterial burden in glands when compared with the non-AMP-containing counterpart, GFP (Figure 6A). This difference can be explained by the presence of the GWH1 antimicrobial peptide, which validates the functionality of this format in the milk environment and its efficacy as a promising anti-mastitis candidate. However, in S. aureus challenged animals, GWH1-GFP PNPs reduced bacterial load at the same level as control GFP (UPs) (Figure 7). In accordance with this, antimicrobial activity of GWHI-GFP PNPs in in vitro experiments against S. aureus was lower than that observed against E. coli (Figure 2A). As previously mentioned, the differences in the structure and physical properties of the bacterial membrane might account for the dissimilar activity of the same AMP in Gram-negative and Gram-positive bacteria [54]. On the other hand, in E. coli, GWH1-IFN-γUPs were not as effective as GWH1-GFP PNPs (Figure 6A), which supports the idea that the multimeric format is of utmost significance for reducing the local concentration of AMP necessary to achieve a deleterious membrane disruption, and, consequently, to increase the efficacy of the compound. Pharmaceutics 2020, 12, 1217 12 of 16 The IB format provides a protective environment against short-time degradation and a sustainable release of recombinant protein that may improve the effect over time [55–57]. However, as it has been described before [12], the amount of protein that can be released from IBs can vary depending on the IB-forming protein and the used experimental conditions during protein production process [58]. All this, associated with the fact that IFN-γ IBs have previously demonstrate activity, even at higher level than the soluble counterpart [12], urged us to test the efficacy of this format in an in vivo approach. The antimicrobial efficacy for GWH1-GFP IBs was partially reduced, showing a more disperse pattern which was not significant when compared to GFP IBs. This observation could be in accordance with the studied characteristics of GWH1-GFP IBs. The low availability of protein due to the poor release (Figure 1C) associated with the fact that part of the released protein can be found in a monomeric state (Figure 1E), which has been demonstrated here whereby bactericidal activity is reduced, may explain the low efficacy of this format in terms of antimicrobial activity. GWH1-IFN-γ and IFN-γ IBs slightly reduced the bacterial burden, being this latter statistically significant when compared to GFP IBs. This behavior could be associated with the increased release showed by both protein designs from IBs, especially for IFN-γ IBs (Figure 1C). Additionally, it is well known that IFN-γ has a very short half-life and therefore its immunological function in the mammary gland is limited [3]. The protective behavior provided by this oligomeric format may enhance its stability and merits further research. In fact, previous results showed that the activity of IFN- γ is preserved in protein released from IBs after a 96 h incubation at 37 ◦ C [12]. The cytokine embedded in IBs could be less sensitive towards proteolytic degradation, which could then result in a better immunological performance inside the mammary gland. In fact, in the in vivo assays, the activity of the proteins containing IFN-γ displayed a higher activity when administered in IB format. Altogether, the results presented in this work show a better antimicrobial performance of the multimeric AMP format against the monomeric configuration, and, most importantly, provided significant data for considering the AMP-containing nanoparticles as a promising alternative for treatment of mastitis. However, it should be noted that efficacy studies in mice should only be considered as an intermediate proxy to predict efficacy in dairy cows [51]. Overall, the versatility of the nanoparticle self-assembling format provides a valuable tool for testing a diversity of AMP with different scaffold proteins that could generate possible synergies or provide antimicrobial activity against other pathogens of interest. 