Leishmania infantum UBC1 in Metacyclic Promastigotes from Phlebotomus perniciosus, a Vaccine Candidate for Zoonotic Visceral Leishmaniasis

Leishmania parasites cause outstanding levels of morbidity and mortality in many developing countries in tropical and subtropical regions. Numerous gene expression profiling studies have been performed comparing different Leishmania species’ life-cycles and stage forms in regard to their distinct infective ability. Based on expression patterns, homology to human orthologues, in silico HLA-binding predictions, and annotated functions, we were able to select several vaccine candidates which are currently under study. One of these candidates is the Leishmania infantum ubiquitin-conjugating enzyme E2 (LiUBC1), whose relative levels, subcellular location, in vitro infectivity in the U937 myeloid human cell model, and protection levels in Syrian hamsters against L. infantum infection were studied herein. LiUBC1 displays a low level of similarity with the mammalian orthologs and relevant structure differences, such as the C-terminal domain, which is absent in the human ortholog. LiUBC1 is present in highly infective promastigotes. Knock-in parasites overexpressing the enzyme increased their infectivity, according to in vitro experiments. Syrian hamsters immunized with the recombinant LiUBC1 protein did not show any parasite burden in the spleen, unlike the infection control group. The IFN-γ transcript levels in splenocytes were significantly higher in the LiUBC1 immunized group. Therefore, LiUBC1 induced partial protection against L. infantum in the Syrian hamster model.


Introduction
Leishmaniasis is a disease caused by trypanosomatid parasites from the genus Leishmania. Human leishmaniasis is classified according to the clinical manifestations into cutaneous (CL), mucocutaneous (MCL), and visceral leishmaniasis (VL), as well as according to the species involved. VL caused by Leishmania infantum is a zoonosis (ZVL), whereas VL caused by L. donovani is anthroponotic. In both cases, VL is fatal without treatment, with about 50,000 annual deaths [1]. HIV-Leishmania coinfections have been reported [2][3][4][5][6]. Canids are described as L. infantum (syn. L. chagasi) reservoirs in the Northern Mediterranean basin, Central Asia, and South America [7,8]. The vector in the Mediterranean basin is Phlebotomus perniciosus (Psychodidae: Phlebotominae). A human L. infantum outbreak declared more than a decade ago in central Spain is still active [9][10][11][12]. In contrast to human VL, dogs display variable cutaneous and visceral clinical signs which appear simultaneously. There are also variable profiles for the clinical signs that range from asymptomatic to severe. These include onicogriposis, weight loss, asthenia, cutaneous lesions, conjunctivitis, the transfer of ubiquitin from E2 to lysine amino acid residues in the substrate protein [37]. There is growing evidence for the possible relation between the downregulation of this system and the appearance of diseases related to the erroneous folding of proteins, mainly neurodegenerative diseases, such as Lewy bodies in Parkinson's disease, which display an increase in ubiquitin bound to aggregated proteins [38,39]. Despite these suggested functions, little is known about their role in cellular physiological mechanisms. Therefore, the study of ubiquitin-related proteins deserves more attention, and lower eukaryotes should provide valuable information. In fact, this protein seems to be related to the clearance of defective proteins from the cell nucleus in Saccharomyces cerevisiae [40]. Parasitic protozoa are complex unicellular organisms that undergo distinct modifications to adapt themselves to the different physicochemical environments corresponding to their distinct hosts during their biological cycles and, therefore, may be a useful model to study these regulation processes. One of the main features of Leishmania parasites are the morphological changes they undergo from the insect vector midgut to the interior of the phagocyte vacuoles in the mammalian host. These changes in shape and size require the cytoskeleton to adapt and strictly regulate the proteolysis mechanisms [41]. In fact, several processes regulated by the proteasome complex have been described in Leishmania spp. In L. donovani, it has been noted that the key enzyme methionine adenosyl transferase is negatively regulated by the proteasome complex. In the same way, ubiquitination has been associated with mRNA regulation in L. major [42] or with resistance to antimonial treatment in L. tropica [43].
The present work describes the structural modeling, relative levels, subcellular localization, and biological role in infectivity of LiUBC1, and presents evidence supporting the conclusion that LiUBC1 is a vaccine candidate that protects Syrian hamsters against L. infantum infection.
At each experimental time point, 2 × 10 8 promastigotes were centrifuged at 200× g for 5 min and washed three times with PBS. The sediment was resuspended in 100 µL of lysis buffer (8.4 M urea, 2.4 M thiourea, 4% CHAPS p/v, 50 mM DTT, 1% Triton X-100, one tablet of protease inhibitor cocktail (Roche, Basel, Switzerland), and 1 unit of DNAse (New England Biolabs, Ipswich, MA, USA)). After gently shaking for 20 min, the mixture was centrifuged at 8000× g for 5 min at 4 • C. The supernatant was recovered and stored at −80 • C before use.
Peanut lectin nonagglutinating (PNA) promastigotes were obtained in stationary phase. First, they were centrifuged at 2000× g for 10 min in CM at a cell density of 2 × 10 8 cells/mL and incubated with 50 µg/mL of PNA (Sigma-Aldrich, St. Louis, MO, USA) at room temperature (RT) for 30 min. Agglutination and sedimentation was checked with light microscopy. The pellet obtained was resuspended in 1 mL of the remaining supernatant and taken to the initial volume of 10 mL with CM at the same concentration of PNA and centrifuged at 200× g for 10 min. The centrifugation step was repeated to obtain the PNA+ promastigotes. The last supernatant was centrifuged at 2000× g to obtain the PNA-promastigotes. The whole process was checked by light microscopy.

