AcetyLeish: acetylation-driven control of stage differentiation and virulence in Leishmania mexicana

AcetyLeish: acetylation-driven control of stage differentiation and virulence in Leishmania mexicana | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article AcetyLeish: acetylation-driven control of stage differentiation and virulence in Leishmania mexicana Nilmar Moretti, Suellen Rodrigues Maran, Anelise Gonçalves Marino, and 19 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7520632/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Protein acetylation regulates essential processes across eukaryotes. In trypanosomatids, stage-specific acetylation suggests roles in parasite differentiation. Here, we functionally characterized zinc-dependent lysine deacetylases (DAC1, DAC3, DAC4, and DAC5) in Leishmania mexicana . CRISPR-Cas9-mediated disruption revealed that DAC1 and DAC3 are essential for procyclics, while DAC4 and DAC5 are dispensable. DAC1 and DAC5 are localized in the cytoplasm, and DAC3 and DAC4 in the nucleus. Functional analysis implicates DAC1, DAC3, and DAC5 in procyclic proliferation, whereas DAC1 and DAC5 drive promastigote-to-metacyclic differentiation. DAC5 was required for metacyclogenesis in the sand flies, the promastigote–amastigote transition, and amastigote intracellular replication. Notably, DAC5-null parasites failed to induce lesions in mice, displaying an attenuated phenotype. Proteomic profiling uncovered altered acetylation patterns in DAC mutants, linking DAC5 to cytoskeleton regulation and cell cycle control. These findings identify acetylation as a central regulator of Leishmania stage differentiation and highlight DAC5 as a key factor in parasite virulence. Biological sciences/Microbiology/Parasitology/Parasite host response Biological sciences/Microbiology/Parasitology/Parasite biology Leishmania acetylation acetylome lysine deacetylases parasite differentiation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Leishmaniases are a group of neglected tropical diseases caused by Leishmania species, present as flagellated promastigotes in the sandfly vector and as intracellular amastigotes in mammalian host phagocytes. The disease manifests in various clinical forms, including cutaneous and visceral leishmaniasis. Approximately 1 billion people reside in areas at risk of infection. In recent decades, the global incidence of leishmaniases has surged, with an estimated 1 million new cases and over 20,000 deaths annually 1 . During its digenetic life cycle, as the parasite transitions between the invertebrate and vertebrate hosts, it must rapidly adjust to altered environmental conditions. These adaptations involve rapid changes in gene expression, metabolism, and morphology, and are essential for Leishmania survival 2 , 3 Protein post-translational modifications (PTMs) are efficient mechanisms that enable cells to rapidly adapt to changing environments. While protein phosphorylation has long been recognized as a key regulator of protein function 4 , recent advancements in proteomic techniques have highlighted the significance of other PTMs, such as lysine/arginine methylation and lysine acetylation (Kac), which occurs in both histone and non-histone proteins 5 – 8 Kac involves adding an acetyl group to the ε-amino group of lysine residues, neutralizing its positive charge. This modification can alter protein function by affecting enzyme activity, protein-protein interactions, and protein subcellular localization, playing a crucial role in cellular adaptation to environmental changes 7 , 8 . We were pioneers in characterizing the first acetylome, a repertoire of lysine-acetylated proteins, of a trypanosomatid 9 , validated later by others 10 . Reduced levels of Kac were observed in glycolytic enzymes of Trypanosoma brucei bloodstream forms, which relies on glycolysis for ATP generation, compared to procyclic forms, which utilize amino acids as an energy source 9 . In a recent study, we confirmed the regulatory role of acetylation in T. brucei aldolase function, demonstrating that acetylation of residue K157 inhibits its enzymatic activity 11 . In Trypanosoma cruzi epimastigotes, we identified acetylation of several antioxidant enzymes, including the crucial defense enzyme superoxide dismutase A (TcSODA). Using various approaches, we confirmed that acetylation regulates TcSODA activity, contributing to the parasite's oxidative stress response 12 . These findings support the idea that Kac likely has a pivotal role in regulating cellular processes involved in Leishmania stage differentiation. The addition, removal, and recognition of acetyl groups on lysines are coordinated by lysine acetyltransferases (KATs), lysine deacetylases (KDACs), and bromodomain-containing proteins (BDPs), respectively 13 . Trypanosomatid genomes typically harbor seven KDAC genes, including four DACs and three sirtuins 14 . All KDACs from T. brucei and T. cruzi have been characterized and are involved in various cellular processes, such as parasite differentiation, DNA repair, cell cycle regulation, and infectivity 15 – 22 . For instance, T. cruzi DAC1 and 2 are essential for cell cycle progression and proliferation 19 , while T. brucei TbDAC1 and TbDAC3, are essential for evading the host immune response 16 , 17 . In contrast, TcDAC4 and TbDAC4 are not essential, and their absence affects parasite morphology and G2/M progression, respectively 17 , 22 . In Leishmania , the three sirtuins (LmSir2rp1-3) have been characterized, with LmSir2rp1 and LmSir2rp2 identified as essential for parasite survival 20 , 21 . LmSir2rp1 is cytoplasmic and primarily regulates tubulin acetylation levels 21 , while LmSir2rp2 and rp3 are mitochondrial and are linked to NAD + metabolism 20 . In contrast, the roles of DAC family members (1, 3, 4, and 5) have been underexplored in Leishmania . In this study, we systematically characterized the function of L. mexicana DACs in parasite stage differentiation and virulence. Using CRISPR/Cas9, we generated DAC1/3 and DAC4/5, hemi and null mutants, respectively, to assess their impact in survival, differentiation, and infection success in vitro and in vivo in both Leishmania hosts. Additionally, we employed multi-omics tools to identify proteins and pathways regulated by DACs, which may contribute to parasite adaptation. Methods Ethics statement This research project and all the proposed procedures were submitted to and approved by the Ethics Committee on the Use of Animals and the Research Ethics Committee of the Ethics Committee of the Federal University of São Paulo under registration numbers 9407210519/2019 and 9869091118/2019, respectively and by the René Rachou Institute Ethics Committee under license number LW38-23. In silico L. mexicana DACs analyses The amino acid sequences of DAC1, 3, 4, and 5 in L. mexicana , T. cruzi , T. brucei , and humans were retrieved from TritrypDB ( https://tritrypdb.org/tritrypdb/app ) and UniProt ( https://www.uniprot.org ). The HDCA domains for each protein were determined using the InterPro Classification tool ( https://www.ebi.ac.uk/interpro/ ), and amino acid sequences alignment and phylogenetic trees were done using the Geneious software. The predicted three-dimensional models of L. mexicana DACs were generated using AlphaFold2 23 , and all structural analyses were performed using Pymol 3.12 software ( https://pymol.org/ ). Final images were generated with Adobe Photoshop 2025 or Adobe Illustrator 2025. Parasite axenic maintenance and differentiation assays Promastigote-stage L. mexicana (MHOM/GT/2001/U11032) T7/Cas9 cells were cultured in M199 medium supplemented with 4.62 mM NaHCO 3 , 40 mM HEPES, 0.1 mM adenine, 0.0001% biotin, 10% heat-inactivated fetal bovine serum (FBS), and 50 µg/mL hygromycin B at 26°C. Knockout mutant parasites were cultured in the same way in the presence of specific antibiotics (20 µg/mL blasticidin and/or 50 µg/mL puromycin. For fluorescently tagged and add-back cells, 20 µg/mL blasticidin or 20 µg/mL G418, were added, respectively. To assess promastigote growth, 1 × 10⁵ cells/mL in the exponential phase were inoculated into M199 supplemented medium and incubated at 26°C. Cell proliferation was monitored daily for five days using a Muse Cell Analyzer (Merck Millipore). Metacyclogenesis was induced by inoculating 1.5 × 10⁶ procyclic cells/mL into Grace's medium (Sigma-Aldrich), pH 5.5, supplemented with 4.62 mM NaHCO₃, 1× BME vitamins (Sigma-Aldrich), 10% FBS, and penicillin/streptomycin. The parasites were incubated at 26°C for seven days. Metacyclic parasites were purified using a Percoll gradient. Briefly, parasites were collected by centrifugation at 3,000 × g for 10 min at room temperature, resuspended in 3 mL of Grace’s medium, and layered onto a 10–100% Percoll gradient. The gradient was centrifuged at 1,300 × g for 10 min at room temperature. The 10% fraction, containing metacyclic parasites, was collected, washed twice with PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4 and 2 mM KH2PO4, pH 7.4), and counted using a Neubauer chamber. The metacyclogenesis rate was calculated as the ratio of metacyclic parasites to the initial number of parasites in the Percoll gradient. Promastigote-to-axenic amastigote differentiation was induced by incubating 1 × 10⁶ procyclic cells/mL in Grace's medium, pH 5.5, supplemented with 4.62 mM NaHCO₃, 1× BME vitamins, and 10% FBS at 33°C and 5% CO₂ for four days. Axenic amastigote to procyclic differentiation was induced by inoculating 1 × 10⁶ cells/mL into an M199 supplemented medium. Parasite growth was monitored daily for four days using a Neubauer chamber. Generation of cell lines We employed the CRISPR/Cas9 method described in 24 to select DAC knockout and fluorescent-tagged L. mexicana mutants. For each DAC gene, specific primers for homologous recombination fragment (HR) and sgRNA synthesis were designed using the LeishGEdit tool ( http://www.leishgedit.net ) (primer sequences provided in Supplementary Table 1). The HR and sgRNA fragments were amplified by PCR using the conditions described in [23], and all PCR products were confirmed by 1% agarose gel electrophoresis before transfection. To obtain the constructs used to generate the add-back cells the full-length sequences corresponding to L. mexicana DAC1, 3, 4 and 5, were obtained via PCR with specific oligonucleotides (see Supplementary Table 1), using as a template the parasite’s genomic DNA. The fragments of each DAC were further cloned in the pSPa-Neo-a plasmid using the BamH I and Hind III restriction enzyme sites introduced in the oligonucleotides. Also, we added the V5-tag sequence at the N-terminal region of each DAC sequence for further analysis. For transfection, 1 × 10⁷ exponentially growing T7/Cas9 cells were collected by centrifugation (2,500 × g, 5 min) and resuspended in transfection cytomix buffer (66.7 mM Na₂HPO₄, 23.3 mM NaH₂PO₄, 5 mM KCl, 50 mM HEPES, pH 7.3, 150 µM CaCl₂). The appropriate sgRNA and HR fragments or the pSPa-Neo-a constructs for each DAC were added to the cell suspension, followed by transfection using the Amaxa Nucleofector™ IIb (Lonza). After 24h, cultures were supplemented with specific antibiotics for selection. Selected transfectants were later cloned by serial dilution in 96-well plates. Confirmation of mutant parasites To confirm the selection of DAC knockout mutants, PCR was performed using genomic DNA (gDNA) as a template. Primers were designed to target the 5' UTR and internal regions of each DAC gene (Supplementary Table 2). Additionally, primers specific for resistance markers were included in the analysis. Quantitative PCR (qPCR) was also employed to quantify gene copy number and mRNA expression levels of specific DACs in knockout mutants. For gene copy number analysis, gDNA was used as a template with SYBR Green and specific DAC primers. Reactions were performed in a 20 µL volume containing 10 pmol of each primer, 1X SYBR Green, and 1 µL of gDNA (100 ng/µL). PCR conditions were: 95° C for 5 min, followed by 45 cycles of 95°C for 15 s, 60°C for 15 s, and 72°C for 30 s, with a final extension at 72°C for 5 min. Gene copy number was calculated using the 2-ΔΔCt method, with GAPDH as the endogenous control. For mRNA expression analysis, total RNA was extracted from parental and knockout mutant parasites using TRIzol and treated with DNase. cDNA was synthesized using a High-Capacity cDNA Reverse Transcription Kit with a spliced leader (SL) primer to specifically convert mature mRNAs. qPCR was performed using the same conditions as above. All primers used for mutant genotyping are listed in Supplementary Table 3. Fluorescent-tagged DAC mutants were confirmed using flow cytometry and Western blot. For flow cytometry analysis, 1 × 10⁶ parasites from parental and fluorescent-tagged cell lines were collected by centrifugation (2,500 × g , 2 min) and resuspended in 1 mL of 1× PBS. Cell fluorescence was measured using an Accuri™ flow cytometer (BD Biosciences) with the FL-1 filter. Data were analyzed using BD Accuri™ C6 v1.0.264.21 software. For Western blot analysis, 1 × 10⁷ cells from each condition were collected and lysed in 4× Laemmli sample buffer. Proteins were separated by 10% SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was blocked with 1× PBS containing 0.1% Tween-20 and 5% non-fat milk for 1h at room temperature. Subsequently, the membrane was incubated overnight at 4°C with an anti-c-Myc tag antibody (Merck, #05-724) diluted 1:3,000 in blocking solution. After washing with 1× PBS containing 0.1% Tween-20, the membrane was incubated with an IRDye® 800CW Goat anti-Mouse IgG Secondary Antibody (1:10,000) for 1h at room temperature. Following additional washes, the membrane was imaged using an Odyssey CLx Imaging System (LI-COR). The add-back cells were confirmed by qPCR with specific oligonucleotides or Western blot with anti-V5, following the approaches described above. Confocal microscopy analysis To determine the subcellular localization of each fluorescently tagged DAC protein, procyclic, metacyclic, and amastigote stages of the mutant parasites were analyzed by fluorescence microscopy. Briefly, 1 × 10⁵ cells of each parasite stage were collected, washed once with 1× PBS, and incubated on a poly-L-lysine-coated slide for 10 min at room temperature. Cells were then fixed with 1% paraformaldehyde for 15 min at room temperature and stained with 10 µM DAPI for 10 min. After washing with 1× PBS, the slides were mounted with glycerol-p-phenylenediamine. Images were acquired using a TCS SP5 II Tandem Scanner microscope (Leica) and processed with Imaris v6 software. In vitro macrophage and in vivo mice infection assays Bone marrow-derived macrophages (BMDMs) were obtained from susceptible BALB/c mice. Briefly, tibias and femurs were aseptically