5. Conclusions The extensive use of antibiotics for the treatment and prevention of bacterial infections increases the selective pressure for appearance of resistant bacteria. Previously, we have described the antimicrobial activity of peptides fused to a scaffold protein and displayed in various copies on soluble PNPs. We have also analyzed the potential of naturally occurring protein supramolecular complexes, in particular IBs, to release biologically active soluble protein nanoparticles in in vivo approaches. In this work, we explored the relevance of the oligomeric state of antimicrobial peptides in such protein nanoparticles and studied their efficiency in vitro and in vivo against different mastitis causing pathogens. The results presented here indicate the potential of multiple displays of peptides in protein nanoparticles for the development of novel antimicrobial peptide-based formulations. Supplementary Materials: The following are available online at http://www.mdpi.com/1999-4923/12/12/1217/s1, Table S1: Solubility and productivity of recombinant proteins produced in the study. Soluble versions were produced at 20 ◦ C for 5 h and IBs were produced at 37 ◦ C for 3 h. Values represent mean and SEM when available. Not determined (n.d.). Author Contributions: Conceptualization, J.V.C., N.F.-M. and F.M.; methodology, J.V.C., E.B., N.S., A.S.-C. and J.M.S.; formal analysis, J.V.C., N.F.-M. and F.M.; investigation, J.V.C., E.B., N.S., A.S.-C. and J.M.S.; resources, A.V., N.F.-M. and F.M.; writing—original draft preparation, J.V.C., N.F.-M. and F.M.; writing—review and editing, all authors contributed; visualization, J.V.C., A.S.-C., J.M.S.; supervision, N.F.-M. and F.M.; project administration, N.F.-M. and F.M.; funding acquisition, A.V., A.A., E.G.-F., N.F.-M. and F.M. All authors have read and agreed to the published version of the manuscript. Pharmaceutics 2020, 12, 1217 13 of 16 Funding: This work was funded by grants from INIA (MINECO, Spain) to N.F.-M. and E.G.-F. (RTA2015- 00064-C02-02 and RTA2015-00064-C02-01, respectively), AGAUR to A.V. (2017 SGR-229), from Bioengineering, Biomaterials and Nanomedicine Networking Biomedical Research Centre (CIBER-BBN), financed by the Carlos III Health Institute, Spain, with assistance from the European Regional Development and by the Natural Sciences and Engineering Research Council of Canada (NSERC grant No. 2020-04811) to F.M. Acknowledgments: The authors are also indebted to the CERCA Programme (Generalitat de Catalunya), European Social Fund and the FRQNT strategic network Op+lait pour un lait de qualité optimale (Université de Montréal) for supporting their research. The authors acknowledge ICTS “NANBIOSIS”, more specifically the Protein Production Platform of CIBER-BBN/IBB, at the UAB sePBioEs scientific-technical service (http://www.nanbiosis.es/unit/u1- protein-production-platform-ppp/) and the UAB scientific-technical services SM and SCAC (https://www.uab.cat/ web/research/scientific-technical-services/all-scientific-technical-services--1345667278676.html). J.V.C. received a pre-doctoral fellowship and a short-term research grant from UAB, and E.G.