Ethics
All possible animal welfare measures were applied to 14 Mesocricetus auratus specimens required for the challenge experiment, such as optimal temperature, ad libitum feeding, refinement, disease follow-up, and environmental enrichment. The experimental design, the procedures, and the safety measures were approved by the Instituto de Salud Carlos III Ethical Advisory Committee for Animal Experimentation following the EU Directive 2010/63 and the Spanish regulation RD53/2013. Manipulation of rabbits was also carried out according to the animal welfare rules and supervised by the Consejo Superior de Investigaciones Científicas (CSIC) Ethical Committee (EU Directive 2010/63 and Spanish regulation RD53/2013).

LiUBC1 Expression and Purification
The LiUBC1 gene CDS, including an upstream 6xHis-tag coding sequence was PCRamplified (BamHI-LiUBC1-Fw 5 -TAAGGATCCCCTCTACGGCGGCT-3 and BamHI-LiUBC1-Rv 5 -CAAAAGCTTTCAGATGCGGCGCAGC-3 ) and cloned in the pQE30 expression vector using XL1-blue chemically competent cells as the recipient E. coli strain. E. coli M15 cells were transformed with pQE30-LiUBC1 and a 10 mL preinoculum grown at 37 • C in LB medium containing ampicillin (100 µg/mL) and kanamycin (50 µg/mL). A 0,5 L flask was seeded with 5 mL of inoculum and grown to an OD 595 value of 0.5. Heterologous expression was then induced by adding 1 mM IPTG and incubating for 4 h at 37 • C. Thereafter, the bacterial cells were centrifuged at 6000× g for 15 min at 4 • C and washed with 0.9% NaCl solution. Per gram of pellet, 10 mL of lysis buffer (1 M KCl, 60 mM imidazole, 1% Brij 58, 10% glycerol, and 1 mM benzamidine) was added. The lysate was stored at −80 • C until usage. Once thawed, it was centrifuged at 20,000× g for 1 h at 4 • C. LiUBC1 contains the 6xHis tag in the N-terminus and was found mainly in the pellet, which was resuspended in 3 mL of 5 M GuHCl for subsequent purification in denaturing conditions. After centrifugation, the extract was subject to affinity chromatography in a 6xHis-loaded HiTrap FF 1 mL column (GE Healthcare, Little Chalfon, UK) using an FPLC ÄKTA system (GE Healthcare, Little Chalfon, UK). After washing with increasing concentrations of imidazole, 6xHis-LiUBC1 elution was performed with 700 mM imidazole and 5 M GuHCl. The purified protein fractions were analyzed by SDS-PAGE.

Antibody Obtainment
The specific anti-LiUBC1 polyclonal antibody was obtained using New Zealand rabbits, each inoculated with four doses of 500 µg of the purified LiUBC1 protein in 0.5 mL PBS buffer. The LiUBC1 protein was obtained by excision of the purified band and subsequent gel diffusion after SDS-PAGE using prestained TGX gels (BioRad ® , Hercules, CA, USA). The initial dose was emulsified with Freund's complete adjuvant (1:1) and the following three doses with incomplete Freund's adjuvant, each inoculated at three-week intervals.

Protein Expression Level Determination by Western Blot
Relative expression levels of proteins between different cellular stages were carried out by Western blot experiments, according to a previously described protocol [44]. In short, previously obtained extracts from different days of promastigote culture as well as amastigote-like cells were quantified by the Bradford method [45], and 20 µg of each sample was run through SDS-PAGE [46] and transferred to nitrocellulose membranes (0.45 µm, BioRad labs). LiUBC1 was detected using the specific polyclonal antibody generated (1:100) and HRP-conjugated goat antirabbit IgG (1:2000) as the secondary antibody (Dako, Agilent Technologies, Santa Clara, CA, USA). A primary antibody against gGAPDH (1:10000) was used for the internal standard [47]. Detection was carried out by using an ECL Western Blotting Detection Kit (GE Healthcare) and a LAS-3000 Image Reader (GE Healthcare).

Generation of L. infantum LiUBC1 Knock-In Promastigote Line
The obtainment of an L. infantum cell line overexpressing LiUBC1 was carried out with the vector pIRmcs3 [48]. The LinJ.33.2910 gene was initially cloned in the DH5α E. coli strain. L. infantum promastigotes were washed in electroporation buffer (132 mM NaCl, 8 mM KCl, 8 mM Na 2 HPO 4 , 1.5 mM KH 2 PO 4 , 0.5 mM potassium acetate, 90µM calcium acetate, pH 7.0), sterilized by filtration (0.22 µm), and resuspended in 400 µL of the same buffer to a final concentration of 7 × 10 6 cells/mL. The electric pulse (1 KV/24 Ω/28 ms) was delivered in the presence of the linearized plasmid pIRmcs3 containing the LiUBC1 gene (3 µg DNA). The same conditions were used with the pIRmcs3 plasmid without insert for the obtainment of a control promastigote line. During the initial 24 h of culture, transfected promastigotes and controls were resuspended in CM supplemented with 20% HIFBS and 10% stationary promastigote culture medium sterilized by filtration. After 24 h, 100 µg/mL of the selection agent nourseothricin (Jena Bioscience, Jena, Germany) was added to the cultures. After one week, the amount of nourseothricin was increased to 120 µg/mL and transfected promastigotes were cultured in the presence of the antibiotic. In all experiments, promastigotes transfected with the pIRmcs3 plasmid without the LinJ. 33.2910 gene were used as controls.