removed, and bone marrow cells were flushed with RPMI 1640 medium supplemented with 10% FBS. Cells were centrifuged (2,500 × g , 5 min), resuspended in ACK lysis buffer (155 mM NH₄Cl, 10 mM KHCO₃, 100 µM EDTA), and incubated at room temperature for 5 min to lyse red blood cells. To obtain BMDMs cells, the population was seeded on glass coverslips and incubated in a differentiation medium (RPMI supplemented with 10% inactivated horse serum, 30% L929 cell conditioned medium, 100 U/ml penicillin and 100 µg/ml streptomycin) at 37°C with 5% CO 2 . After 5 days, BMDMs were maintained in RPMI 1640 supplemented with 10% FBS (R10 medium) for 24h before infection. Axenic amastigotes from parental and knockout mutant parasites were used to infect BMDMs at a ratio of 1 parasite per macrophage. After a 2h incubation period, cells were washed to remove extracellular parasites and incubated for 12, 24, or 48h. To quantify intracellular amastigote proliferation, cells were stained with Giemsa. Images were acquired using a Nikon Eclipse Ti-U microscope and analyzed using ImageJ software to count the number of cells and intracellular amastigotes to obtain the macrophage infection rate and the number of amastigotes per infected macrophage. In vivo infection assays were performed using female 8–10 weeks old BALB/c mice at the Instituto René Rachou - Fiocruz, Belo Horizonte, Minas Gerais, Brazil. Briefly, 1 × 10⁷ stationary-phase parasites from parental (T7/Cas9) and DAC knockout mutants were injected into the right footpads of each mouse with a final volume of 30 µL (n = 6 per group), and footpad swelling was monitored daily for 60 days using a caliper (Mitutoyo Corp., Japan). For the challenge infection assays, five weeks after primary infection with DAC5 −/− cells, parental parasites were injected into the left footpads (1 × 10 6 stationary phase parasites) and infection was monitored as above for 48 days. Data were analyzed using GraphPad Prism software to compare the parental groups with DAC mutant groups. In vivo Lutzomyia longipalpis infection assays Promastigote forms of parental, DACs knockout mutant, and add-back cells were added to heat-inactivated heparinized mouse blood at a final concentration of 5 × 10⁶ parasites/mL. Female L. longipalpis sand flies were fed on this blood meal through a chick membrane in an artificial feeder. Approximately 100 female sandflies were used per group. On the following day, the fed females were transferred to a new container. To monitor infection rates, approximately 10–15 fed females were dissected on days 1, 4, and 8 post-infection. Midgut contents were extracted in PBS using a disposable plastic pestle, and the total number of parasites in each midgut was counted in a hemocytometer and the frequency of differentiating promastigotes, including procyclics, nectomonads, and metacyclics, determined by morphology as described in 25 . Cell-cycle progression analyses EdU incorporation assays and ‘click’ chemistry reaction Exponentially growing L. mexicana promastigotes were incubated with 100 µM 5-ethynyl-2’-deoxyuridine (EdU) for different time periods (ranging from 30 min to 1.15h) at 26°C. The parasites were then harvested (~ 1 x 10 7 cells) by centrifugation at 2,500 g for 5 min, washed three times in PBS, fixed for 20 min with 1% sterile paraformaldehyde diluted in PBS, washed twice, and re-suspended in 200 µL of PBS. Then, 20 µL of the cell suspension was loaded onto poly-L-lysine pre-treated microscope slides and washed three times with 3% BSA diluted in PBS. To detect incorporated EdU, we used the Click-iT EdU detection solution for 45 min protected from light. This solution is composed of 1 mM CuSO 4 , 10 µM Alexa Fluor Azide 488, and 100 mM C 6 H 8 O 6 , diluted in distilled water. Next, the cells were washed five times with PBS. Vectashield mounting medium (Vector) containing DAPI was used as an anti-fade mounting solution and to stain the organelles containing DNA. Images were acquired using the fluorescent microscope (Nikon Eclipse 80i). The percentage of parasites in cytokinesis and mitosis was calculated for each group relative to the total number of DAPI-positive parasites. Cell cycle analysis DAPI-stained exponentially growing promastigotes were examined under a fluorescent microscope (Nikon) to observe the profile of the nuclei (N) and kinetoplast (K). The profile 2N2K was used to estimate the percentage of cells in cytokinesis (C). The profile containing one nucleus being divided was used to estimate the percentage of performing mitosis (M). To estimate the G2 + M phases length, we added EdU in the medium containing exponentially growing promastigotes and collected samples every 15 min, proceeding with the ‘click’ chemistry reaction, until a parasite containing two EdU-labeled nuclei (2N2K or 2N1K) was observed (this time corresponds to the length of G2 + M phases). To monitor DNA replication, we measured the proportion of cells EdU-labeled after 1h EdU pulse. The cell cycle phases duration were estimated after all these parameters (together with doubling time extracted from the procyclic growth curves) using the online software CeCyD, available at https://cecyd.vital.butantan.gov.br/ 26 . RNAseq experiments RNA samples and library preparation Exponentially growing procyclic parasites of parental and DAC knockout mutants were collected in quadruplicate and triplicate, respectively. Total RNA was extracted using the RNeasy Mini Kit, followed by DNase I treatment. RNA quality and concentration were assessed using Qubit™ RNA IQ Assay Kits and agarose gel electrophoresis. Paired-end sequencing libraries were prepared from total RNA using the Stranded mRNA Prep Kit by Illumina. Finally, the libraries were sequenced on an Illumina NovaSeq™ 6000 sequencer using the 100-cycle SP v1.5 kit by Illumina. RNAseq analysis The quality of reads was assessed using the FastQC tool 27 . Each sample library was mapped against the L. mexicana genome (strain MHOM/GT/2001/U1103) with the STAR mapping tool 28 . The count data for each sample was then structured as a matrix in the R environment, and differential expression analysis was performed using the DESeq2 package 29 . We considered only genes represented by at least five reads across all replicates (parental or knockout strains). The gene expression ratio between the parental and the modified strain was calculated using a base-2 logarithmic transformation (log2 fold change). Genes with an absolute log2 fold change ≥0.6 (positive for upregulated genes and negative for downregulated genes) and corrected p -values ≤0.05 were considered differentially expressed genes (DEGs). Gene enrichment analysis Gene enrichment analysis was conducted using the TriTrypDB database 30 . The selected DEGs were used for an ID search in the Gene Ontology (GO) database for Biological Process (BP) and Cellular Component (CC) enrichment, filtering for curated evidence using Benjamini method and a p -value cutoff of ≤0.05. Metabolic pathway enrichment was performed using the KEGG database 31 with the same p -value cutoff of ≤0.05. Acetylome experiments Protein Sample Preparation Exponentially growing procyclic parasites of parental and DAC knockout mutants were collected in triplicate and subjected to phenol protein extraction as described in 10 . Parasites were resuspended in phenol extraction buffer (0.7 M sucrose, 0.1 M KCl, 0.5 M Tris-HCl, pH 7.5, 1% TritonX-100, 10 mM DTT) supplemented with 1% protease inhibitors, 3 µM of trichostatin A, 50 mM nicotinamide, 2 mM EDTA, and 2% b-mercaptoethanol. The mixture was sonicated, phenol-extracted, and proteins from the supernatant were precipitated overnight with 0.1 M ammonium acetate/methanol, washed with acetone and methanol, and dissolved in 8 M urea. Trypsin Digestion and Affinity Enrichment The protein solution was reduced with 5 mM DTT for 30 min at 56°C and alkylated with 11 mM iodoacetamide for 15 min at room temperature in darkness. The protein sample was then diluted by adding 200 mM TEAB to a urea concentration less than 2 M. Finally, trypsin was added at a 1:50 trypsin-to-protein mass ratio for the first digestion overnight and a 1:100 trypsin-to-protein mass ratio for a second 4 h-digestion. Finally, the peptides were desalted by the Strata X SPE column. To enrich the acetylated peptides, tryptic peptides dissolved in NETN buffer (100 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl, 0.5% NP-40, pH 8.0) were incubated with beads conjugated with anti-acetyllysine antibodies (PTM Bio) at 4°C overnight with gentle shaking. Then the beads were washed for four times with NETN buffer and twice with H 2 O 2 . The bound peptides were eluted from the beads with 0.1% trifluoroacetic acid. Finally, the eluted fractions were combined and vacuum-dried. For LC-MS/MS analysis, the resulting peptides were desalted with C18 ZipTips. LC-MS/MS Analysis Tryptic peptides were dissolved in solvent A and loaded onto a custom-made reversed-phase analytical column (25 cm length, 100 µm ID). Mobile phases A (0.1% formic acid, 2% acetonitrile in water) and B (0.1% formic acid in acetonitrile) were used to separate peptides with the following gradient: 0–40 min, 7–24% B; 40–52 min, 24–32% B; 52–56 min, 32–80% B; 56–60 min, 80% B, at a flow rate of 450 nL/min on a NanoElute UHPLC system. Peptides were analyzed by capillary source-timsTOF Pro2 mass spectrometry. The electrospray voltage was set to 1.5 kV. Precursors and fragments were detected by the TOF detector, with an MS/MS scan range of 100–1700 m/z. The timsTOF Pro was operated in PASEF mode, selecting precursors with charge states 0–5 for fragmentation. Ten PASEF-MS/MS scans were acquired per cycle, with a dynamic exclusion time of 24 s. Database Processing The resulting MS/MS data were processed using MaxQuant (v.1.6.15.0). Peptides were searched against the UniProtKB L. mexicana database, concatenated with a reverse decoy and contaminant database. Trypsin/P was specified as the cleavage enzyme, allowing up to two missed cleavages. Minimum peptide length was set to 7, and a maximum of five modifications per peptide was allowed. The mass tolerance for precursor ions was set to 20 ppm in both the first and main searches, and the mass tolerance for fragment ions was set to 20 ppm. Carbamidomethylation on cysteine was specified as a fixed modification. Acetylation on protein N-terminus, methionine oxidation, and lysine acetylation were specified as variable modifications. The false discovery rate (FDR) for proteins, peptides, and PSMs was adjusted to < 1%. Label-free quantification (LFQ) was selected for quantification. Data availability The raw sequencing files (fastq) for all RNA samples from T7/Cas9 and DAC mutants have been deposited in the Sequence Read Archive (SRA) under accession code: PRJNA1062791 (for T7/Cas) and PRJNA1312851 (for DACs). The processed RNAseq data are provided in the Supplementary Table 4. The raw proteomic files have been deposit in ProteomeXchange. The processed acetylome data is provided in the Supplementary Tables 5,6 and 7. Results L. mexicana genome encodes four zinc-dependent lysine deacetylase orthologs The L. mexicana genome contains four genes encoding zinc-dependent lysine deacetylases, a feature shared with other Trypanosomatids species such as T. cruzi and T. brucei 14 . Phylogenetic analysis revealed that L. mexicana DAC1 and DAC2 cluster closely with human HDAC class I (HDAC1-3 and 8), while DAC3 and 4 could not be definitively assigned to a specific family but exhibit greater similarity to human HDAC class IIa (HDAC4, 5, 7, and 9) (Fig. 1 A). In contrast to human class IIa HDACs, neither DAC3 nor DAC4 possesses N- or C-terminal extensions. However, like T. cruzi DAC3 19 , L. mexicana DAC3 contains insertions within its catalytic domain (Fig. 1 B). Comparative analysis of the catalytic domain amino acid sequences between L. mexicana and human HDACs revealed low identity (< 49%) across all Leishmania DACs, with DAC4 and DAC5 exhibiting the highest divergence (~ 41%) (Supplementary Data 1A). Further sequence analysis of L. mexicana HDAC domains in comparison to their T. brucei and T. cruzi orthologs demonstrated high identity for DAC1, DAC33, and DAC4 (53–73%) (Fig. 1 C). In contrast, L. mexicana DAC2 shared very low identity (< 26%) with its trypanosome orthologs (Fig. 1 C), leading us to rename this enzyme as DAC5. This significant divergence was also evident in phylogenetic analysis of Leishmania and trypanosome DACs (Supplementary Data 1B). Moreover, multiple sequence alignments of L. mexicana DACs with human HDAC classes I and II revealed conservation of residues critical for zinc binding and the catalytic tyrosine residue (Fig. 1 D and Supplementary Data 1C). Despite the phylogenetic proximity of DAC3 and 4 to human HDAC class IIa, the characteristic substitution of the catalytic tyrosine (Y) to histidine (H) is absent in these parasite enzymes, which retain a tyrosine residue (Supplementary Data 1C). To gain insights in L. mexicana DACs, we compared their predicted protein structures to those of human HDAC1 and T. cruzi DAC2. Figure 1 D illustrates a high degree of structural conservation among these enzymes, including the catalytic site region. L. mexicana DACs subcellular localization and expression in different parasite stages To functionally characterize L. mexicana DACs, we generated parasite cell lines expressing DAC1, DAC3, DAC4, or DAC5, each endogenous fused to mNeonGreen and a 3xMyc tags. To confirm successful transfection, we performed flow cytometry analysis to measure mNeonGreen fluorescence in procyclic forms of parental (Cas9) and transfected (DAC-tag) parasites. All transfected parasites exhibited higher fluorescence levels compared to their counterpart Cas9 control, confirming mNeonGreen expression (Supplementary Data 2A). Next, to verify the correct expression of DAC-tagged fusion proteins, Western blot analysis was performed using anti-c-Myc antibody. As expected, we detected the DAC-tagged proteins exclusively in the transfected parasites, with the expected molecular weights of 75, 93, 91, and 84 kDa for DAC1, DAC3, DAC4, and DAC5, respectively (Fig. 1 E). Using the endogenous DAC-tagged cell lines, we determined the subcellular localization of these enzymes in the major parasite stages: procyclic, metacyclic, and amastigote forms. We found that DAC1 and DAC5 are primarily localized to specific regions of the cytoplasm, while DAC3 and DAC4 are nuclear proteins in all parasite stages (Fig. 1FC and Supplementary Data 2B). Interestingly, although both DAC3 and DAC4 are nuclear, DAC3 is predominantly found in euchromatin regions, while DAC4 is primarily localized to the nucleolar region (Fig. 1 G). To investigate the indirect expression of DACs in procyclic, metacyclic, and axenic amastigote stages of L. mexicana , we