-F. received a post-doctoral fellowship from INIA (DOC-INIA). The authors acknowledge Microsoft and Servier Medical Art for the bacteria and mouse design in the graphical abstract. Conflicts of Interest: The authors declare no conflict of interest. References 1. Ciumac, D.; Gong, H.; Hu, X.; Lu, J.R. Membrane targeting cationic antimicrobial peptides. J. Colloid Interface Sci. 2019, 537, 163–185. [CrossRef] [PubMed] 2. Seo, M.D.; Won, H.S.; Kim, J.H.; Mishig-Ochir, T.; Lee, B.J. Antimicrobial peptides for therapeutic applications: A review. Molecules 2012, 17, 12276–12286. [CrossRef] [PubMed] 3. Aitken, S.L.; Corl, C.M.; Sordillo, L.M. Immunopathology of mastitis: Insights into disease recognition and resolution. J. Mammary Gland Biol. Neoplasia 2011, 16, 291–304. [CrossRef] [PubMed] 4. Sordillo, L.M.; Babiuk, L.A. Controlling acute Escherichia coli mastitis during the periparturient period with recombinant bovine interferon gamma. Vet. Microbiol. 1991, 28, 189–198. [CrossRef] 5. Pighetti, G.; Sordillo, L. Specific immune responses of dairy cattle after primary inoculation with recombinant bovine interferon-gamma as an adjuvant when vaccinating against mastitis. Am. J. Vet. Res. 1996, 57, 819–824. 6. Lei, R.; Hou, J.; Chen, Q.; Yuan, W.; Cheng, B.; Sun, Y.; Jin, Y.; Ge, L.; Ben-Sasson, S.A.; Chen, J.; et al. Self- assembling myristoylated human α-defensin 5 as a next-generation nanobiotics potentiates therapeutic efficacy in bacterial infection. ACS Nano 2018, 12, 5284–5296. [CrossRef] 7. Serna, N.; Sánchez-García, L.; Sánchez-Chardi, A.; Unzueta, U.; Roldán, M.; Mangues, R.; Vázquez, E.; Villaverde, A. Protein-only, antimicrobial peptide-containing recombinant nanoparticles with inherent built-in antibacterial activity. Acta Biomater. 2017, 60, 256–263. [CrossRef] [PubMed] 8. Serna, N.; Céspedes, M.V.; Saccardo, P.; Xu, Z.; Unzueta, U.; Álamo, P.; Pesarrodona, M.; Sánchez-Chardi, A.; Roldán, M.; Mangues, R.; et al. Rational engineering of single-chain polypeptides into protein-only, BBB-targeted nanoparticles. Nanomed. Nanotechnol. Biol. Med. 2016, 12, 1241–1251. [CrossRef] [PubMed] 9. Serna, N.; Céspedes, M.V.; Sánchez-García, L.; Unzueta, U.; Sala, R.; Sánchez-Chardi, A.; Cortés, F.; Ferrer-Miralles, N.; Mangues, R.; Vázquez, E.; et al. Peptide-based nanostructured materials with intrinsic proapoptotic activities in CXCR4+ solid tumors. Adv. Funct. Mater. 2017, 27, 1–9. [CrossRef] 10. Unzueta, U.; Ferrer-Miralles, N.; Cedano, J.; Zikung, X.; Pesarrodona, M.; Saccardo, P.; García-Fruitós, E.; Domingo-Espín, J.; Kumar, P.; Gupta, K.C.; et al. Non-amyloidogenic peptide tags for the regulatable self-assembling of protein-only nanoparticles. Biomaterials 2012, 33, 8714–8722. [CrossRef] 11. Chou, H.; Kuo, T.; Chiang, J.; Pei, M.; Yang, W.; Yu, H.; Lin, S.; Chen, W. Design and synthesis of cationic antimicrobial peptides with improved activity and selectivity against Vibrio spp. Int. J. Antimicrob. Agents 2008, 32, 130–138. [CrossRef] [PubMed] 12. Carratalá, J.V.; Cano-garrido, O.; Sánchez, J.; Membrado, C.; Pérez, E.; Conchillo-solé, O.; Daura, X.; Sánchez-chardi, A. Aggregation-prone peptides modulate activity of bovine interferon gamma released from naturally occurring protein nanoparticles. N. Biotechnol. 2020, 57, 11–19. [CrossRef] [PubMed] 13. Pesarrodona, M.; Jauset, T.; Díaz-Riascos, Z.V.; Sánchez-Chardi, A.; Beaulieu, M.-E.; Seras-Franzoso, J.; Sánchez-García, L.; Baltà-Foix, R.; Mancilla, S.; Fernández, Y.; et al. Targeting antitumoral proteins to breast cancer by local administration of functional inclusion bodies. Adv. Sci. 2019, 6, 1900849. [CrossRef] [PubMed] 14. Céspedes, M.V.; Cano-Garrido, O.; Álamo, P.; Sala, R.; Gallardo, A.; Serna, N.; Falgàs, A.; Voltà-Durán, E.; Casanova, I.; Sánchez-Chardi, A.; et al. Engineering secretory amyloids for remote and highly selective destruction of metastatic foci. Adv. Mater. 