LiUBC1 Cellular Detection by Indirect Immunofluorescence (IIF)
L. infantum promastigotes (2 × 10 5 ) were sedimented onto a 0.2 cm 2 area on a glass slide and fixed with 4% paraformaldehyde solution for 5 min at RT. After three washes with PBS in a Coplin jar for 5 min, L. infantum promastigotes were permeabilized with a 0.5% Triton X-100 solution in PBS for 5 min. Blocking was carried out with a 0.1% solution of Tween-20 in PBS containing 5% w/v skimmed milk in a wet chamber. Promastigotes were incubated with specific polyclonal antibody anti-LiUBC1 in blocking buffer (1:50) for 1 h. After a single wash, cells were incubated with the secondary antibody antirabbit IgG conjugated to the fluorophore Alexa Fluor 488 (Jackson Immunoresearch, West Grove, PA, USA) (1:200) in blocking buffer for 1 h in the dark. Ten minutes before the end of this incubation, 10 µL of 10 µg/mL DAPI (Thermo Fisher Scientific, Waltham, MA, USA) in PBS was added. The cells were finally washed three times with PBS in the dark and preparations were mounted with Mowiol 4-88 (Sigma-Aldrich, Burlington, MA, USA). Images were obtained in a Spectrum Laser confocal Leica TCS-SP5-OABS microscope previously adjusted to the negative control (preimmune serum 1:50).

Transmission Electron Microscopy (TEM) Detection of LiUBC1
Promastigotes were fixed with 2% p-formaldehyde and 0.2% glutaraldehyde in 0.1 M sodium kakodylate, 4.5% sucrose, pH 7.4, for 30 min at 4 • C. The samples were dehydrated in an ethanol-ethyleneglycol sequential mixture (50-100%) in a temperature range of 0 • C to −35 • C at 30 min intervals. Then, promastigotes were transferred to a Lowycril K4M solution in ethanol (1:1 and 1:2) and finally to pure Lowicryl K4M overnight. Subsequently, the samples were introduced into gelatin capsules and polymerized by UV light for 72 h and then cut with an ultramicrotome.
The promastigote preparations were incubated with the anti-LiUBC1 antibody (see Section 2.4) for 60 min at 37 • C. After four washes of 5 min, the samples were incubated with antirabbit IgG secondary antibody conjugated with colloidal gold (1:10). After three additional washes with buffer containing 0.1% Triton X-100 in milliQ water, the samples were incubated with 1% uranyle acetate in bidistilled water for 10 min, washed twice with water for 1 min, mounted, and observed with a JEOL JEM-1230 electron microscope.

Mass Spectrometry LC-MS/MS Analysis
LiUBC1 samples were dissolved in Laemmli sample buffer and run through a 10% SDS-PAGE gel. Protein bands were excised from the gel and digested in-gel with trypsin, as described in [49,50]. Peptides were separated with an Easy-nLC 1000 Nano System (Thermo Fisher Scientific, Waltham, MA, USA) using a precolumn Acclaim PepMap 100 (Thermo Fisher Scientific) and an RSLC PepMap C18, 15 cm long, 75 µm inner diameter, and 3 µm particle size column (Thermo Fisher Scientific). The flow rate was 300 nL/min using 0.1% formic acid in water (FAW) and 0.1% formic acid in 100% acetonitrile (FAA). The gradient profile was set as follows: 0-35% FAA for 90 min, 35-100% FAA for 4 min, 100% FAA for 8 min. The sample input was 4 µL.
A Q Exactive LC-MS/MS system (Thermo Fisher Scientific) was used to perform the MS analysis. Ionization was set at a liquid junction voltage of 1800 V and a capillary temperature of 270 • C. The software scan method was set with m/z values equal to 400-1500, a resolution value of 70,000 for the Orbitrap analyzer (for m/z 200), target automatic gain control (AGC) equal to 3 × 10 6 , and an injection time up to 100 ms. The 10 most intense precursor ions were chosen for MS/MS fragmentation, which was performed with a normalized collision energy of 27 eV and the following settings applied for acquisition: starting m/z value of 100; AGC target of 2 × 10 5 with a 8 × 10 3 threshold; 17,500 resolution (m/z 200); 2 m/z isolation window; and 100 ms maximum IT. The ions that were unassigned, singly charged, or bore ≥7 protonations were excluded from subsequent analysis, applying a dynamic exclusion time of 20 s to discriminate previously selected ions.
Whole protein samples were obtained on days 1, 3, and 5 of growth using a lysis buffer containing 8.4 M urea, 2.4 M thiourea, 4% w/v CHAPS, 50 mM DTT, 1% Triton X-100, one tablet of a protease inhibitor cocktail (Roche), and 50 µg/mL benzonase (Merck, Kenilworth, NJ, USA). The protein samples were precipitated with methanol:chloroform:water (4:1:3), resuspended in 50 mM triethyl ammonium bicarbonate (TEAB), and labelled using an 8-Plex iTRAQ kit (AB SCIEX) according to manufacturer instructions. The labelled samples were grouped and purified using MCX cartridges (Waters) to remove excess iTRAQ reagent and nonpeptidic residual materials. Peptides were eluted with 1 mL of methanol:ammonium hydroxide (9:1), dried, and fractionated by liquid chromatography. After desalting with C18 OMIX (Agilent Technologies), samples were kept at −80 • C until their LC-MS/MS analysis. All peptide separation was carried out using an Easy-nLC 100 (Thermo Fisher Scientific) system. The elution stream was 300 nL/min using formic acid in acetonitrile from 0.1% to 100%. MS analysis was conducted using an OrbiTrap Mass Spectrometer (Thermo Fisher Scientific). For quantification, labeled peptide mixtures were reconstituted in formic acid 0.1%, trapped in a precolumn Acclaim PepMap 100 (Thermo Fisher Scientific), and eluted in an Acclaim PepMap C18 column. Each analysis was carried out in duplicate.
Proteome Discoverer software (version1.4.1.14) (Thermo Fisher Scientific) was used for iTRAQ analysis. The *.raw files containing the mass spectra were analyzed with SEQUEST against the reference L. infantum JPCM5 genome, which contains 8062 protein entries. Mass tolerance was adjusted to 10 ppm for precursors and 0.02 Da for fragments. Other settings were 2 missed cleavages, carbamidomethylation of cysteines (fixed), methionine oxidation, and N-terminal acetylation (variable). The Percolator algorithm 9 [51] was used to filter identified peptides, setting 0.01 as q-value threshold.