evaluated mNeonGreen fluorescence levels by flow cytometry. We observed a decreasing expression for DAC3 and DAC4 from promastigote to metacyclic and axenic amastigote stages, while DAC1 and DAC5 showed a slight increase (Supplementary Data 2C). DAC1 and DAC3 are probably essential in L. mexicana promastigotes To further characterize L. mexicana DACs, we employed a CRISPR/Cas9-based loss-of-function approach to generate knockout mutant parasites. Following selection of transfected parasites, diagnostic PCR reactions were performed using total genomic DNA and oligonucleotide primers flanking the specific gene locus of each DAC (Supplementary Data 3A). PCR amplification products confirmed the generation of hemizygous knockout parasites (one disrupted allele) for DAC1 (DAC1 −/+ ) and DAC3 (DAC3 −/+ ), and homozygous knockout parasites (both alleles disrupted) for DAC4 (DAC4 −/− ) and DAC5 (DAC5 −/− ) (Supplementary Data 3B), suggesting that DAC1 and DAC3 may be essential for the promastigote stage of L. mexicana . To further validate these findings, we quantified gene copy number and mRNA expression levels of each DAC in the knockout mutants by qPCR, which confirmed the results obtained by PCR (Supplementary Data 3C and D). At least four rounds of transfection were performed for each DAC, yielding consistent results. In addition, we confirmed the re-expression of each DAC in add-back transfectants (DAC1-AB, DAC3-AB, DAC4-AB, and DAC5-AB) by Western blot, RT-qPCR, and immunofluorescence (Supplementary Data 4). DAC1, DAC3, and DAC5 influence L. mexicana promastigote multiplication Compared to Cas9 parental cells, DAC1 −/+ , DAC3 −/+ , and DAC5 −/− mutants exhibited a reduction in promastigote multiplication varying from 25 to 40% (Fig. 2 A). In contrast, DAC4 null mutants showed no significant effect (Fig. 2 A). This phenotype was partially restored in DAC5-AB but not in DAC1-AB or DAC3-AB parasites (Fig. 2 B). DAC1 and DAC5 affect in vitro and in vivo metacyclogenesis Next, we investigated the impact of DACs on in vitro and in vivo differentiation into metacyclics. To assess in vitro metacyclogenesis, we compared the number of non-metacyclic forms before differentiation to the number of metacyclic forms after the differentiation protocol. We found that DAC5 −/− and DAC1 −/+ parasites exhibited a nearly 80% and 40% reduction in metacyclogenesis, compared to the Cas9 parental cell line (Fig. 3 A, left panel). These phenotypes were rescued by re-expressing DAC1 and 5 in add-back parasites (Fig. 3 A, right panel). We did not observe any significant differences in metacyclogenesis of DAC3 −/+ and DAC4 −/− mutants. Given the observed effects on in vitro metacyclogenesis, we investigated the impact of DACs on metacyclogenesis during in vivo infection with L. longipalpis . Female L. longipalpis were fed with procyclic forms of parental and DAC mutant parasites. Metacyclogenesis was assessed by counting parasite stages (procyclic, nectomonads, and metacyclics) at 1, 4, and 8-days post-infection. As observed in vitro , DAC5 −/− parasites exhibited impaired differentiation to metacyclics compared to the parental cell line (Fig. 3 B). Interestingly, DAC3 −/+ showed a decreased differentiation rate compared to parental cells, while DAC1 −/+ had no significant effect on in vivo differentiation, contrasting with the in vitro observations (Fig. 3 A-B). Unfortunately, we did not observe a reversion of the DAC3 −/+ and DAC5 −/− phenotypes in the DAC3-AB and DAC5-AB cells (Supplementary Data 5). Detailed metacyclogenesis data for 1-, 4-, and 8-days post-infection of all DACs mutants are provided in Supplementary Data 5. DAC3 and DAC5 impair axenic amastigote multiplication but not differentiation To investigate the role of DACs in promastigote-to-axenic amastigote differentiation and multiplication, we performed differentiation kinetics of promastigote to axenic amastigotes using DAC mutants (Fig. 3 C). We found no significant effect of DACs on the differentiation process, as the frequency of promastigotes, intermediate axenic amastigotes, and axenic amastigote (axAMA) forms remained unchanged in the first 24 h comparing DACs mutant cells and Cas9 (Fig. 3 C). However, we observed a major deleterious effect on axAMA multiplication in DAC5 −/− , about a ~ 70% decrease, and DAC3 −/+ (about 50% reduction) compared to Cas9 cells after 24 h, while DAC1 −/+ and DAC4 −/− mutants showed no significant effect (Fig. 3 D). The observed deleterious phenotype in axAMA multiplication observed in DAC3 −/+ and DAC5 −/− was restored to similar levels of Cas9 in the DAC3-AB and DAC5-AB parasites (Fig. 3 D). Effect of DACs on in vitro macrophage infection Using BMDMs from BALB/c mice, we performed in vitro infection assays with axenic amastigotes from parental and DAC knockout mutant parasites. After infection, we monitored intracellular amastigote multiplication at 12-, 24-, and 48h post-infection. A significant reduction in the infection index was observed for all mutants, as measured by the number of intracellular parasites and infected cells (Fig. 4 A). The most pronounced reduction (~ 90%) was observed for DAC5 −/− after 48h of infection, while DAC1 −/+ , DAC3 −/+ , and DAC4 −/− exhibited reductions of approximately 50%, 70%, and 60%, respectively (Fig. 4 A). Consistent with these results, microscopy images revealed more intracellular amastigotes in macrophages infected with Cas9 than those infected with DAC mutants, particularly for DAC5 −/− (Fig. 4 B). The infection index was restored in DAC5-AB parasites (Fig. 4 C and Supplementary Data 6A). DAC5 impairs in vivo infection of L. mexicana Female BALB/c mice were infected with stationary-phase procyclic forms of parental and DACs knockout mutant parasites in the left hind footpad. Footpad thickness was measured daily for 48 days to monitor parasite-induced lesion development. We observed no significant lesion development in mice infected with DAC5 −/− parasites compared to the control group, indicating that disruption of the DAC5 gene attenuates parasite virulence (Fig. 4 D). No significant differences in lesion development were observed for DAC1 −/+ , DAC3 −/+ , and DAC4 −/− parasites compared to the parental strain (Fig. 4 D). These findings were further supported by parasite load quantification and representative photographs of BALB/c mice at day 48 post-infection (Supplementary Date 6B-C). No phenotype reversion was observed when we performed the same experiments comparing DAC5 −/− and DAC5-AB parasites (data not shown). Given the attenuated phenotype observed in DAC5 −/− parasites, we investigated their potential to confer protection against subsequent infection. To address this, BALB/c mice previously infected with DAC5 −/− parasites were re-challenged with Cas9 parasites in the contralateral paw, and footpad thickness was monitored for additional 60 days. No significant difference in footpad thickness was observed between mice previously infected with DAC5 −/− and re-challenged with Cas9 parasites compared to PBS-treated control mice up to 44 days post-infection (Fig. 4 E), suggesting a protective phenotype. DAC5 delays axenic amastigote-to-procyclic differentiation Axenic amastigotes from parental and DACs knockout mutants were inoculated into M199 medium at 28°C to induce differentiation into procyclic forms. After differentiation, we monitored parasite multiplication for four days and observed a similar growth phenotype (Fig. 5 A) to that seen in our previous procyclic multiplication experiments (Fig. 2 ). Markedly, we observed a significant delay in procyclic differentiation in DAC5 −/− compared to parental cells at 24h post-differentiation (Fig. 5 A), which prompted us to evaluate DAC5 −/− differentiation at a time series from 2 to 24h. We observed that compared to parental cells, most of DAC5 −/− still resemble as axenic amastigotes with a rounded morphology after 24h (Supplementary Data 7), indicating a delay in parasite differentiation. To link this phenotype to DAC5 function, we repeated the experiments using DAC5-AB parasites, and as expected, the axenic amastigote-procyclic differentiation phenotype was rescued, as detected in the number and morphology of procyclic cells after 24h (Fig. 5 B and C). DAC mutants express the procyclic, metacyclic and amastigote markers To confirm the expression of stage-specific markers in the parasite stages quantified in our phenotype differentiation experiments, we collected total RNA samples from each stage of DAC mutants and measured the expression of histone h4 (procyclic), sherp (metacyclic), and amastin (axenic amastigote) genes by RT-qPCR 2 . We successfully validated the stages of L. mexicana in our analysis, and no significant differences in the expression of these markers were observed between DAC mutants and the parental cell line (Supplementary Data 8). DACs effect on the transcriptome of procyclic stages To investigate the mechanisms regulated by DACs that may influence L. mexicana stage differentiation, we first performed RNA-Seq analyses comparing procyclic DAC knockout mutants with parental cells. We identified 516, 165, 641, and 381 upregulated genes in the DAC1, DAC3, DAC4, and DAC5 mutants, respectively, and 763, 437, 941, and 1935 downregulated genes in the same mutants (Supplementary Data 9A and Supplementary Table 4). Assessment of all differentially expressed genes across the DAC mutants revealed only 21 commonly upregulated and 40 commonly downregulated genes (Supplementary Data 9B and Supplementary Table 4). Comparing only cytosolic (DAC1 and DAC5) or nuclear (DAC3 and DAC4) DAC mutants we identified 36 commonly upregulated and 111 commonly downregulated genes between DAC1 and DAC5, and 159 commonly upregulated and 393 commonly downregulated genes between DAC3 and DAC4 (Supplementary Data 9C). Differentially expressed genes (DEGs) from the DAC mutant RNA-Seq data were subjected to functional enrichment analysis using the Gene Ontology (GO) terms "Cellular Compartment (CC)" and "Biological Process (BP)". A stringent p -value cutoff of 0.05 was applied to identify significantly enriched GO terms. Consequently, not all mutants yielded significant enrichment results. Analysis of the "Cellular Compartment (CC)" GO category revealed distinct enrichment patterns for each DAC mutant (Supplementary Data 10). In DAC1 −/+ , upregulated genes were enriched for membrane-related terms (membrane, intrinsic component of membrane, and integral component of membrane), while downregulated genes were enriched for general cellular component terms (cellular component, cellular anatomical entity, and intracellular anatomical structure). In DAC3 −/+ , upregulated genes were enriched for membrane, ribosome, and chromosome terms, whereas downregulated genes were enriched for cellular component and intracellular anatomical structure terms, as well as terms related to the nucleus and chromatin. Both upregulated and downregulated genes in DAC4 −/− were enriched for nucleus- and chromatin-related terms. In DAC5 −/− , upregulated genes were enriched for cell projection, cilium, plasma membrane-bounded cell projection, and cytoskeleton terms, while downregulated genes were enriched for ribonucleoprotein complex, ribosomal subunit, and mitochondrial matrix terms. Analyses of the "Biological Process (BP)" GO category revealed that in DAC1 −/+ , upregulated genes were enriched for autophagy-related terms (autophagy and process utilizing autophagy mechanisms), while downregulated genes were enriched for metabolic process terms (cellular nitrogen compound metabolic process, organic cyclic compound metabolic process, and heterocycle metabolic process) (Supplementary Data 10). In DAC3 −/+ , upregulated genes were enriched for biosynthetic process terms (biosynthetic process, organic substance biosynthetic process, and cellular biological process), and downregulated genes were enriched for catabolic process terms (catabolic process, organic substance catabolic process, and proteolysis). No significantly enriched terms were identified among upregulated genes in DAC4 −/− ; however, downregulated genes were enriched for metabolic processes related to nitrogen compounds, nucleobases, and RNA (cellular nitrogen compound metabolic process, nucleobase-containing compound metabolic process, and RNA metabolic process). In DAC5 −/− , upregulated genes were enriched for terms related to cellular movement and microtubules (movement of cell or subcellular component, microtubule-based process, and microtubule-based movement). Among the downregulated genes we found cellular component, cellular anatomical entity and intracellular anatomical structure as the most enriched terms in DAC5 mutants ((Supplementary Data 10). DAC mutants have altered protein acetylation levels To further investigate the mechanisms associated with DAC regulatory function, we performed proteomic analyses of lysine-acetylated enriched (Kac) and non-modified procyclic protein fractions, comparing DAC mutants to Cas9 control cells. We identified differentially expressed proteins (DEPs) with a log 2 fold change > 1.5: 210 upregulated and 262 downregulated in DAC1 −/+ , 230 upregulated and 151 downregulated in DAC3 −/+ , 120 upregulated and 183 downregulated in DAC4 −/− , and 630 upregulated and 624 downregulated in DAC5 −/− compared to Cas9 (Supplementary Data 11A-C and Supplementary Table 5). Comparison of DEPs across all mutants revealed only 19 commonly downregulated and 36 commonly upregulated proteins (Supplementary Data 11D). However, comparison of cytoplasmic DAC mutants (DAC1 −/+ vs. DAC5 −/− ) identified 119 commonly upregulated and 104 commonly downregulated proteins, while comparison of nuclear DAC mutants (DAC3 −/+ vs. DAC4 −/− ) identified 54 commonly downregulated and 130 commonly upregulated proteins (Supplementary Fig. 11D). We observed no preferential distribution of DEPs according to subcellular localization, except for DAC3 −/+ and DAC4 −/− , with a tendency of DEPs to be nuclear (Supplementary Fig. 11E). Analysis of the top 30 differentially expressed proteins (DEPs) revealed the five most downregulated proteins for each DAC mutant. In the DAC1 −/− , there were: protein of unknown function (DUF1077, putative), RNA recognition motif (putative), GP63 (leishmanolysin), cytochrome c oxidase VIII (COX VIII, putative), and hypothetical protein (LmxM.34.0620). For DAC3 −/+ , the five most downregulated proteins were: ring-box protein 1, protein of unknown function (DUF1077), galactokinase-like protein, GP63, leishmanolysin and xylulokinase. For DAC4 −/− , we found DnaJ domain containing protein, hypothetical predicted multi-pass transmembrane protein, aldehyde dehydrogenase, mitochondrial precursor, GP63, leishmanolysin and hypothetical protein ( LmxM.11.0750 ) as the most downregulated