2020, 32, e1907348. [CrossRef] [PubMed] Pharmaceutics 2020, 12, 1217 14 of 16 15. Ferrer-Miralles, N.; Saccardo, P.; Corchero, J.L.; Xu, Z.; García-Fruitós, E. General introduction: Recombinant protein production and purification of insoluble proteins. Methods Mol. Biol. 2015, 1258, 1–24. [CrossRef] [PubMed] 16. Seras-Franzoso, J.; Sánchez-Chardi, A.; Garcia-Fruitós, E.; Vázquez, E.; Villaverde, A. Cellular uptake and intracellular fate of protein releasing bacterial amyloids in mammalian cells. Soft Matter 2016, 12, 3451–3460. [CrossRef] [PubMed] 17. Cano-Garrido, O.; Sánchez-Chardi, A.; Parés, S.; Giró, I.; Tatkiewicz, W.I.; Ferrer-Miralles, N.; Ratera, I.; Natalello, A.; Cubarsi, R.; Veciana, J.; et al. Functional protein-based nanomaterial produced in microorganisms recognized as safe: A new platform for biotechnology. Acta Biomater. 2016, 43, 230–239. [CrossRef] 18. Céspedes, M.V.; Fernández, Y.; Unzueta, U.; Mendoza, R.; Seras-Franzoso, J.; Sánchez-Chardi, A.; Álamo, P.; Toledo-Rubio, V.; Ferrer-Miralles, N.; Vázquez, E.; et al. Bacterial mimetics of endocrine secretory granules as immobilized in vivo depots for functional protein drugs. Sci. Rep. 2016, 6, 1–10. [CrossRef] 19. Genís, S.; Sánchez-Chardi, A.; Bach, À.; Fàbregas, F.; Arís, A. A combination of lactic acid bacteria regulates Escherichia coli infection and inflammation of the bovine endometrium. J. Dairy Sci. 2017, 100, 479–492. [CrossRef] 20. De Pinho Favaro, M.T.; Sánchez-García, L.; Sánchez-Chardi, A.; Roldán, M.; Unzueta, U.; Serna, N.; Cano-Garrido, O.; Azzoni, A.R.; Ferrer-Miralles, N.; Villaverde, A.; et al. Protein nanoparticles are nontoxic, tuneable cell stressors. Nanomedicine 2018, 13, 255–268. [CrossRef] 21. Cano-Garrido, O.; Garcia-Fruitós, E.; Villaverde, A.; Sánchez-Chardi, A. Improving Biomaterials Imaging for Nanotechnology: Rapid Methods for Protein Localization at Ultrastructural Level. Biotechnol. J. 2018, 13, 1–8. [CrossRef] 22. Unzueta, U.; Cespedes, M.V.; Sala, R.; Alamo, P.; Sánchez-Chardi, A.; Pesarrodona, M.; Sánchez-García, L.; Cano-Garrido, O.; Villaverde, A.; Vázquez, E.; et al. Release of targeted protein nanoparticles from functional bacterial amyloids: A death star-like approach. J. Control. Release 2018, 279, 29–39. [CrossRef] [PubMed] 23. Sánchez, J.M.; López-Laguna, H.; Álamo, P.; Serna, N.; Sánchez-Chardi, A.; Nolan, V.; Cano-Garrido, O.; Casanova, I.; Unzueta, U.; Vazquez, E.; et al. Artificial inclusion bodies for clinical development. Adv. Sci. 2019, 1902420, 1–7. [CrossRef] 24. Malu, S.; Srinivasan, S.; Maiti, P.K.; Rajagopal, D.; John, B.; Nandi, D. IFN-γ bioassay: Development of a sensitive method by measuring nitric oxide production by peritoneal exudate cells from C57BL/6 mice. J. Immunol. Methods 2003, 272, 55–65. [CrossRef] 25. Brouillette, E.; Malouin, F. The pathogenesis and control of Staphylococcus aureus-induced mastitis: Study models in the mouse. Microbes Infect. 2005, 7, 560–568. [CrossRef] [PubMed] 26. Asli, A.; Brouillette, E.; Ster, C.; Ghinet, M.G.; Brzezinski, R.; Lacasse, P.; Jacques, M.; Malouin, F. Antibiofilm and antibacterial effects of specific chitosan molecules on Staphylococcus aureus isolates associated with bovine mastitis. PLoS ONE 2017, 12, e0176988. [CrossRef] [PubMed] 27. Mordmuang, A.; Brouillette, E.; Voravuthikunchai, S.P.; Malouin, F. Evaluation of a Rhodomyrtus tomentosa ethanolic extract for its therapeutic potential on Staphylococcus aureus infections using in vitro and in vivo models of mastitis. Vet. Res. 2019, 50, 49. [CrossRef] 28. Singh, A.; Upadhyay, V.; Upadhyay, A.K.; Singh, S.M.; Panda, A.K. Protein recovery from inclusion bodies of Escherichia coli using mild solubilization process. Microb. Cell Fact. 2015, 14, 1–10. [CrossRef] 29. Boireau, C.; Cazeau, G.; Jarrige, N.; Calavas, D.; Madec, J.