L. infantum In Vitro Infection Experiments
The U937 cells (CRL-1593.2 TM , ATCC ® , Manassas, Virginia, USA) were maintained in Roux flasks at 37 • C in the presence of a 5% CO2 atmosphere. On an 8-well cell culture slide, 150 µL of cell suspension per well was fixed at a cell density of 2 × 10 8 /cm 2 . Cells were stimulated with 20 ng/mL of phorbol myristate acetate (PMA) for 72 h and infections were carried out by adding wild-type and LiUBC1-transfected L. infantum promastigotes to the cell cultures at a promastigote:U937 cell ratio of 10:1 for 2 h. After infection, cells were washed with CM. All the biological replicates were carried out in triplicate. After 24 or 48 h, the culture slides were washed and 150 µL of fixing solution (ethanol:acetic acid (3:1)) was added to each well. Wells were removed and the slides were stained with Diff-Quik solution (Dade Behring, Deerfield, IL, USA). The percentage of infected cells as well as the number of amastigotes per cell were calculated in all biological replicates by using the Student's t-test implemented in Sigmaplot 11 software (Sigmaplot ® , San Jose, CA, USA).

Animal Challenge Experiments
Of a total of 14 animals (M. auratus), 6 were used as controls and 8 were inoculated intraperitoneally with 20 µg of recombinant LiUBC1 protein in 1 mL of PBS. Controls were inoculated with PBS. The second inoculation was given three weeks later. After two weeks, animals were challenged with 10 7 L. infantum metacyclic promastigotes (MCAN/ES98/10445) in 200 µL of PBS. The animals were euthanized according to the EU rules for animal welfare (EU Directive 2010/63 and Spanish regulation RD52/2013), and spleen and blood were obtained for analysis.

Parasite Burden Evaluation and Immune IgG Response Determination in M. auratus Model
At the end of the challenge experiments with M. auratus, the spleens were extracted aseptically, weighed, homogenized, and resuspended in 3 mL of Schneider s Drosophila medium supplemented with 10% HIFBS (HyClone, Thermo Fisher Scientific), penicillin (100 U/mL), streptomycin (50 mg/mL, Lonza ® ), 20 mM HEPES (Sigma-Aldrich), and 1% sterile urine. Four samples of the homogenized suspension were seeded and diluted six times in a 96-well plate. Plates were incubated for two weeks and parasites were detected in the well cultures by optical microscopy (200×). Parasite burden was calculated as the geometric mean of the parasites detected at the maximum dilution described [52].
The blood sample was allowed to clot at RT for 20 min and was centrifuged at 10,000× g at room temperature for 10 min. The serum was stored at −80 • C and used later for a total IgG antibody determination against total L. infantum soluble antigen (SLA) by ELISA. First, a flat bottom plate was lined with 100 µL of a 10 µg/mL SLA solution in 3.36 mM carbonate-10 mM bicarbonate buffer overnight at 4 • C. Subsequently, it was blocked with a 1% BSA solution in PBS and washed three times with a 0.1% BSA and 0.03% Tween 20 solution in PBS. Next, 100 µL of a 1:20 serum sample was added to each well and left for 30 min at RT. Meanwhile, protein A (Sigma-Aldrich, Burlington, MA, USA) was prepared at a concentration of 0.19 µg/mL using a wash solution. The samples were washed before and after incubation, which was performed with 100 µL of the protein A solution for 2 h at RT. The last wash was carried out with PBS. An o-phenylenediamine dihydrochloride (OPD) tablet was dissolved in 12 mL of citrate buffer (5.2 mM citric acid, 12.7 mM Na 2 HPO 4 , pH 5) and 15 µL H 2 O 2 . To each well was added 100 µL of said solution, and the colorimetric reaction was allowed to develop for 20 min. The reaction was stopped by adding 50 µL of a 1% SDS solution. The absorbance was read at 450 nm using a Microplate Reader 680 (BioRad ® , Hercules, CA, United States) and Microplate Manager 5.2.1 software (BioRad ® ).