proteins. Finally, in the DAC5 −/− , the most downregulated proteins were ascorbate peroxidase, Kinesin-13 3, thymine-7-hydroxylase, 3-hydroxy-3-methylglutaryl-CoA synthase, metallo-peptidase, Clan MH, Family M18 and aminopeptidase-like protein, metallo-peptidase, Clan MA(E), Family M1 (Supplementary Data 11F and Supplementary Table 6). Conversely, the five most upregulated proteins varied across the DAC mutants. In the DAC1 −/+ mutant, these were: hypothetical proteins ( LmxM.31.0470; and LmxM.31.3650 ), rab-like GTPase activating protein, conserved hypothetical protein ( LmxM.17.0990 ), and surface antigen-like protein (Supplementary Data 11F and Supplementary Table 6). For the DAC3 −/+ mutant, the five most upregulated proteins were: zinc transporter 3, ferrous iron transport protein, rab-like GTPase activating protein, cytochrome b5-like heme/steroid binding domain-containing protein, and peptidylprolyl isomerase-like protein (Supplementary Data 11F and Supplementary Table 6). In the DAC4 −/− mutant, the five most upregulated proteins were: putative ribosomal protein L29, hypothetical protein ( LmxM.31.3650 ), acyl-CoA binding protein, rab-like GTPase activating protein, and polyketide cyclase/dehydrase and lipid transport protein (Supplementary Data 11F and Supplementary Table 6). Finally, in the DAC5 −/− mutant, the five most upregulated proteins were: hypothetical protein ( LmxM.23.0840 ), acyl-CoA binding protein, microtubule-binding stalk of dynein motor, protein of unknown function (DUF3437), and 1,2-dihydroxy-3-keto-5-methylthiopentene dioxygenase (Supplementary Data 11F and Supplementary Table 6). Our analysis of the lysine-acetylated enriched fraction identified 7,515 Kac sites on 2,895 proteins. The replicates of the same cell line cluster closely in the principal component analysis (PCA) (Supplementary Data 12A and B). We defined a high confidence "common dataset" shared between the DAC mutants and the Cas9 control, which required the detection of a given peptide in at least three biological replicates. This dataset consisted of 2,390 Kac sites on 1,061 proteins. Considering all these findings, we could define the L. mexicana procyclic acetylome containing 5,125 Kac sites in 1,834 proteins (22.5% of the total proteome), which is like T. brucei , T. evansi and T. cruzi published acetylomes 9 , 10 (Supplementary Data 12C). Subsequent analysis of individual mutants showed that protein acetylation was predominantly upregulated in DAC1 −/+ (1,502 Kac sites/763 proteins) and DAC3 −/+ (1,365 Kac sites/708 proteins). In contrast, the DAC4 −/− (150 sites/134 proteins) and DAC5 −/− (32 sites/25 proteins) displayed far fewer upregulated sites (Fig. 6 A, Supplementary Table 7). Downregulation of protein acetylation was more pronounced in DAC5 −/− (505 sites/344 proteins) and DAC4 −/− (105 sites/134 proteins) (Fig. 6 A, Supplementary Table 7). Notably, the majority of these upregulated or downregulated proteins in any given mutant contained only one modified lysine site (Fig. 6 B-C). Comparing the differentially acetylated proteins (DAPs) across all mutants showed limited overlap, with only 8 commonly downregulated and 18 commonly upregulated proteins (Fig. 6 D). A more focused comparison between the cytoplasmic mutants (DAC1 −/+ vs. DAC5 −/− ) yielded 22 upregulated and 18 downregulated proteins. A similar analysis of the nuclear mutants (DAC3 −/+ vs. DAC4 −/− ) identified 18 commonly downregulated and 122 commonly upregulated proteins (Fig. 6 D). We validated our acetylome data by targeting histones, successfully identifying Kac-sites in all four canonical histones (H3, H4, H2A, and H2B) as well as the variants H2Az and H2Bv (Fig. 6 E). Also, in parallel we analyzed the effect of DACs on protein implicated in glycolysis, protein kinases, cell cycle progression and microtubule organization, which might be regulated by these enzymes impacting parasite stage differentiation. Glycolysis In the glycolytic pathway, we found that nine of the ten core enzymes were acetylated, with pyruvate kinase (PK) being the only exception (Supplementary Data 13A). Notably, acetylated phosphoglycerate kinase (PGK), phosphoglycerate mutase (PGM), and enolase (ENO) were not detected in DAC4 −/− and DAC5 −/− . The number of Kac sites on commonly acetylated enzymes also differed among the mutants. For instance, three enzymes – glucose phosphate isomerase (PGI), phosphofructokinase (PFK), and aldolase (ALD) – exhibited a greater number of Kac sites in DAC5 −/− compared to the other mutants (Supplementary Data 13B). Furthermore, analysis of specific residues revealed significant differences in acetylation levels (Supplementary Data 13C). The ALD catalytic sites K51 and K239, which are conserved and known to play a role regulating the enzymatic activity in other organisms 6 , 32 , were hyperacetylated in the DAC5 −/− mutant (Supplementary Data 13C-D). This finding suggests that lysine acetylation may exert a negative regulatory effect on ALD activity in L. mexicana and might influence parasite differentiation. Protein kinases In eukaryotes, signal transduction via phosphorylation is fundamental to the regulation of protein activity. In Leishmania , this process is strongly implicated in driving differentiation, as indicated by stage-specific phosphorylation patterns and recent comprehensive analyses of the L. mexicana kinome 33 , 34 . Among the whole kinome, which comprises 204 proteins kinases, 15 and 29 were identified as required for colonization of the sand fly and for survival as amastigotes in vivo and in vitro , respectively 33 . We hypothesized that acetylation could regulate the activity of protein kinases involved in L. mexicana differentiation. We assessed our DACs acetylome and identified 15 differentially acetylated protein kinases, where six are among those identified as required for parasite survival (AEK1, PKAC1, CK1.2, CRK1, GSK3 and TOR2) and two (CK2A2 and PKAC3) as crucial for amastigote in vitro and in vivo infection (Fig. 6 F). Target of rapamycin (TOR) is a serine/threonine kinase that acts as a master regulator of multiple signaling pathways in eukaryotes 35 – 37 . In trypanosomatids, four TOR paralogs — TOR1, TOR2, TOR3 and TOR4 — have been identified. TOR1 is associated with cell cycle progression and protein synthesis 38 , whereas TbTOR2 has been linked to the regulation of cell polarity and cytokinesis 39 . In our study, TOR2 was found to be hypoacetylated specifically in the DAC5 −/− mutant (K1299), but not in the other mutants, suggesting a potential regulatory mechanism that may contribute to the altered parasite proliferation and differentiation phenotypes observed in DAC5 −/− (Fig. 6 F). Microtubule organization and cell cycle progression Microtubule organization, together with the coordinated action of associated proteins, plays a critical role in regulating the cell cycle and maintaining cellular morphology in Leishmania 40 , 41 . Protein acetylation has emerged as an important regulatory mechanism influencing microtubule dynamics, cell cycle progression, and morphological transitions 42 , 43 . In this context, analysis of the DAC mutant acetylomes revealed differential acetylation of several key proteins, including α-tubulin, actin, cyclins, and Kharon (Supplementary Table 7). Notably, α-tubulin, actin, and Kharon were hyperacetylated in the DAC5 −/− mutant compared to other lines, while no acetylated cyclins were detected (Fig. 6 G). Depletion of DAC1, DAC3, and DAC5 alters G1 and S phase lengths Given the altered acetylation of proteins involved in cell cycle and cytokinesis in our DAC mutant acetylomes, we evaluated cell cycle phase durations. Doubling times were 6.25 h (Cas9), 6.8 h (DAC1 −/+ ), 6 h (DAC3 −/+ ), 6.5 h (DAC4 −/− ), and 8.5 h (DAC5 −/− ), indicating impaired proliferation in DAC5 −/− (Fig. 2 ). Next, based on the doubling time, we estimated the duration of each cell cycle phase. Cytokinesis duration was similar across all lines — 0.45 h (Cas9), 0.4 h (DAC1 −/+ ), 0.41 h (DAC3 −/+ ), 0.43 h (DAC4 −/− ), and 0.36 h (DAC5 −/− ) — with a slight, non-significant reduction in DAC5 −/− (Supplementary Data 14). Mitosis also showed no significant variation, lasting ~ 0.2 h in most lines and 0.34 h in DAC5 −/− (Supplementary Data 14). G2 phase, estimated by subtracting M from G2 + M duration, was comparable across mutants: 0.55 h (Cas9), 0.54 h (DAC1 −/+ ), 0.53 h (DAC3 −/+ ), 0.58 h (DAC4 −/− ), and 0.66 h (DAC5 −/− ). S-phase, however, showed distinct changes: 0.92 h (Cas9), 0.48 h (DAC1 −/+ ), 0.36 h (DAC3 −/+ ), 1 h (DAC4 −/− ), and 2.2 h (DAC5 −/− ). DAC1-/+ and DAC3 −/+ showed shortened S phase, while DAC5 −/− displayed a marked increase. As S-phase length largely reflects replication fork progression, our data suggest that DAC1 and DAC3 may act as negative regulators, and DAC5 as a positive regulator of replication fork progression. Finally, G1-phase durations were 4.13 h (Cas9), 5.17 h (DAC1 −/+ ), 4.48 h (DAC3 −/+ ), 4.32 h (DAC4 −/− ), 4.94 h (DAC5 −/− ), with DAC1 −/+ and DAC3 −/+ showing increases, and DAC5 −/− a modest reduction (Supplementary Data 14). These shifts are consistent with compensatory changes in response to altered S-phase duration. Discussion Adapting to diverse host environments requires Leishmania to rapidly modulate cellular processes. Post-translational modifications, such as acetylation, offer a rapid and efficient mechanism for regulating the proteins involved in these processes. Here, we characterized the DACs of L. mexicana and their roles in parasite life cycle progression and differentiation. Our findings reveal that specific DACs are crucial for distinct life stages of L. mexicana , strengthening the importance of protein acetylation in regulating key cellular processes and pointing out DACs as potencial drug targets. Consistent with observations in trypanosomatids T. brucei and T. cruzi 17 , 19 , 22 , bioinformatic analyses identified four DAC-encoding genes (DAC1, DAC3, DAC4, and DAC5) in the L. mexicana genome. However, unlike those species, L. mexicana possesses a divergent DAC2 ortholog, designated here as DAC5. Functional analysis revealed that L. mexicana DAC1 and DAC3 are essential for procyclic forms, whereas DAC4 and DAC5 are not, indicating a lack of redundance in the deacetylation machinery. DAC1 and DAC5 are cytosolic, while DAC3 and 4 are nuclear, mirroring the general localization and essentiality patterns in T. brucei and T. cruzi 16 , 17 , 19 , except for TbDAC1, which is nuclear in bloodstream forms and shifts to the cytoplasm in procyclic forms, and TbDAC4, which is cytosolic 16 . Subcellular localization data showed that TcDAC1 is nuclear/cytosolic (Sielecki et al., 2024, personal communication). To elucidate the role of DACs in parasite adaptation throughout the life cycle, we conducted a detailed phenotypic analysis, which revealed that each enzyme contributes uniquely to parasite stage differentiation. Assessment of procyclic multiplication showed that disruption of DAC1, DAC3, and DAC5 resulted in reduced growth compared to parental cells, with DAC5 −/− exhibiting the most pronounced effect. These mutants did not exhibit significant cell-cycle alterations (Supplementary Data 9). This contrasts with T. brucei , where disruption of DAC1, DAC3, or DAC4 impairs parasite multiplication and TbDAC4 affects mitotic entry 16 , 17 , and with Tc DAC1 and Tc DAC2 single-knockout epimastigotes, which show reduced proliferation and abnormal cell-cycle progression 19 . Like the lack of effect observed in TcDAC4 −/− epimastigotes 22 , we found that disruption of L. mexicana DAC4 did not affect procyclic growth. Transmission of Leishmania parasites by the insect vector depends on the metacyclic stage, which evolves through a developmental process called metacyclogenesis 44 . Metacyclogenesis involves a significant reduction in RNA, protein, and lipid turnover compared to highly replicative procyclics 2 , 45 , reflecting the decreased cell volume and non-replicative state of metacyclics. In vitro metacyclogenesis assays revealed that DAC1 −/+ and DAC5 −/− parasites exhibited 40% and 80% reductions in metacyclic formation compared to parental cells. This phenotype was rescued using DAC1-AB and DAC5-AB cells and was also observed in the in vivo L. longipalpis infections with DAC5 −/− parasites. Given the cytosolic localization of DAC1 and DAC5 and the established role of alpha-tubulin acetylation in maintaining microtubule organization 42 , we hypothesize that the reduction of both enzymes might affect parasite tubulin acetylation, thereby impairing the procyclic-to-metacyclic transition. In T. cruzi , overexpression of Tc ATAT, the a-tubulin acetyltransferase, induced morphological changes that are associated with cell division impairment and ultrastructural alterations in the mitochondrial branches and kDNA topology 46 . Moreover, TcDAC4 null mutants display morphological alterations in metacyclics, including changes in nucleus/kinetoplast distance and a thinner cell body compared to wild-type parasites 22 . Also, Tc Sir2rp1 sirtuin overexpression impaired epimastigote to metacyclic transition 15 . The involvement of DAC1 and DAC5 in a-tubulin acetylation and microtubule organization is yet to be described. Intracellular replication of L. mexicana amastigotes is crucial for parasite propagation and infection of new host cells. To investigate the role of DACs in amastigote differentiation, we first evaluated procyclic-to-axenic amastigote differentiation and found no significant differences in the DAC mutants. However, we observed a reduction in axenic amastigote multiplication in DAC3 −/+ and DAC5 −/− parasites after initial differentiation, a phenotype that was rescued upon re-expression of the corresponding genes. This growth defect in DAC5 −/− mutants was also observed in in vitro and in vivo infection assays. The precise mechanisms underlying the reduction of amastigote multiplication observed in DAC3 −/+ and DAC5 −/− parasites remain unclear and warrant additional exploitation. In T. cruzi , TcDAC1 overexpression reduces host cell infection by approximately 40% (Sielecki et al., personal communication), while overexpression of the sirtuin TcSir2rp3 enhances intracellular amastigote multiplication 15 , 47 . Furthermore, pan-HDAC inhibitors targeting TcDAC2 (quisinostat and in-house TB compounds) exhibit only a modest anti- T. cruzi effect in vitro and in vivo mouse infection assays 19 . A finding consistent with studies using other FDA-approved inhibitors (vorinostat [SAHA], romidepsin, belinostat, and panobinostat) against intracellular amastigotes of L. amazonensis and L. donovani 48 . Despite these findings, exploring DACs as potential drug targets deserves further consideration. As observed during the promastigote-to-axenic amastigote