-Y.; Leblond, A.; Haenni, M.; Gay, É. Antimicrobial resistance in bacteria isolated from mastitis in dairy cattle in France, 2006–2016. J. Dairy Sci. 2018, 101, 9451–9462. [CrossRef] 30. Carratalá, J.V.; Serna, N.; Villaverde, A.; Vázquez, E.; Ferrer-Miralles, N. Nanostructured antimicrobial peptides: The last push towards clinics. Biotechnol. Adv. 2020, 44, 107603. [CrossRef] 31. Bray, B.L. Large-scale manufacture of peptide therapeutics by chemical synthesis. Nat. Rev. Drug Discov. 2003, 2, 587–593. [CrossRef] [PubMed] 32. Meiyalaghan, S.; Latimer, J.M.; Kralicek, A.V.; Shaw, M.L.; Lewis, J.G.; Conner, A.J.; Barrell, P.J. Expression and purification of the antimicrobial peptide GSL1 in bacteria for raising antibodies. BMC Res. Notes 2014, 7, 777. [CrossRef] [PubMed] 33. Sun, B.; Wibowo, D.; Sainsbury, F.; Zhao, C.-X. Design and production of a novel antimicrobial fusion protein in Escherichia coli. Appl. Microbiol. Biotechnol. 2018, 102, 8763–8772. [CrossRef] [PubMed] Pharmaceutics 2020, 12, 1217 15 of 16 34. Yu, H.; Li, H.; Gao, D.; Gao, C.; Qi, Q. Secretory production of antimicrobial peptides in Escherichia coli using the catalytic domain of a cellulase as fusion partner. J. Biotechnol. 2015, 214, 77–82. [CrossRef] 35. Soundrarajan, N.; Cho, H.-S.; Ahn, B.; Choi, M.; Thong, L.M.; Choi, H.; Cha, S.-Y.; Kim, J.-H.; Park, C.-K.; Seo, K.; et al. Green fluorescent protein as a scaffold for high efficiency production of functional bacteriotoxic proteins in Escherichia coli. Sci. Rep. 2016, 6, 20661. [CrossRef] 36. Zhou, Q.-F.; Luo, X.-G.; Ye, L.; Xi, T. High-level production of a novel antimicrobial peptide perinerin in Escherichia coli by fusion expression. Curr. Microbiol. 2007, 54, 366–370. [CrossRef] 37. Li, J.F.; Zhang, J.; Zhang, Z.; Kang, C.T.; Zhang, S.Q. SUMO mediating fusion expression of antimicrobial peptide CM4 from two joined genes in Escherichia coli. Curr. Microbiol. 2011, 62, 296–300. [CrossRef] 38. Wei, X.; Wu, R.; Zhang, L.; Ahmad, B.; Si, D.; Zhang, R. Expression, Purification, and Characterization of a Novel Hybrid Peptide with Potent Antibacterial Activity. Molecules 2018, 23, 1491. [CrossRef] 39. Lee, J.H.; Kim, J.H.; Hwang, S.W.; Lee, W.J.; Yoon, H.K.; Lee, H.S.; Hong, S.S. High-level expression of antimicrobial peptide mediated by a fusion partner reinforcing formation of inclusion bodies. Biochem. Biophys. Res. Commun. 2000, 277, 575–580. [CrossRef] 40. Sánchez-García, L.; Serna, N.; Álamo, P.; Sala, R.; Céspedes, M.V.; Roldan, M.; Sánchez-Chardi, A.; Unzueta, U.; Casanova, I.; Mangues, R.; et al. Self-assembling toxin-based nanoparticles as self-delivered antitumoral drugs. J. Control. Release 2018, 274, 81–92. [CrossRef] 41. Gray, P.W.; Goeddel, D.V. Cloning and expression of murine immune interferon cDNA. Proc. Natl. Acad. Sci. USA 1983, 80, 5842–5846. [CrossRef] 42. Ahmed, C.M.I.; Burkhart, M.A.; Subramaniam, P.S.; Mujtaba, M.G.; Johnson, H.M. Peptide Mimetics of Gamma Interferon Possess Antiviral Properties against Vaccinia Virus and Other Viruses in the Presence of Poxvirus B8R Protein. J. Virol. 2005, 79, 5632–5639. [CrossRef] [PubMed] 43. Magazine, H.I.; Carter, J.M.; Russell, J.K.; Torres, B.A.; Dunn, B.M.; Johnson, H.M. Use of synthetic peptides to identify an N-terminal epitope on mouse γ interferon that may be involved in function. Proc. Natl. Acad. Sci. USA 1988, 85, 1237–1241. [CrossRef] 44. Torcato, I.M.; Huang, Y.