Relative IFN-γ Expression Levels in Splenocytes by qRT-PCR
Whole RNA samples were obtained using TRIzol ® reagent (Life Technologies, Carlsbad, CA, USA). First-strand cDNA synthesis was performed starting from 10 µg of total RNA. First, the RNA was mixed with 6 µg of random hexamer primers (Life Technologies). The mixture was denatured at 70 • C for 2 min and immediately cooled on ice. Then, the samples were incubated at 46 • C for 3 h with 570 µM of each dNTP, 10 µM DTT, and 600 U SuperScript III Reverse Transcriptase (Life Technologies) in 30 µL of reaction volume. Next, RNA degradation was performed using 1U RNaseH (Life Technologies) at 37 • C for 30 min. Enzyme denaturation was performed at 65 • C for 10 min. The resulting cDNA samples were purified using QiaQuick PCR Purification Kit (Qiagen, Hilden, Germany). Syrian hamster IFN-γ TaqMan assay no. Cg04455412_m1 (primers and FAM-NFQ MGB probe, Applied Biosystems, Waltham, MA, USA) were mixed with cDNA samples (10, 2, and 0.4 ng cDNA per reaction set by serial dilutions) and with TaqMan Universal Master Mix 2X (Life Technologies) in 10 µL reaction volume. Thermal cycling runs were performed in a 7900HT Fast Real-Time PCR system. Thermal cycling conditions set in SDS 4.1. software (Life Technologies) were: 95 • C, 5 min; 40 × (95 • C, 30 sec; 60 • C, 1 min, data acquisition). The β-actin taqman assay no. Cg04424027_gH (Applied Biosystems) was used as the refer-ence gene for quantification using the ∆∆Ct [53]. The Mann-Whitney U test was applied to the 2 −∆∆Ct differential expression value at 0.01 significance level.

Sequence Alignment and Molecular Modeling
Protein sequences were retrieved from the NCBI database and the alignments were performed using the Clustal Omega software.
The molecular model predictions were obtained with i-Tasser. The structures were taken from Protein Data Bank (PDB) and the final comparative analysis was performed using PyMol program.

Primary Structure of LiUBC1
The comparative analysis of the LiUBC1 sequence (encoded by the LinJ.33.2910 gene) with several ortholog proteins showed high identity (>80%) within the genus Leishmania, including L. braziliensis, a species from the Viannia subgenus. The UBC1 from Trypanosoma cruzi is evolutionarily more distant, as 50% sequence identity was observed. The similarity with mammalian orthologs is lower, with values ranging between 30% and 40% in the human, hamster, and rabbit orthologs. Arabidopsis thaliana showed an amino acid identity value in the same range as mammalians ( Figure 1, Table 1 The results indicated a high level of LiUBC1 conservation within the genus, in both the Leishmania and Viannia subgenera. Identity decreases to 50% within the Trypanosoma genus and further in the higher eukaryotes, especially mammals. However, the role of the LiUBC1 orthologs is presumably maintained, because the N-terminal domain is the most conserved region between these species ( Figure S1). Taking into account that percentages of identity only provide partial information about the tertiary or quaternary structure of the proteins, as well as about the final functional performance of the proteins, we determined the predicted structure of the enzyme based on known structures.
The predicted LiUBC1 structure is composed of a C-terminal domain and a UBC catalytic core. This structure is similar to the structure of the S. cerevisiae E2 ubiquitin-conjugating enzyme ( Figure 2B), which has been described as one homodimer [53]. The human ortholog enzyme lacks the C-terminal domain ( Figure 2C) present in LiUBC1 and S. cerevisiae proteins, but the N-terminal end, which contains the enzyme's active center, also  The results indicated a high level of LiUBC1 conservation within the genus, in both the Leishmania and Viannia subgenera. Identity decreases to~50% within the Trypanosoma genus and further in the higher eukaryotes, especially mammals. However, the role of the LiUBC1 orthologs is presumably maintained, because the N-terminal domain is the most conserved region between these species ( Figure S1). Taking into account that percentages of identity only provide partial information about the tertiary or quaternary structure of the proteins, as well as about the final functional performance of the proteins, we determined the predicted structure of the enzyme based on known structures.
The predicted LiUBC1 structure is composed of a C-terminal domain and a UBC catalytic core. This structure is similar to the structure of the S. cerevisiae E2 ubiquitinconjugating enzyme ( Figure 2B), which has been described as one homodimer [53]. The human ortholog enzyme lacks the C-terminal domain ( Figure 2C) present in LiUBC1 and S. cerevisiae proteins, but the N-terminal end, which contains the enzyme's active center, also displays a very similar structure ( Figure 2D).

LiUBC1 Purification
The LinJ.33.2910 gene, which encodes LiUBC1, was cloned in the pQE30 expression vector in the E. coli XL1-blue strain, and the M15 strain for LiUBC1 overexpression in a heterologous system. LiUBC1 was expressed in the presence of 1 mM IPTG at an optimal temperature of 37 °C ( Figure 3A). Most of the overexpressed protein was located in the nonsoluble fraction ( Figure 3B). The LiUBC1 protein was solubilized with a buffer containing 5 M GuHCl and 40 mM imidazole and purified in batch using a Ni 2+ -NTA agarose column. Discontinuous elution was performed in three steps using 100, 400, and 700 mM imidazole, respectively. Most of the LiUBC1 protein was eluted at an imidazole concentration of 700 mM. The purified protein presented a complex pattern with bands that may correspond to the molecular weights of the LiUBC1 protein (26 kDa plus the histidine tag),