differentiation, the reverse transition (axenic amastigote to procyclic) was also impaired in DAC5 −/− mutants compared to parental cells, potentially due to defects in microtubule reorganization. Transcriptomic analysis of DAC5 −/− revealed enrichment of genes related to cell projection, cytoskeleton, microtubule-associated complexes, and dynein complexes, which may underlie this phenotype. Alterations in global protein acetylation levels have been linked to parasite stage transitions and physiological adaptations, as previously shown by us in T. brucei 9 . Proteomic and acetylome profiling of DAC mutants confirmed extensive remodeling, with DAC5 deficiency causing the most pronounced changes. Notably, DAC5 loss led to hyperacetylation of cytoskeletal and metabolic proteins, including aldolase, α-tubulin, and actin, potentially contributing to defects in cell cycle progression and differentiation. Furthermore, the differential acetylation of kinases essential for L. mexicana development, such as TOR2, supports a role for acetylation in modulating signaling pathways critical for stage progression. The acetylation of cytoskeletal components and TOR2 in DAC5 −/− is particularly intriguing, given the known involvement of TOR2 in cytokinesis in T. brucei 39 . This, along with the altered cell cycle phase durations and differentially acetylation of Kharon, a well-known player involved in trypanosomatids cell cycle, observed in DAC5 −/− parasites 49 – 52 , suggests that DAC5 regulates both morphological transitions and cell cycle progression in promastigote forms through acetylation-mediated control of cytoskeletal organization and signaling. In conclusion, our study highlights the critical role of lysine acetylation and the regulatory functions of zinc-dependent lysine deacetylases (DACs) in Leishmania mexicana stage differentiation and virulence. Through functional, phenotypic, transcriptomic and proteomic analyses, we demonstrate that specific DACs are essential for parasite development, proliferation, and morphological transitions. These findings not only emphasize the importance of acetylation dynamics in the regulation of key cellular processes but also open new avenues of research into the broader role of acetylation in trypanosomatid biology. In this sense, the attenuation driven by DAC5 disruption together with the in vivo protection against a new infection can be further explored to develop live-attenuated parasite vaccines against leishmaniases. Moreover, our results suggest that targeting DACs may represent a promising strategy for therapeutic intervention in leishmaniases. Declarations Acknowledgments We thank Dr. Carolina Catta-Preta for scientific support during the generation of DACs mutant parasites. The authors also acknowledge the Fiocruz Core Facilities (Next Generation Sequencing Facility-RPT 01I at Aggeu Magalhães Institute). The authors thank Fiocruz’s Network of Technological Platforms (https://plataformas.fiocruz.br/) and the animal facility at René Rachou Institute, Oswaldo Cruz Founation – IRR/Fiocruz Minas, Belo Horizonte, Minas Gerais, Brazil, for providing technical assistance and research support services. Funding Statement This study was financed, in part, by the São Paulo Research Foundation (FAPESP), Brasil. Process Number 2018/09948-0, 2020/07870-4 and 2022/03075-0 to N.M.; 2019/13765-1 and 2021/13477-6 to S.R.M; 2023/16672-0 to ABL; 2025/03898-5 to MANG; 2019/10753-2 and 2020/10277-3 to MSdS). NM and RLMN are CNPq research fellows (grant numbers CNPq 312353/2023-5 and 306191/2024-5). A.M.R. was supported by Instituto Aggeu Magalhães/FIOCRUZ - CNPq grant 400739/2019-4. Research coordinated by RLMN is supported by “Fundação de Amparo à Pesquisa do Estado de Minas Gerais – Fapemig, from “Rede Mineira de Imunobiológicos Aplicada à Vacinas, Biofármacos e Diagnóstico para Leishmaniose Visceral” grant #RED-0003222 and “ReMinD - Rede Mineira de Diagnóstico de Doenças Infecciosas” #RED-00196–23; and “Programa Inova Fiocruz” from Oswaldo Cruz Foundation. National Natural Science Foundation of China (32402916 to N.Z. and 32072880 to Q.C.). This research was supported in part by the Intramural Research Program of the National Institutes of Health (NIH). The contributions of the NIH authors were made as part of their official duties as NIH federal employees, are in compliance with agency policy requirements, and are considered Works of the United States Government. However, the findings and conclusions presented in this paper are those of the author(s) and do not necessarily reflect the views of the NIH or the U.S. Department of Health and Human Services. 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Mitochondrial Sirtuin TcSir2rp3 Affects TcSODA Activity and Oxidative Stress Response in Trypanosoma cruzi. Front Cell Infect Microbiol 11 , (2021). Choudhary, C. et al. Lysine acetylation targets protein complexes and co-regulates major cellular functions. Science (1979) 325 , (2009). Maran, S. R. et al. Protein acetylation in the critical biological processes in protozoan parasites. Trends Parasitol 37 , 815–830 (2021). Moretti, N. S. et al. Characterization of Trypanosoma cruzi Sirtuins as Possible Drug Targets for Chagas Disease. Antimicrob Agents Chemother 59 , 4669–79 (2015). Wang, Q.-P., Kawahara, T. & Horn, D. Histone deacetylases play distinct roles in telomeric VSG expression site silencing in African trypanosomes. Mol Microbiol 77 , 1237–45 (2010). Ingram, A. K. & Horn, D. Histone deacetylases in Trypanosoma brucei: two are essential and another is required for normal cell cycle progression. Mol Microbiol 45 , 89–97 (2002). Alsford, S., Kawahara, T., Isamah, C. & Horn, D. A sirtuin in the African trypanosome is involved in both DNA repair and telomeric gene silencing but is not required for antigenic variation. Mol Microbiol 63 , 724–36 (2007). Marek, M. et al. Species-selective targeting of pathogens revealed by the atypical structure and active site of Trypanosoma cruzi histone deacetylase DAC2. Cell Rep 37 , 110129 (2021). Vergnes, B., Gazanion, E. & Grentzinger, T. Functional divergence of SIR2 orthologs between trypanosomatid parasites. Mol Biochem Parasitol 207 , 96–101 (2016). Tavares, J. et al. The Leishmania infantum cytosolic SIR2-related protein 1 (LiSIR2RP1) is an NAD+ -dependent deacetylase and ADP-ribosyltransferase. Biochem J 415 , 377–86 (2008). Picchi-Constante, G. F. A. et al. Metacyclogenesis defects and gene expression hallmarks of histone deacetylase 4-deficient Trypanosoma cruzi cells. Sci Rep 11 , 21671 (2021). Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596 , (2021). Beneke, T. et al. 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KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res 28 , 27–30 (2000). Lundby, A. et al. Proteomic Analysis of Lysine Acetylation Sites in Rat Tissues Reveals Organ Specificity and Subcellular Patterns. Cell Rep 2 , (2012). Baker, N. et al. Systematic functional analysis of Leishmania protein kinases identifies regulators of differentiation or survival. Nat Commun 12 , 1244 (2021). Tsigankov, P., Gherardini, P. F., Helmer-Citterich, M., Späth, G. F. & Zilberstein, D. Phosphoproteomic analysis of differentiating Leishmania parasites reveals a unique stage-specific phosphorylation motif. J Proteome Res 12 , 3405–12 (2013). Yang, Q. & Guan, K.-L. Expanding mTOR signaling. Cell Res 17 , 666–81 (2007). Gingras, A. C., Raught, B. & Sonenberg, N. mTOR signaling to translation. Curr Top Microbiol Immunol 279 , 169–97 (2004). Dazert, E. & Hall, M. N. mTOR signaling in disease. Curr Opin Cell Biol 23 , 744–55 (2011). Barquilla, A., Crespo, J. L. & Navarro, M. Rapamycin inhibits trypanosome cell growth by preventing TOR complex 2 formation. Proc Natl Acad Sci U S A 105 , 14579–84 (2008). Barquilla, A. & Navarro, M. Trypanosome TOR complex 2 functions in cytokinesis. Cell Cycle 8 , 697–9 (2009). Kelly, F. D. et al. A cytoskeletal protein complex is essential for division of intracellular amastigotes of Leishmania mexicana. J Biol Chem 295 , 13106–13122 (2020). Corrales, R. M. et al. Tubulin detyrosination shapes Leishmania cytoskeletal architecture and virulence. Proc Natl Acad Sci U S A 122 , e2415296122 (2025). Janke, C. & Montagnac, G. Causes and Consequences of Microtubule Acetylation. Curr Biol 27 , R1287–R1292 (2017). Janke, C. & Magiera, M. M. The tubulin code and its role in controlling microtubule properties and functions. Nat Rev Mol Cell Biol 21 , 307–326 (2020). Sacks, D. L. Metacyclogenesis in Leishmania promastigotes. Exp Parasitol 69 , 100–3 (1989). Cortazzo da Silva, L., Aoki, J. I. & Floeter-Winter, L. M. Finding Correlations Between mRNA and Protein Levels in Leishmania Development: Is There a Discrepancy? Front Cell Infect Microbiol 12 , 852902 (2022). Alonso, V. L. et al. Alpha-Tubulin Acetylation in Trypanosoma cruzi: A Dynamic Instability of Microtubules Is Required for Replication and Cell Cycle Progression. Front Cell Infect Microbiol 11 , 642271 (2021). Ritagliati, C., Alonso, V. L., Manarin, R., Cribb, P. & Serra, E. C. Overexpression of cytoplasmic TcSIR2RP1 and mitochondrial TcSIR2RP3 impacts on Trypanosoma cruzi growth and cell invasion. PLoS Negl Trop Dis 9 , e0003725 (2015). Chua, M. J. et al. Effect of clinically approved HDAC inhibitors on Plasmodium, Leishmania and Schistosoma parasite growth. Int J Parasitol Drugs Drug Resist 7 , 42–50 (2017). Santi, A. M. M. et al. Growth arrested live-attenuated Leishmania infantum KHARON1 null mutants display cytokinesis defect and protective immunity in mice. Sci Rep 8 , 11627 (2018). Saenz-Garcia, J. L. et al. Kharon Is Crucial for Trypanosoma cruzi Morphology but Does Not Impair In Vitro Infection. Pathogens 14 , (2025). Kelly, F. D. et al. A cytoskeletal protein complex is essential for division of intracellular amastigotes of Leishmania mexicana. J Biol Chem 295 , 13106–13122 (2020). Sanchez, M. A. et al. KHARON Is an Essential Cytoskeletal Protein Involved in the Trafficking of Flagellar Membrane Proteins and Cell Division in African Trypanosomes. J Biol Chem 291 , 19760–73 (2016). Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryTable1.xlsx Supplementary Table 1. List of primers for generate the homologous recombination (HR) and sgRNA fragments. SupplementaryTable2.xlsx Supplementary Table 2. List of PCR primers for knockout and add-back parasites confirmation. SupplementaryTable3.xlsx Supplementary Table 3. List of RT-qPCR primers for knockout and add-back parasites confirmation. SupplementaryTable4.xlsx Supplementary Table 4. List of DE genes in DACs mutants compared to parental cells. SupplementaryTable5.xlsx Supplementary Table 5. List of DE proteins in DACs mutants compared to parental cells. SupplementaryTable6.xlsx Supplementary Table 6. Top 30 DE proteins in DACs mutants compared to Cas9 cells. SupplementaryTable7.xlsx Supplementary Table 7. List of DE acetylated proteins in DACs mutants compared to parental cells. SupplementaryInformation.docx Supplementary Data Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7520632","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":516061806,"identity":"80d7d5f0-e43c-4999-a6f4-977eddd5f809","order_by":0,"name":"Nilmar 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19:20:54","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7520632/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7520632/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":91495617,"identity":"c4a993bb-c595-49cf-98f5-08106928bff3","added_by":"auto","created_at":"2025-09-17 06:18:57","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":253565,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eL. mexicana\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e DACs. A.\u003c/strong\u003e Phylogenetic analysis of human HDACs and trypanosomatid DACs. \u003cem\u003eL. mexicana\u003c/em\u003e DAC1 and DAC5 cluster with human HDAC class I, while DAC3 and DAC4 are more closely related to class IIa. \u003cstrong\u003eB.\u003c/strong\u003e Schematic representation of \u003cem\u003eL. mexicana\u003c/em\u003e DAC protein domain architecture. DAC3 contains a unique internal insertion within its HDAC domain (blue), which is not found in the other DACs. \u003cstrong\u003eC.\u003c/strong\u003e Pairwise amino acid sequence identity matrix of \u003cem\u003eL. mexicana\u003c/em\u003e DAC HDAC domains compared to their \u003cem\u003eT. brucei\u003c/em\u003e and \u003cem\u003eT. cruzi\u003c/em\u003e orthologs. \u003cstrong\u003eD.\u003c/strong\u003eStructural comparison of human HDAC1 and \u003cem\u003eL. mexicana\u003c/em\u003e DACs. Top panel: Overall protein structures of HDAC domains. Bottom panel: Detailed view of the zinc-binding site with the key residues important to deacetylase activity and the zinc molecule (grey). \u003cstrong\u003eE.\u003c/strong\u003e Western blot analysis of DAC-tag fusion proteins. Arrowheads indicate the expected sizes of DAC1 (75 kDa), DAC3 (93 kDa), DAC4 (91 kDa), and DAC5 (84 kDa). Aldolase was used as a loading control (bottom panel). Data shown are representative of three independent experiments. \u003cstrong\u003eF\u003c/strong\u003e. Subcellular localization of DACs in procyclic parasites. DAC1/5 are cytosolic, while DAC3/4 are nuclear. DACs fused to mNeonGreen (FITC - green) and nuclear/kinetoplast DNA (DAPI). K: kinetoplast; N: nucleus. Scale bar: 5 µm. \u003cstrong\u003eG.\u003c/strong\u003e Three-dimensional images at different angles showing the differential nuclear localization of DAC3 and DAC4 in the euchromatin and nucleolar regions, respectively.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7520632/v1/ef4b85787c78da32e59bb414.png"},{"id":91496534,"identity":"429f394c-ae04-44f5-85ef-85ce27a04b32","added_by":"auto","created_at":"2025-09-17 06:26:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1095284,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDAC1, DAC3, and DAC5 are required for optimal promastigote multiplication.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e. Growth curves of promastigotes of \u003cem\u003eL. mexicana\u003c/em\u003e Cas9 and DAC1\u003csup\u003e-/+\u003c/sup\u003e, DAC3\u003csup\u003e-/+\u003c/sup\u003e, DAC4\u003csup\u003e-/-\u003c/sup\u003e,\u003csup\u003e \u003c/sup\u003eand DAC5\u003csup\u003e-/-\u003c/sup\u003e mutants. A significant reduction in parasite multiplication was observed for DAC1\u003csup\u003e-/+\u003c/sup\u003e, DAC3\u003csup\u003e-/+\u003c/sup\u003e,\u003csup\u003e \u003c/sup\u003eand DAC5\u003csup\u003e-/-\u003c/sup\u003e mutants compared to parental cells. \u003cstrong\u003eB.\u003c/strong\u003e The observed phenotype was rescued in DAC5-AB but not in DAC1-AB or DAC3-AB parasites. The area under the curve (AUC) is shown in the inset in each graph. Two clones of each DACs knockout mutant were analyzed. Statistical significance was determined by two-way ANOVA with Tukey's multiple comparisons test. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001. All experiments were performed in triplicate (n=6 for each cell line). Ribbons around the curve indicate the standard error of the mean (SEM). C1 and C2: independent cloned cell lines.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7520632/v1/05a329f4170c08e339fbf65c.png"},{"id":91496532,"identity":"6c5118a7-9775-4d77-a3da-6ea338e39df9","added_by":"auto","created_at":"2025-09-17 06:26:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2141724,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDACs impact metacyclogenesis and amastigogenesis. A.\u003c/strong\u003e Percentage of metacyclic parasites after 7 days of \u003cem\u003ein vitro\u003c/em\u003e differentiation. Two clones of each DAC knockout (left panel) and one clone of add-back cells (right panel) were analyzed. DAC1\u003csup\u003e-/+\u003c/sup\u003e and DAC5\u003csup\u003e-/- \u003c/sup\u003epresented an impaired metacyclogenesis compared to Cas9, a phenotype that was recovered in the add-backs (DAC1-AB and DAC5-AB). The Cas9 metacyclic number was assigned as 100% for comparison with the other cell lines. Statistical significance was determined by one-way ANOVA with Dunnett's multiple comparisons test. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001. The experiments were done in biological triplicates (n=6 technical replicates) \u003cstrong\u003eB.\u003c/strong\u003e \u003cem\u003eIn vivo\u003c/em\u003e metacyclogenesis in \u003cem\u003eL. longipalpis\u003c/em\u003e. Metacyclogenesis was assessed at 1-, 4-, and 8-days post-infection. Statistical significance was determined by two-way ANOVA with Tukey's multiple comparisons test. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001. All experiments were performed in triplicate. \u003cstrong\u003eC.\u003c/strong\u003e Kinetics of procyclic-to-axenic amastigote differentiation of DACs knockout mutants. The number of procyclic, intermediate, and axAMA forms was monitored at different time points (0, 30 min, 1 h, 1.5h, 2h, 4h, and 24h). No significant changes in differentiation rates were observed between DAC mutants and Cas9. \u003cstrong\u003eD-E.\u003c/strong\u003e Axenic amastigote multiplication of DACs knockout and add-back parasites. Logarithmic procyclic promastigotes were differentiate into axAMA, and multiplication rates were monitored daily. Significant reduction in axAMA multiplication was observed in DAC3\u003csup\u003e-/+\u003c/sup\u003e and DAC5\u003csup\u003e-/-\u003c/sup\u003e compared to parental cells after 24 h \u003cstrong\u003e(D)\u003c/strong\u003e, a phenotype that was rescued in DAC3-AB and DAC5-AB \u003cstrong\u003e(E)\u003c/strong\u003e. The area under the curve (AUC) is shown within each plot. Statistical significance was determined by two-way ANOVA with Tukey's multiple comparisons test. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001. All experiments were performed in triplicate (n=6 technical replicates). Ribbons around the curve indicate the standard error of the mean (SEM). C1 and C2: independent cloned cell lines.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7520632/v1/4fe5c47fad183da1694c4d5b.png"},{"id":91495623,"identity":"0cc31027-c903-4dd1-91ae-01d5f6dc94f7","added_by":"auto","created_at":"2025-09-17 06:18:57","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1852197,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe absence of DAC5 affects macrophage \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e amastigote intracellular multiplication.\u003c/strong\u003e \u003cstrong\u003eA-B.\u003c/strong\u003e Bone marrow-derived macrophages (BMDMs) from BALB/c mice were infected with axenic amastigotes of parental and DACs mutants. The number of intracellular amastigotes per macrophage was quantified using ImageJ software (n=200 macrophages per condition), and a pronounced reduction was observed for DAC5\u003csup\u003e-/-\u003c/sup\u003e. Orange arrowheads indicates intracellular amastigotes. \u003cstrong\u003eC.\u003c/strong\u003e The same analysis was done for DAC5-AB parasites compared to parental and DAC5\u003csup\u003e-/-\u003c/sup\u003e. A reversion in the phenotype observed before was detected. Scale bar: 50 μm. Error bars: Mean ± SD. Statistical significance: One-way ANOVA with Tukey's multiple comparisons test. GP: 0.1234 (ns), 0.0332 (*), 0.0021 (**), 0.0002 (**). n=3 independent experiments. \u003cstrong\u003eD. \u003c/strong\u003eLesion progression of BALB/c mice infected with stationary promastigotes Cas9 and DACs knockout mutants. Footpad thickness was monitored daily for 48 days, and no apparent lesion was detected for DAC5\u003csup\u003e-/-\u003c/sup\u003e parasites, while DAC1\u003csup\u003e-/+\u003c/sup\u003e, DAC3\u003csup\u003e-/+\u003c/sup\u003e, and DAC4\u003csup\u003e-/-\u003c/sup\u003e infected mice presented similar lesions compared to Cas9. \u003cstrong\u003eE.\u003c/strong\u003e DAC5\u003csup\u003e-/-\u003c/sup\u003e challenge experiment. Experimental overview (upper panel). BALB/c mice were inoculated in the left hind footpad with DAC5\u003csup\u003e-/-\u003c/sup\u003e stationary-phase promastigotes 48 days after infection, re-challenge with parental or with PBS as a control in the contralateral paw, and footpad thickness was measured every two days for an additional 60 days. No apparent lesion was detected up to 44 days after the challenge, suggesting a protective effect of DAC5\u003csup\u003e-/-\u003c/sup\u003e parasites. As a positive control, mice were infected with Cas9 parasites. Data representation: Each group consisted of 6 mice, and a single clone of each strain was used. Error bars represent mean ± SD. Statistical significance was determined by one-way ANOVA with Sidak's multiple comparisons test. GP: 0.1234 (ns), 0.0332 (*), 0.0021 (**), 0.0002 (***), \u0026lt;0.0001 (****).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7520632/v1/dfe3a20763a78ae0b42eeeee.png"},{"id":91495625,"identity":"c83cb58e-e9a0-4918-b82d-adb418c79926","added_by":"auto","created_at":"2025-09-17 06:18:57","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":893672,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDAC5 knockout presents a delayed axenic amastigote-procyclic differentiation. A.\u003c/strong\u003e Axenic amastigotes from parental and DACs knockout cell lines were induced to differentiate into procyclic forms in M199 medium. After 24h, a significant reduction in the number of differentiated parasites was observed in DAC5\u003csup\u003e-/-\u003c/sup\u003e (left panel) compared to the parental line (left panel). Procyclic multiplication was monitored for four days after differentiation (right panel), revealing a similar multiplication for DACs mutant as observed before in the procyclic multiplication (Figure 3). \u003cstrong\u003eB-C.\u003c/strong\u003e The observed phenotype was rescued in the DAC5-AB as observed in the number of procyclic cells \u003cstrong\u003e(B)\u003c/strong\u003e and morphology \u003cstrong\u003e(C)\u003c/strong\u003e.\u0026nbsp; The area under the curve (AUC) is shown within each plot. Statistical significance was determined by one-way and two-way ANOVA with Dunnett's and Tukey's multiple comparisons tests. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001. All experiments were performed in triplicate.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7520632/v1/9bc2853f82d60e064129b2c3.png"},{"id":91496538,"identity":"5475e1e9-8658-4dc1-b5b8-364319e4e771","added_by":"auto","created_at":"2025-09-17 06:26:57","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":332293,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGlobal protein acetylation profiles in DAC mutant parasites. A.\u003c/strong\u003e Number of differentially acetylated (Kac) proteins and sites identified in DAC mutants compared to Cas9. \u003cstrong\u003eB–C.\u003c/strong\u003e Distribution of proteins based on the number of Kac sites with decreased (downregulated) or increased (upregulated) acetylation in each DAC mutant relative to Cas9. \u003cstrong\u003eD.\u003c/strong\u003e Venn diagrams showing the overlap of downregulated (left) and upregulated (right) acetylated proteins across DAC mutants. Right panel also highlights shared hypo- and hyperacetylated proteins between cytosolic DACs (DAC1 and DAC5) and nuclear DACs (DAC3 and DAC4). \u003cstrong\u003eE.\u003c/strong\u003e Differentially acetylated histone lysine residues in DAC mutants compared to Cas9. \u003cstrong\u003eF.\u003c/strong\u003e Acetylated protein kinases previously reported to be essential for \u003cem\u003eL. mexicana\u003c/em\u003e stage differentiation (Baker et al., 2021) detected in DAC mutants. \u003cstrong\u003eG.\u003c/strong\u003e Proteins involved in microtubule organization and cell cycle regulation identified as differentially acetylated in DAC mutants relative to Cas9.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7520632/v1/723abb83b678b1227c4b1c31.png"},{"id":91497606,"identity":"d2f0ac91-c6fb-46ee-a931-e92ded52ef33","added_by":"auto","created_at":"2025-09-17 06:43:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8433384,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7520632/v1/2a5b37ad-3f44-4bc8-bd85-b7ca0ac16ed3.pdf"},{"id":91495615,"identity":"d871b49a-3ba9-418d-ab9a-7433e8f80ce7","added_by":"auto","created_at":"2025-09-17 06:18:57","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":12173,"visible":true,"origin":"","legend":"Supplementary Table 1. List of primers for generate the homologous recombination (HR) and sgRNA fragments.","description":"","filename":"SupplementaryTable1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7520632/v1/347be61eca712f691ff46b1d.xlsx"},{"id":91496533,"identity":"4f10faec-2345-4f45-bc4f-3db90d9303df","added_by":"auto","created_at":"2025-09-17 06:26:57","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":10420,"visible":true,"origin":"","legend":"Supplementary Table 2. List of PCR primers for knockout and add-back parasites confirmation.","description":"","filename":"SupplementaryTable2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7520632/v1/653bdeb3070ba0ceb879b922.xlsx"},{"id":91495622,"identity":"d9f6ff69-cac1-4518-b4cf-c15a0a0a6869","added_by":"auto","created_at":"2025-09-17 06:18:57","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":10884,"visible":true,"origin":"","legend":"Supplementary Table 3. List of RT-qPCR primers for knockout and add-back parasites confirmation.","description":"","filename":"SupplementaryTable3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7520632/v1/bdbc7bef0f6d4e41a3a0eec5.xlsx"},{"id":91496535,"identity":"4aebfac7-e24d-4dfe-afe8-de449386418a","added_by":"auto","created_at":"2025-09-17 06:26:57","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":633435,"visible":true,"origin":"","legend":"Supplementary Table 4. List of DE genes in DACs mutants compared to parental cells.","description":"","filename":"SupplementaryTable4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7520632/v1/d105ce7d1c312069d5efb96e.xlsx"},{"id":91495629,"identity":"30e71336-ae7b-46f9-8dc2-99033c31ede6","added_by":"auto","created_at":"2025-09-17 06:18:57","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":1402981,"visible":true,"origin":"","legend":"Supplementary Table 5. List of DE proteins in DACs mutants compared to parental cells.","description":"","filename":"SupplementaryTable5.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7520632/v1/577cc7a1939fe3a5ef82b9f1.xlsx"},{"id":91496779,"identity":"1c728f9d-8991-4009-9623-4c04f68d4b8c","added_by":"auto","created_at":"2025-09-17 06:34:57","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":18396,"visible":true,"origin":"","legend":"Supplementary Table 6. Top 30 DE proteins in DACs mutants compared to Cas9 cells.","description":"","filename":"SupplementaryTable6.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7520632/v1/a869a445734376bfbe664f62.xlsx"},{"id":91495637,"identity":"c4475827-b67d-4f42-b342-9b5f2d39c06c","added_by":"auto","created_at":"2025-09-17 06:18:57","extension":"xlsx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":2048360,"visible":true,"origin":"","legend":"Supplementary Table 7. List of DE acetylated proteins in DACs mutants compared to parental cells.","description":"","filename":"SupplementaryTable7.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7520632/v1/05dc16b7633514f1ce9e3c59.xlsx"},{"id":91495653,"identity":"d28e8561-0170-4317-b0cf-5d815204a022","added_by":"auto","created_at":"2025-09-17 06:18:58","extension":"docx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":33055324,"visible":true,"origin":"","legend":"Supplementary Data","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-7520632/v1/f135f42cc00caf52f17acd27.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"AcetyLeish: acetylation-driven control of stage differentiation and virulence in Leishmania mexicana","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLeishmaniases are a group of neglected tropical diseases caused by \u003cem\u003eLeishmania\u003c/em\u003e species, present as flagellated promastigotes in the sandfly vector and as intracellular amastigotes in mammalian host phagocytes. The disease manifests in various clinical forms, including cutaneous and visceral leishmaniasis. Approximately 1\u0026nbsp;billion people reside in areas at risk of infection. In recent decades, the global incidence of leishmaniases has surged, with an estimated 1\u0026nbsp;million new cases and over 20,000 deaths annually \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eDuring its digenetic life cycle, as the parasite transitions between the invertebrate and vertebrate hosts, it must rapidly adjust to altered environmental conditions. These adaptations involve rapid changes in gene expression, metabolism, and morphology, and are essential for \u003cem\u003eLeishmania\u003c/em\u003e survival \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eProtein post-translational modifications (PTMs) are efficient mechanisms that enable cells to rapidly adapt to changing environments. While protein phosphorylation has long been recognized as a key regulator of protein function \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e, recent advancements in proteomic techniques have highlighted the significance of other PTMs, such as lysine/arginine methylation and lysine acetylation (Kac), which occurs in both histone and non-histone proteins \u003csup\u003e\u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e Kac involves adding an acetyl group to the ε-amino group of lysine residues, neutralizing its positive charge. This modification can alter protein function by affecting enzyme activity, protein-protein interactions, and protein subcellular localization, playing a crucial role in cellular adaptation to environmental changes \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eWe were pioneers in characterizing the first acetylome, a repertoire of lysine-acetylated proteins, of a trypanosomatid \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, validated later by others \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Reduced levels of Kac were observed in glycolytic enzymes of \u003cem\u003eTrypanosoma brucei\u003c/em\u003e bloodstream forms, which relies on glycolysis for ATP generation, compared to procyclic forms, which utilize amino acids as an energy source \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. In a recent study, we confirmed the regulatory role of acetylation in \u003cem\u003eT. brucei\u003c/em\u003e aldolase function, demonstrating that acetylation of residue K157 inhibits its enzymatic activity \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. In \u003cem\u003eTrypanosoma cruzi\u003c/em\u003e epimastigotes, we identified acetylation of several antioxidant enzymes, including the crucial defense enzyme superoxide dismutase A (TcSODA). Using various approaches, we confirmed that acetylation regulates TcSODA activity, contributing to the parasite's oxidative stress response \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. These findings support the idea that Kac likely has a pivotal role in regulating cellular processes involved in \u003cem\u003eLeishmania\u003c/em\u003e stage differentiation.