-H.; Franquelim, H.G.; Gaspar, D.; Craik, D.J.; Castanho, M.A.R.B.; Troeira Henriques, S. Design and characterization of novel antimicrobial peptides, R-BP100 and RW-BP100, with activity against Gram-negative and Gram-positive bacteria. Biochim. Biophys. Acta 2013, 1828, 944–955. [CrossRef] [PubMed] 45. Melo, M.N.; Ferre, R.; Castanho, M.A.R.B. Antimicrobial peptides: Linking partition, activity and high membrane-bound concentrations. Nat. Rev. Microbiol. 2009, 7, 245–250. [CrossRef] [PubMed] 46. Aghamohammadi, M.; Haine, D.; Kelton, D.F.; Barkema, H.W.; Hogeveen, H.; Keefe, G.P.; Dufour, S. Herd-Level Mastitis-Associated Costs on Canadian Dairy Farms. Front. Vet. Sci. 2018, 5, 100. [CrossRef] [PubMed] 47. Apparao, M.D.; Ruegg, P.L.; Lago, A.; Godden, S.; Bey, R.; Leslie, K. Relationship between in vitro susceptibility test results and treatment outcomes for gram-positive mastitis pathogens following treatment with cephapirin sodium. J. Dairy Sci. 2009, 92, 2589–2597. [CrossRef] 48. Demon, D.; Ludwig, C.; Breyne, K.; Guédé, D.; Dörner, J.-C.; Froyman, R.; Meyer, E. The intramammary efficacy of first generation cephalosporins against Staphylococcus aureus mastitis in mice. Vet. Microbiol. 2012, 160, 141–150. [CrossRef] 49. Li, L.; Wang, L.; Gao, Y.; Wang, J.; Zhao, X. Effective antimicrobial activity of plectasin-derived antimicrobial peptides against Staphylococcus aureus infection in mammary glands. Front. Microbiol. 2017, 8, 2386. [CrossRef] 50. Wei, W.; Dejie, L.; Xiaojing, S.; Tiancheng, W.; Yongguo, C.; Zhengtao, Y.; Naisheng, Z. Magnolol inhibits the inflammatory response in mouse mammary epithelial cells and a mouse mastitis model. Inflammation 2015, 38, 16–26. [CrossRef] 51. Schmelcher, M.; Powell, A.M.; Camp, M.J.; Pohl, C.S.; Donovan, D.M. Synergistic streptococcal phage λSA2 and B30 endolysins kill streptococci in cow milk and in a mouse model of mastitis. Appl. Microbiol. Biotechnol. 2015, 99, 8475–8486. [CrossRef] 52. Notebaert, S.; Demon, D.; Vanden Berghe, T.; Vandenabeele, P.; Meyer, E. Inflammatory mediators in Escherichia coli-induced mastitis in mice. Comp. Immunol. Microbiol. Infect. Dis. 2008, 31, 551–565. [CrossRef] [PubMed] Pharmaceutics 2020, 12, 1217 16 of 16 53. Yu, Y.; Fang, J.T.; Sun, J.; Zheng, M.; Zhang, Q.; He, J.S.; Liao, X.P.; Liu, Y.H. Efficacy of cefquinome against Escherichia coli environmental mastitis assessed by pharmacokinetic and pharmacodynamic integration in lactating mouse model. Front. Microbiol. 2017, 8, 1–9. [CrossRef] [PubMed] 54. Li, J.; Koh, J.-J.; Liu, S.; Lakshminarayanan, R.; Verma, C.S.; Beuerman, R.W. Membrane Active Antimicrobial Peptides: Translating Mechanistic Insights to Design. Front. Neurosci. 2017, 11, 73. [CrossRef] [PubMed] 55. Garcia-Fruitós, E. Inclusion bodies: A new concept. Microb. Cell Fact. 2010, 9, 80. [CrossRef] 56. Villaverde, A.; Corchero, J.L.; Seras-Franzoso, J.; Garcia-Fruitós, E. Functional protein aggregates: Just the tip of the iceberg. Nanomedicine 2015, 10, 2881–2891. [CrossRef] [PubMed] 57. Rinas, U.; Garcia-Fruitós, E.; Corchero, J.L.; Vázquez, E.; Seras-Franzoso, J.; Villaverde, A. Bacterial inclusion bodies: Discovering their better half. Trends Biochem. Sci. 2017, 42, 726–737. [CrossRef] 58. Peternel, Š.; Grdadolnik, J.; Gaberc-Porekar, V.; Komel, R. Engineering inclusion bodies for non denaturing extraction of functional proteins. Microb. Cell Fact. 2008, 7, 1–9. [CrossRef] Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).