LiUBC1 Purification
The LinJ.33.2910 gene, which encodes LiUBC1, was cloned in the pQE30 expression vector in the E. coli XL1-blue strain, and the M15 strain for LiUBC1 overexpression in a heterologous system. LiUBC1 was expressed in the presence of 1 mM IPTG at an optimal temperature of 37 • C ( Figure 3A). Most of the overexpressed protein was located in the nonsoluble fraction ( Figure 3B). The LiUBC1 protein was solubilized with a buffer containing 5 M GuHCl and 40 mM imidazole and purified in batch using a Ni 2+ -NTA agarose column. Discontinuous elution was performed in three steps using 100, 400, and 700 mM imidazole, respectively. Most of the LiUBC1 protein was eluted at an imidazole concentration of 700 mM. The purified protein presented a complex pattern with bands that may correspond to the molecular weights of the LiUBC1 protein (26 kDa plus the histidine tag), as well as bands that may correspond to aggregates ( Figure 3C).  The two bands correspond to the LiUBC1, confirming the obtainment of the purified recombinant enzyme, and suggest the presence of the protein in bands corresponding to aggregates that could be dimers. The presence of faint bands with intermediate molecular weights may also indicate the existence of intermediate forms, probably due to the sensitivity of LiUBC1 to degradation agents even in the presence of protease inhibitors.

LiUBC1 Expression Levels throughout the Growth Curve of IPER/ES/2013/ATE1FL6 Strain of L. infantum Promastigotes in Axenic Culture
We studied the expression of LiUBC1 in the highly infective IPER/ES/2013/ATE1FL6 strain obtained from P. perniciosus gut during a human outbreak of the disease in Spain (Section 2.1). Figure 4 shows the growth curve, as well as the expression of the LiUBC1 protein. The strain from the human outbreak ( Figure 4A) has a lag phase that extends to day three, followed by a sharp logarithmic phase that reaches a maximum at day 6 and a short stationary phase before the rapid start of cell death on day 7.
The Western blot analysis of the extracts obtained from each day of culture also showed the different protein expression patterns of LiUBC1. The strain from the human outbreak displayed two bands that may correspond to the monomer and dimer forms of the enzyme, with a slight decrease at the stationary phase of growth as indicated by the gGADPH controls, as well as the presence of faint intermediate forms.
The LiUBC1 expression pattern was similar in the PNA selection extracts, though there was a lower expression of the monomeric form in the PNA extracts. To confirm the presence of LiUBC1 aggregates, the bands were excised, in-gel SDS-PAGE trypsin-digested, and analyzed by nLC-MS/MS (Table 2). Table 2. LiUBC1 protein band identification by nLC-MS/MS. The protein bands were excised from Table 3. C. Proteins were extracted from the gel and analyzed by nLC-MS/MS. The MASCOT score is a statistical parameter that indicates that the identification is reliable (p < 0.05) when values are higher than 52. The coverage indicates the percentage of the protein sequence which has been detected in each case. The two bands correspond to the LiUBC1, confirming the obtainment of the purified recombinant enzyme, and suggest the presence of the protein in bands corresponding to aggregates that could be dimers. The presence of faint bands with intermediate molecular weights may also indicate the existence of intermediate forms, probably due to the sensitivity of LiUBC1 to degradation agents even in the presence of protease inhibitors.

LiUBC1 Expression Levels throughout the Growth Curve of IPER/ES/2013/ATE1FL6 Strain of L. infantum Promastigotes in Axenic Culture
We studied the expression of LiUBC1 in the highly infective IPER/ES/2013/ATE1FL6 strain obtained from P. perniciosus gut during a human outbreak of the disease in Spain (Section 2.1). Figure 4 shows the growth curve, as well as the expression of the LiUBC1 protein. The strain from the human outbreak ( Figure 4A) has a lag phase that extends to day three, followed by a sharp logarithmic phase that reaches a maximum at day 6 and a short stationary phase before the rapid start of cell death on day 7. The Western blot analysis of the extracts obtained from each day of culture also showed the different protein expression patterns of LiUBC1. The strain from the human outbreak displayed two bands that may correspond to the monomer and dimer forms of the enzyme, with a slight decrease at the stationary phase of growth as indicated by the gGADPH controls, as well as the presence of faint intermediate forms.
The LiUBC1 expression pattern was similar in the PNA selection extracts, though there was a lower expression of the monomeric form in the PNA extracts.

Subcellular Localization of LiUBC1 in the L. infantum Parasite
IIF experiments with the anti-LiUBC1 polyclonal antibody displayed a particulate distribution of the protein in two parasite areas: the nucleus and the kinetoplast area ( Figure 5). This technique did not allow us to differentiate between the nuclear membrane and the perinuclear cytoplasmic zone. The same happened with the presence of the protein in the flagellar pocket area. To clarify this point, we carried out TEM experiments. TEM using the same LiUBC1 primary antibody and the antirabbit IgG conjugated to colloidal gold was carried out to obtain more accurate information about the LiUBC1 subcellular localization. This experiment revealed that the protein is located in the nuclear envelope area, as well as in the kinetoplast and adjacent to the flagellar pocket membrane (Figure 6). the flagellar pocket area. To clarify this point, we carried out TEM experiments. TEM using the same LiUBC1 primary antibody and the antirabbit IgG conjugated to colloidal gold was carried out to obtain more accurate information about the LiUBC1 subcellular localization. This experiment revealed that the protein is located in the nuclear envelope area, as well as in the kinetoplast and adjacent to the flagellar pocket membrane ( Figure 6).
The TEM experiments confirmed the presence of LiUBC1 in the nuclear membrane area, as well as the flagellar pocket and the kinetoplast.