\u003c/p\u003e\u003cp\u003eThe addition, removal, and recognition of acetyl groups on lysines are coordinated by lysine acetyltransferases (KATs), lysine deacetylases (KDACs), and bromodomain-containing proteins (BDPs), respectively \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Trypanosomatid genomes typically harbor seven KDAC genes, including four DACs and three sirtuins \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. All KDACs from \u003cem\u003eT. brucei\u003c/em\u003e and \u003cem\u003eT. cruzi\u003c/em\u003e have been characterized and are involved in various cellular processes, such as parasite differentiation, DNA repair, cell cycle regulation, and infectivity \u003csup\u003e\u003cspan additionalcitationids=\"CR16 CR17 CR18 CR19 CR20 CR21\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. For instance, \u003cem\u003eT. cruzi\u003c/em\u003e DAC1 and 2 are essential for cell cycle progression and proliferation \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, while \u003cem\u003eT. brucei\u003c/em\u003e TbDAC1 and TbDAC3, are essential for evading the host immune response \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. In contrast, TcDAC4 and TbDAC4 are not essential, and their absence affects parasite morphology and G2/M progression, respectively \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. In \u003cem\u003eLeishmania\u003c/em\u003e, the three sirtuins (LmSir2rp1-3) have been characterized, with LmSir2rp1 and LmSir2rp2 identified as essential for parasite survival \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. LmSir2rp1 is cytoplasmic and primarily regulates tubulin acetylation levels \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, while LmSir2rp2 and rp3 are mitochondrial and are linked to NAD\u003csup\u003e+\u003c/sup\u003e metabolism \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. In contrast, the roles of DAC family members (1, 3, 4, and 5) have been underexplored in \u003cem\u003eLeishmania\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eIn this study, we systematically characterized the function of \u003cem\u003eL. mexicana\u003c/em\u003e DACs in parasite stage differentiation and virulence. Using CRISPR/Cas9, we generated DAC1/3 and DAC4/5, hemi and null mutants, respectively, to assess their impact in survival, differentiation, and infection success \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e in both \u003cem\u003eLeishmania\u003c/em\u003e hosts. Additionally, we employed multi-omics tools to identify proteins and pathways regulated by DACs, which may contribute to parasite adaptation.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eEthics statement\u003c/h2\u003e\u003cp\u003e This research project and all the proposed procedures were submitted to and approved by the Ethics Committee on the Use of Animals and the Research Ethics Committee of the Ethics Committee of the Federal University of S\u0026atilde;o Paulo under registration numbers 9407210519/2019 and 9869091118/2019, respectively and by the Ren\u0026eacute; Rachou Institute Ethics Committee under license number LW38-23.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn silico\u003c/b\u003e \u003cb\u003eL. mexicana\u003c/b\u003e \u003cb\u003eDACs analyses\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe amino acid sequences of DAC1, 3, 4, and 5 in \u003cem\u003eL. mexicana\u003c/em\u003e, \u003cem\u003eT. cruzi\u003c/em\u003e, \u003cem\u003eT. brucei\u003c/em\u003e, and humans were retrieved from TritrypDB (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://tritrypdb.org/tritrypdb/app\u003c/span\u003e\u003cspan address=\"https://tritrypdb.org/tritrypdb/app\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and UniProt (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.uniprot.org\u003c/span\u003e\u003cspan address=\"https://www.uniprot.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The HDCA domains for each protein were determined using the InterPro Classification tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ebi.ac.uk/interpro/\u003c/span\u003e\u003cspan address=\"https://www.ebi.ac.uk/interpro/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and amino acid sequences alignment and phylogenetic trees were done using the Geneious software. The predicted three-dimensional models of \u003cem\u003eL. mexicana\u003c/em\u003e DACs were generated using AlphaFold2 \u003csup\u003e23\u003c/sup\u003e, and all structural analyses were performed using Pymol 3.12 software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pymol.org/\u003c/span\u003e\u003cspan address=\"https://pymol.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Final images were generated with Adobe Photoshop 2025 or Adobe Illustrator 2025.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eParasite axenic maintenance and differentiation assays\u003c/h3\u003e\n\u003cp\u003ePromastigote-stage \u003cem\u003eL. mexicana\u003c/em\u003e (MHOM/GT/2001/U11032) T7/Cas9 cells were cultured in M199 medium supplemented with 4.62 mM NaHCO\u003csub\u003e3\u003c/sub\u003e, 40 mM HEPES, 0.1 mM adenine, 0.0001% biotin, 10% heat-inactivated fetal bovine serum (FBS), and 50 \u0026micro;g/mL hygromycin B at 26\u0026deg;C. Knockout mutant parasites were cultured in the same way in the presence of specific antibiotics (20 \u0026micro;g/mL blasticidin and/or 50 \u0026micro;g/mL puromycin. For fluorescently tagged and add-back cells, 20 \u0026micro;g/mL blasticidin or 20 \u0026micro;g/mL G418, were added, respectively.\u003c/p\u003e\u003cp\u003eTo assess promastigote growth, 1 \u0026times; 10⁵ cells/mL in the exponential phase were inoculated into M199 supplemented medium and incubated at 26\u0026deg;C. Cell proliferation was monitored daily for five days using a Muse Cell Analyzer (Merck Millipore). Metacyclogenesis was induced by inoculating 1.5 \u0026times; 10⁶ procyclic cells/mL into Grace's medium (Sigma-Aldrich), pH 5.5, supplemented with 4.62 mM NaHCO₃, 1\u0026times; BME vitamins (Sigma-Aldrich), 10% FBS, and penicillin/streptomycin. The parasites were incubated at 26\u0026deg;C for seven days. Metacyclic parasites were purified using a Percoll gradient. Briefly, parasites were collected by centrifugation at 3,000 \u0026times; g for 10 min at room temperature, resuspended in 3 mL of Grace\u0026rsquo;s medium, and layered onto a 10\u0026ndash;100% Percoll gradient. The gradient was centrifuged at 1,300 \u0026times; g for 10 min at room temperature. The 10% fraction, containing metacyclic parasites, was collected, washed twice with PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4 and 2 mM KH2PO4, pH 7.4), and counted using a Neubauer chamber. The metacyclogenesis rate was calculated as the ratio of metacyclic parasites to the initial number of parasites in the Percoll gradient.\u003c/p\u003e\u003cp\u003ePromastigote-to-axenic amastigote differentiation was induced by incubating 1 \u0026times; 10⁶ procyclic cells/mL in Grace's medium, pH 5.5, supplemented with 4.62 mM NaHCO₃, 1\u0026times; BME vitamins, and 10% FBS at 33\u0026deg;C and 5% CO₂ for four days. Axenic amastigote to procyclic differentiation was induced by inoculating 1 \u0026times; 10⁶ cells/mL into an M199 supplemented medium. Parasite growth was monitored daily for four days using a Neubauer chamber.\u003c/p\u003e\n\u003ch3\u003eGeneration of cell lines\u003c/h3\u003e\n\u003cp\u003eWe employed the CRISPR/Cas9 method described in \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e to select DAC knockout and fluorescent-tagged \u003cem\u003eL. mexicana\u003c/em\u003e mutants. For each DAC gene, specific primers for homologous recombination fragment (HR) and sgRNA synthesis were designed using the LeishGEdit tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.leishgedit.net\u003c/span\u003e\u003cspan address=\"http://www.leishgedit.net\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (primer sequences provided in Supplementary Table\u0026nbsp;1). The HR and sgRNA fragments were amplified by PCR using the conditions described in [23], and all PCR products were confirmed by 1% agarose gel electrophoresis before transfection. To obtain the constructs used to generate the add-back cells the full-length sequences corresponding to \u003cem\u003eL. mexicana\u003c/em\u003e DAC1, 3, 4 and 5, were obtained via PCR with specific oligonucleotides (see Supplementary Table\u0026nbsp;1), using as a template the parasite\u0026rsquo;s genomic DNA. The fragments of each DAC were further cloned in the pSPa-Neo-a plasmid using the \u003cem\u003eBamH\u003c/em\u003eI and \u003cem\u003eHind\u003c/em\u003eIII restriction enzyme sites introduced in the oligonucleotides. Also, we added the V5-tag sequence at the N-terminal region of each DAC sequence for further analysis.\u003c/p\u003e\u003cp\u003eFor transfection, 1 \u0026times; 10⁷ exponentially growing T7/Cas9 cells were collected by centrifugation (2,500 \u0026times; g, 5 min) and resuspended in transfection cytomix buffer (66.7 mM Na₂HPO₄, 23.3 mM NaH₂PO₄, 5 mM KCl, 50 mM HEPES, pH 7.3, 150 \u0026micro;M CaCl₂). The appropriate sgRNA and HR fragments or the pSPa-Neo-a constructs for each DAC were added to the cell suspension, followed by transfection using the Amaxa Nucleofector\u0026trade; IIb (Lonza). After 24h, cultures were supplemented with specific antibiotics for selection. Selected transfectants were later cloned by serial dilution in 96-well plates.\u003c/p\u003e\n\u003ch3\u003eConfirmation of mutant parasites\u003c/h3\u003e\n\u003cp\u003eTo confirm the selection of DAC knockout mutants, PCR was performed using genomic DNA (gDNA) as a template. Primers were designed to target the 5' UTR and internal regions of each DAC gene (Supplementary Table\u0026nbsp;2). Additionally, primers specific for resistance markers were included in the analysis. Quantitative PCR (qPCR) was also employed to quantify gene copy number and mRNA expression levels of specific DACs in knockout mutants. For gene copy number analysis, gDNA was used as a template with SYBR Green and specific DAC primers. Reactions were performed in a 20 \u0026micro;L volume containing 10 pmol of each primer, 1X SYBR Green, and 1 \u0026micro;L of gDNA (100 ng/\u0026micro;L). PCR conditions were: 95\u0026deg; C for 5 min, followed by 45 cycles of 95\u0026deg;C for 15 s, 60\u0026deg;C for 15 s, and 72\u0026deg;C for 30 s, with a final extension at 72\u0026deg;C for 5 min. Gene copy number was calculated using the 2-ΔΔCt method, with GAPDH as the endogenous control. For mRNA expression analysis, total RNA was extracted from parental and knockout mutant parasites using TRIzol and treated with DNase. cDNA was synthesized using a High-Capacity cDNA Reverse Transcription Kit with a spliced leader (SL) primer to specifically convert mature mRNAs. qPCR was performed using the same conditions as above. All primers used for mutant genotyping are listed in Supplementary Table\u0026nbsp;3.\u003c/p\u003e\u003cp\u003eFluorescent-tagged DAC mutants were confirmed using flow cytometry and Western blot. For flow cytometry analysis, 1 \u0026times; 10⁶ parasites from parental and fluorescent-tagged cell lines were collected by centrifugation (2,500 \u0026times; \u003cem\u003eg\u003c/em\u003e, 2 min) and resuspended in 1 mL of 1\u0026times; PBS. Cell fluorescence was measured using an Accuri\u0026trade; flow cytometer (BD Biosciences) with the FL-1 filter. Data were analyzed using BD Accuri\u0026trade; C6 v1.0.264.21 software. For Western blot analysis, 1 \u0026times; 10⁷ cells from each condition were collected and lysed in 4\u0026times; Laemmli sample buffer. Proteins were separated by 10% SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was blocked with 1\u0026times; PBS containing 0.1% Tween-20 and 5% non-fat milk for 1h at room temperature. Subsequently, the membrane was incubated overnight at 4\u0026deg;C with an anti-c-Myc tag antibody (Merck, #05-724) diluted 1:3,000 in blocking solution. After washing with 1\u0026times; PBS containing 0.1% Tween-20, the membrane was incubated with an IRDye\u0026reg; 800CW Goat anti-Mouse IgG Secondary Antibody (1:10,000) for 1h at room temperature. Following additional washes, the membrane was imaged using an Odyssey CLx Imaging System (LI-COR).\u003c/p\u003e\u003cp\u003eThe add-back cells were confirmed by qPCR with specific oligonucleotides or Western blot with anti-V5, following the approaches described above.\u003c/p\u003e\n\u003ch3\u003eConfocal microscopy analysis\u003c/h3\u003e\n\u003cp\u003eTo determine the subcellular localization of each fluorescently tagged DAC protein, procyclic, metacyclic, and amastigote stages of the mutant parasites were analyzed by fluorescence microscopy. Briefly, 1 \u0026times; 10⁵ cells of each parasite stage were collected, washed once with 1\u0026times; PBS, and incubated on a poly-L-lysine-coated slide for 10 min at room temperature. Cells were then fixed with 1% paraformaldehyde for 15 min at room temperature and stained with 10 \u0026micro;M DAPI for 10 min. After washing with 1\u0026times; PBS, the slides were mounted with glycerol-p-phenylenediamine. Images were acquired using a TCS SP5 II Tandem Scanner microscope (Leica) and processed with Imaris v6 software.