Characterization of an L. infantum Stable LiUBC1 Knock-In Promastigote Cell Line
A knock-in cell line was generated for functional studies using the infective human outbreak strain (Section 2.1). Figure 7A shows the growth pattern of L. infantum parasites overexpressing LiUBC1. Both growth curves, corresponding to the pIRmcs3-LiUBC1-and the pIRmcs3-transfected promastigotes, show similar patterns. The expression levels of the LiUBC1 protein are similar throughout the growth curve in the case of the pIRmcs3-  The TEM experiments confirmed the presence of LiUBC1 in the nuclear membrane area, as well as the flagellar pocket and the kinetoplast.

Characterization of an L. infantum Stable LiUBC1 Knock-In Promastigote Cell Line
A knock-in cell line was generated for functional studies using the infective human outbreak strain (Section 2.1). Figure 7A shows the growth pattern of L. infantum parasites overexpressing LiUBC1. Both growth curves, corresponding to the pIRmcs3-LiUBC1and the pIRmcs3-transfected promastigotes, show similar patterns. The expression levels of the LiUBC1 protein are similar throughout the growth curve in the case of the pIRmcs3-transfected promastigotes ( Figure 7B). The transfected promastigotes overexpressing LiUBC1 showed a higher expression of LiUBC1 at the beginning of the growth curve, mostly in the first 24 h ( Figure 7C). We validated the results shown in Figure 7 using iTRAQ. We analyzed the proteome pattern of the transfected parasite and the possible modifications induced by this noticeable increase in the ubiquitin-conjugating E2 enzyme on other related enzymes of the ubiquitin-conjugating system. Thus, we used the following ubiquitin enzymes (putative) as a reference: the E1-activating E1 enzyme, the ubiquitin-conjugating E2 enzyme, and the ubiquitin-conjugating E3 enzyme, corresponding to the LinJ.23.0710, LinJ.35.1310, and LinJ.13.1320 genes, respectively. Table 3 shows the levels of the relative abundance of these proteins with respect to LiUBC1. The overexpression of LiUBC1 does not affect the pro-duction of these similar conjugating and activating enzymes. Thus, proteins belonging to the ubiquitin proteasome system are not generally overexpressed, while those of ubiquitin modification systems are, specifically, LiUBC1.

Infectivity of LiUBC1 Knock-In Promastigotes in U937 Cells In Vitro
In vitro infection experiments were performed using the PMA stimulated U937 cell line model. Figure 8 shows the percentage of U937 cells infected by L. infantum LiUBC1 knock-in promastigotes (red bars) and pIRmcs3 transfected promastigotes (blue bars). Slight differences are observed ( Figure 8A). A statistically significant (t-test, p < 0.05) 27% average increase in the number of amastigotes per infected cell was observed in the 24 h post-infection timeframe ( Figure 8B), i.e., early infection stage.

Infectivity of LiUBC1 Knock-In Promastigotes in U937 Cells In Vitro
In vitro infection experiments were performed using the PMA stimulated U937 cell line model. Figure 8 shows the percentage of U937 cells infected by L. infantum LiUBC1 knock-in promastigotes (red bars) and pIRmcs3 transfected promastigotes (blue bars). Slight differences are observed ( Figure 8A). A statistically significant (t-test, p < 0.05) 27% average increase in the number of amastigotes per infected cell was observed in the 24 h post-infection timeframe ( Figure 8B), i.e., early infection stage.
An increase of the presence of intracellular amastigotes in U937 cells infected with promastigotes overexpressing the LiUBC1 gene in the early phases ( Figure 8) suggests a probable role of LiUBC1 in the parasite infection process.

Protective Effect of the LiUBC1 Recombinant Protein in M. auratus against L. infantum Challenge
The possible role of the LiUBC1 recombinant protein as a protective molecule against L. infantum infection was evaluated herein. The investigation was performed using the M. auratus model in a challenge experiment with L. infantum promastigotes. Four months post-infection, 50% of the control animals showed the presence of L. infantum amastigotes in the spleen, whereas animals vaccinated with the recombinant LiUBC1 protein did not (Table 4).  (Table 4).
Additionally, we analyzed the IgG anti-SLA and IFN-γ production. As can be seen in Figure 9A, the production of IgG anti-SLA was increased in the animals inoculated with LiUBC1 recombinant protein. Additionally, the IFN-γ production in the spleen cells of the animals inoculated with the recombinant protein showed a statistically significant increase ( Figure 9B).  Additionally, we analyzed the IgG anti-SLA and IFN- production. As can be seen in Figure 9A, the production of IgG anti-SLA was increased in the animals inoculated with LiUBC1 recombinant protein. Additionally, the IFN- production in the spleen cells of the animals inoculated with the recombinant protein showed a statistically significant increase ( Figure 9B). The results of the parasite burden, the IgG anti-SLA, and the IFN- production suggest the possible role of the LiUBC1 recombinant protein as a protective molecule against L. infantum infection.

Discussion
LiUBC1 is a protein involved in degradation and regulation mechanisms essential for the adaptation of the cell to distinct physiological conditions, helping to maintain cell homeostasis. Processes such as DNA repair or protein synthesis are regulated by ubiquitin [34]. This may be relevant for Leishmania parasites, which have to adapt to the different Figure 9. Evaluation of the specific immune response in M. auratus: (a) humoral immunity (IgG anti-SLA); (b) cellular immunity. We used qPCR for the quantification of the relative IFN-γ mRNA levels in splenocytes (IFN-γ). Statistical inference was performed using the Student's t-test (** p < 0.01).
The results of the parasite burden, the IgG anti-SLA, and the IFN-γ production suggest the possible role of the LiUBC1 recombinant protein as a protective molecule against L. infantum infection.

Discussion
LiUBC1 is a protein involved in degradation and regulation mechanisms essential for the adaptation of the cell to distinct physiological conditions, helping to maintain cell homeostasis. Processes such as DNA repair or protein synthesis are regulated by ubiquitin [34]. This may be relevant for Leishmania parasites, which have to adapt to the different environments along its life cycle in two different hosts, from the vector gut to the macrophage parasitophorous vacuole. We characterized the protein in a lower eukaryote, and it displays structural features similar to other members of the Leishmania genus and other lower eukaryotes, such as S. cerevisiae, but dissimilar to other members of the Trypanosomatidae family. The protein structure of LiUBC1, predicted based on the data obtained from S. cerevisiae and H. sapiens orthologs, showed a C-terminal end very similar to that observed in the yeast protein, which is not present in the human enzyme ( Figure 2). This fact suggests that the LiUBC1 enzyme may be a good vaccine or drug target candidate, whose blockage through the C-terminus would only affect the parasite's survival and not their host, providing a good pharmacological advantage for a possible treatment for humans infected with L. infantum. This increases the interest in unraveling the characterization of LiUBC1 from L. infantum, responsible for VL in the Mediterranean basin, China, and South America.
The purified recombinant LiUBC1 protein displayed a complex pattern with the presence of two main bands (Figure 3) that showed molecular weights corresponding to the monomer and the dimer, as confirmed by LC-MS/MS (Table 2). A specific antibody was obtained to study the LiUBC1 expression pattern and subcellular localization throughout the promastigote growth curve.
The immunofluorescence experiments showed localization in the nuclear area and close to the base of the parasite's flagellum. TEM analysis confirmed the presence of LiUBC1 in the nuclear membrane zone, as well as the kinetoplast and the flagellar pocket area. This localization corresponds to the assigned functions of the protein. The protein is only located near protein-synthesis and -modification areas, and not throughout the entire cytoplasm, in a parasite in which autophagy and endosome sorting are essential in metacyclogenesis [56]. In Leishmania, the perinuclear zone has been shown to control post-translational regulation [57], which would require the presence of ubiquitin activity. The internalization of exogenous polypeptides and proteins through the flagellar pocket (cytostome) may be related to ubiquitin targeting for degradation [37]. The generation of knock-in transfectants helps us study the possible relation of the ubiquitin-conjugating E2 protein with the parasite's infectivity. LiUBC1-transfected promastigotes showed a clear predominance of the monomer form over the aggregate. The LiUBC1 knock-in promastigote line reached the highest levels of protein expression at the beginning of the growth curve (Figure 7). This high amount of the monomeric form rapidly decayed at 72 h, reaching normal levels. The iTRAQ-based proteome analysis of the LiUBC1 knock-in promastigotes revealed that LiUBC1 overexpression did not affect the intracellular levels of any other identified protein. Overexpression was confirmed in the pIRmcs3-LiUBC1 stable knock-in promastigote cell line. Growth and morphology were not altered in this cell line and, according to iTRAQ analysis, constitutively induced LiUBC1 expression did not lead to pleiotropic effects consisting of substantial changes in the proteome. The cell line overexpressing the LiUBC1 gene was more infective to PMA-stimulated U937 cells during the 24 h post-infection, as seen by the increase in intracellular amastigotes in the infected cells. The increase in infectivity at an early stage induced by LiUBC1 gene overexpression ( Figure 8) suggests that immunization with this molecule may protect the host against infection. A protection experiment against a challenge with L. infantum in the M. auratus model was performed. In this experiment, the humoral and cellular immune response against the parasite was increased in the vaccinated animals. Furthermore, inoculation with the protein elicited a moderate, although clear, protection against parasite infection. These results agree with those obtained using a closely related E2 enzyme from L. donovani, which showed its ability to protect against infection in the mouse model [58]. Recent data indicate the presence of specific antibodies against the E2 protein in human serum obtained from VL patients [59].
Taken as a whole, these results are encouraging and indicate that further studies should be carried out on the role of the ubiquitin-conjugating E2 enzyme in the key functions of L. infantum metabolism, especially in the regulation processes. The unraveling of the role of the enzyme as a response to the parasite's environmental conditions along its biological cycle would provide basic knowledge on the regulation mechanisms of this protozoa. This knowledge would also be useful for the development of effective drugs or vaccines against the parasite.

Conclusions
LiUBC1 is similar (>80%) to orthologs from the Leishmania spp., but not to the mammalian orthologs included in the comparison (<40%), which lack the C-terminal domain found in Leishmania spp. According to the Western blot experiment, the LiUBC1 gene is expressed throughout L. infantum promastigote growth, including highly infective PNApromastigotes. The Syrian hamster protection experiment against challenge with L. infantum revealed a partial reduction in parasite burden and IFN-γ mRNA levels in the spleen. Therefore, LiUBC1 is a vaccine candidate that confers partial protection in Syrian hamsters against L. infantum infection.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/vaccines10020231/s1, Figure S1: Comparative alignment of the primary structure of the LiUBC1 protein, Figure S2: Raw Western Blot Images.