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003emacrophage and\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e \u003cb\u003emice infection assays\u003c/b\u003e\u003c/p\u003e\u003cp\u003eBone marrow-derived macrophages (BMDMs) were obtained from susceptible BALB/c mice. Briefly, tibias and femurs were aseptically removed, and bone marrow cells were flushed with RPMI 1640 medium supplemented with 10% FBS. Cells were centrifuged (2,500 \u0026times; \u003cem\u003eg\u003c/em\u003e, 5 min), resuspended in ACK lysis buffer (155 mM NH₄Cl, 10 mM KHCO₃, 100 \u0026micro;M EDTA), and incubated at room temperature for 5 min to lyse red blood cells. To obtain BMDMs cells, the population was seeded on glass coverslips and incubated in a differentiation medium (RPMI supplemented with 10% inactivated horse serum, 30% L929 cell conditioned medium, 100 U/ml penicillin and 100 \u0026micro;g/ml streptomycin) at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. After 5 days, BMDMs were maintained in RPMI 1640 supplemented with 10% FBS (R10 medium) for 24h before infection. Axenic amastigotes from parental and knockout mutant parasites were used to infect BMDMs at a ratio of 1 parasite per macrophage. After a 2h incubation period, cells were washed to remove extracellular parasites and incubated for 12, 24, or 48h. To quantify intracellular amastigote proliferation, cells were stained with Giemsa. Images were acquired using a Nikon Eclipse Ti-U microscope and analyzed using ImageJ software to count the number of cells and intracellular amastigotes to obtain the macrophage infection rate and the number of amastigotes per infected macrophage.\u003c/p\u003e\u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e infection assays were performed using female 8\u0026ndash;10 weeks old BALB/c mice at the Instituto Ren\u0026eacute; Rachou - Fiocruz, Belo Horizonte, Minas Gerais, Brazil. Briefly, 1 \u0026times; 10⁷ stationary-phase parasites from parental (T7/Cas9) and DAC knockout mutants were injected into the right footpads of each mouse with a final volume of 30 \u0026micro;L (n\u0026thinsp;=\u0026thinsp;6 per group), and footpad swelling was monitored daily for 60 days using a caliper (Mitutoyo Corp., Japan). For the challenge infection assays, five weeks after primary infection with DAC5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e cells, parental parasites were injected into the left footpads (1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e stationary phase parasites) and infection was monitored as above for 48 days. Data were analyzed using GraphPad Prism software to compare the parental groups with DAC mutant groups.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn vivo Lutzomyia longipalpis\u003c/b\u003e \u003cb\u003einfection assays\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePromastigote forms of parental, DACs knockout mutant, and add-back cells were added to heat-inactivated heparinized mouse blood at a final concentration of 5 \u0026times; 10⁶ parasites/mL. Female \u003cem\u003eL. longipalpis\u003c/em\u003e sand flies were fed on this blood meal through a chick membrane in an artificial feeder. Approximately 100 female sandflies were used per group. On the following day, the fed females were transferred to a new container. To monitor infection rates, approximately 10\u0026ndash;15 fed females were dissected on days 1, 4, and 8 post-infection. Midgut contents were extracted in PBS using a disposable plastic pestle, and the total number of parasites in each midgut was counted in a hemocytometer and the frequency of differentiating promastigotes, including procyclics, nectomonads, and metacyclics, determined by morphology as described in \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eCell-cycle progression analyses\u003c/h2\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003eEdU incorporation assays and \u0026lsquo;click\u0026rsquo; chemistry reaction\u003c/h2\u003e\u003cp\u003eExponentially growing \u003cem\u003eL. mexicana\u003c/em\u003e promastigotes were incubated with 100 \u0026micro;M 5-ethynyl-2\u0026rsquo;-deoxyuridine (EdU) for different time periods (ranging from 30 min to 1.15h) at 26\u0026deg;C. The parasites were then harvested (~\u0026thinsp;1 x 10\u003csup\u003e7\u003c/sup\u003e cells) by centrifugation at 2,500 \u003cem\u003eg\u003c/em\u003e for 5 min, washed three times in PBS, fixed for 20 min with 1% sterile paraformaldehyde diluted in PBS, washed twice, and re-suspended in 200 \u0026micro;L of PBS. Then, 20 \u0026micro;L of the cell suspension was loaded onto poly-L-lysine pre-treated microscope slides and washed three times with 3% BSA diluted in PBS. To detect incorporated EdU, we used the Click-iT EdU detection solution for 45 min protected from light. This solution is composed of 1 mM CuSO\u003csub\u003e4\u003c/sub\u003e, 10 \u0026micro;M Alexa Fluor Azide 488, and 100 mM C\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e8\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e, diluted in distilled water. Next, the cells were washed five times with PBS. Vectashield mounting medium (Vector) containing DAPI was used as an anti-fade mounting solution and to stain the organelles containing DNA. Images were acquired using the fluorescent microscope (Nikon Eclipse 80i). The percentage of parasites in cytokinesis and mitosis was calculated for each group relative to the total number of DAPI-positive parasites.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\n\u003ch3\u003eCell cycle analysis\u003c/h3\u003e\n\u003cp\u003eDAPI-stained exponentially growing promastigotes were examined under a fluorescent microscope (Nikon) to observe the profile of the nuclei (N) and kinetoplast (K). The profile 2N2K was used to estimate the percentage of cells in cytokinesis (C). The profile containing one nucleus being divided was used to estimate the percentage of performing mitosis (M). To estimate the G2\u0026thinsp;+\u0026thinsp;M phases length, we added EdU in the medium containing exponentially growing promastigotes and collected samples every 15 min, proceeding with the \u0026lsquo;click\u0026rsquo; chemistry reaction, until a parasite containing two EdU-labeled nuclei (2N2K or 2N1K) was observed (this time corresponds to the length of G2\u0026thinsp;+\u0026thinsp;M phases). To monitor DNA replication, we measured the proportion of cells EdU-labeled after 1h EdU pulse. The cell cycle phases duration were estimated after all these parameters (together with doubling time extracted from the procyclic growth curves) using the online software CeCyD, available at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cecyd.vital.butantan.gov.br/\u003c/span\u003e\u003cspan address=\"https://cecyd.vital.butantan.gov.br/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e \u003csup\u003e26\u003c/sup\u003e.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eRNAseq experiments\u003c/h2\u003e\u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\u003ch2\u003eRNA samples and library preparation\u003c/h2\u003e\u003cp\u003eExponentially growing procyclic parasites of parental and DAC knockout mutants were collected in quadruplicate and triplicate, respectively. Total RNA was extracted using the RNeasy Mini Kit, followed by DNase I treatment. RNA quality and concentration were assessed using Qubit\u0026trade; RNA IQ Assay Kits and agarose gel electrophoresis. Paired-end sequencing libraries were prepared from total RNA using the Stranded mRNA Prep Kit by Illumina. Finally, the libraries were sequenced on an Illumina NovaSeq\u0026trade; 6000 sequencer using the 100-cycle SP v1.5 kit by Illumina.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eRNAseq analysis\u003c/h2\u003e\u003cp\u003eThe quality of reads was assessed using the FastQC tool \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Each sample library was mapped against the \u003cem\u003eL. mexicana\u003c/em\u003e genome (strain MHOM/GT/2001/U1103) with the STAR mapping tool \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. The count data for each sample was then structured as a matrix in the R environment, and differential expression analysis was performed using the DESeq2 package \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. We considered only genes represented by at least five reads across all replicates (parental or knockout strains). The gene expression ratio between the parental and the modified strain was calculated using a base-2 logarithmic transformation (log2 fold change). Genes with an absolute log2 fold change \u0026ge;0.6 (positive for upregulated genes and negative for downregulated genes) and corrected \u003cem\u003ep\u003c/em\u003e-values \u0026le;0.05 were considered differentially expressed genes (DEGs).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eGene enrichment analysis\u003c/h2\u003e\u003cp\u003eGene enrichment analysis was conducted using the TriTrypDB database \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. The selected DEGs were used for an ID search in the Gene Ontology (GO) database for Biological Process (BP) and Cellular Component (CC) enrichment, filtering for curated evidence using Benjamini method and a \u003cem\u003ep\u003c/em\u003e-value cutoff of \u0026le;0.05. Metabolic pathway enrichment was performed using the KEGG database \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e with the same \u003cem\u003ep\u003c/em\u003e-value cutoff of \u0026le;0.05.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eAcetylome experiments\u003c/h2\u003e\u003cdiv id=\"Sec16\" class=\"Section3\"\u003e\u003ch2\u003eProtein Sample Preparation\u003c/h2\u003e\u003cp\u003eExponentially growing procyclic parasites of parental and DAC knockout mutants were collected in triplicate and subjected to phenol protein extraction as described in \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Parasites were resuspended in phenol extraction buffer (0.7 M sucrose, 0.1 M KCl, 0.5 M Tris-HCl, pH 7.5, 1% TritonX-100, 10 mM DTT) supplemented with 1% protease inhibitors, 3 \u0026micro;M of trichostatin A, 50 mM nicotinamide, 2 mM EDTA, and 2% b-mercaptoethanol. The mixture was sonicated, phenol-extracted, and proteins from the supernatant were precipitated overnight with 0.1 M ammonium acetate/methanol, washed with acetone and methanol, and dissolved in 8 M urea.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eTrypsin Digestion and Affinity Enrichment\u003c/h2\u003e\u003cp\u003eThe protein solution was reduced with 5 mM DTT for 30 min at 56\u0026deg;C and alkylated with 11 mM iodoacetamide for 15 min at room temperature in darkness. The protein sample was then diluted by adding 200 mM TEAB to a urea concentration less than 2 M. Finally, trypsin was added at a 1:50 trypsin-to-protein mass ratio for the first digestion overnight and a 1:100 trypsin-to-protein mass ratio for a second 4 h-digestion. Finally, the peptides were desalted by the Strata X SPE column.\u003c/p\u003e\u003cp\u003eTo enrich the acetylated peptides, tryptic peptides dissolved in NETN buffer (100 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl, 0.5% NP-40, pH 8.0) were incubated with beads conjugated with anti-acetyllysine antibodies (PTM Bio) at 4\u0026deg;C overnight with gentle shaking. Then the beads were washed for four times with NETN buffer and twice with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. The bound peptides were eluted from the beads with 0.1% trifluoroacetic acid. Finally, the eluted fractions were combined and vacuum-dried. For LC-MS/MS analysis, the resulting peptides were desalted with C18 ZipTips.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eLC-MS/MS Analysis\u003c/h2\u003e\u003cp\u003eTryptic peptides were dissolved in solvent A and loaded onto a custom-made reversed-phase analytical column (25 cm length, 100 \u0026micro;m ID). Mobile phases A (0.1% formic acid, 2% acetonitrile in water) and B (0.1% formic acid in acetonitrile) were used to separate peptides with the following gradient: 0\u0026ndash;40 min, 7\u0026ndash;24% B; 40\u0026ndash;52 min, 24\u0026ndash;32% B; 52\u0026ndash;56 min, 32\u0026ndash;80% B; 56\u0026ndash;60 min, 80% B, at a flow rate of 450 nL/min on a NanoElute UHPLC system. Peptides were analyzed by capillary source-timsTOF Pro2 mass spectrometry. The electrospray voltage was set to 1.5 kV. Precursors and fragments were detected by the TOF detector, with an MS/MS scan range of 100\u0026ndash;1700 m/z. The timsTOF Pro was operated in PASEF mode, selecting precursors with charge states 0\u0026ndash;5 for fragmentation. Ten PASEF-MS/MS scans were acquired per cycle, with a dynamic exclusion time of 24 s.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eDatabase Processing\u003c/h2\u003e\u003cp\u003eThe resulting MS/MS data were processed using MaxQuant (v.1.6.15.0). Peptides were searched against the UniProtKB \u003cem\u003eL. mexicana\u003c/em\u003e database, concatenated with a reverse decoy and contaminant database. Trypsin/P was specified as the cleavage enzyme, allowing up to two missed cleavages. Minimum peptide length was set to 7, and a maximum of five modifications per peptide was allowed. The mass tolerance for precursor ions was set to 20 ppm in both the first and main searches, and the mass tolerance for fragment ions was set to 20 ppm. Carbamidomethylation on cysteine was specified as a fixed modification. Acetylation on protein N-terminus, methionine oxidation, and lysine acetylation were specified as variable modifications. The false discovery rate (FDR) for proteins, peptides, and PSMs was adjusted to \u0026lt;\u0026thinsp;1%. Label-free quantification (LFQ) was selected for quantification.\u003c/p\u003e\u003c/div\u003e\u003cp\u003eData availability\u003c/p\u003e\n\u003cp\u003eThe raw sequencing files (fastq) for all RNA samples from T7/Cas9 and DAC mutants have been deposited in the Sequence Read Archive (SRA) under accession code: PRJNA1062791 (for T7/Cas) and PRJNA1312851 (for DACs). The processed RNAseq data are provided in the Supplementary Table 4. The raw proteomic files have been deposit in ProteomeXchange. The processed acetylome data is provided in the Supplementary Tables 5,6 and 7.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eL. mexicana\u003c/b\u003e \u003cb\u003egenome encodes four zinc-dependent lysine deacetylase orthologs\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe \u003cem\u003eL. mexicana\u003c/em\u003e genome contains four genes encoding zinc-dependent lysine deacetylases, a feature shared with other Trypanosomatids species such as \u003cem\u003eT. cruzi\u003c/em\u003e and \u003cem\u003eT. brucei\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Phylogenetic analysis revealed that \u003cem\u003eL. mexicana\u003c/em\u003e DAC1 and DAC2 cluster closely with human HDAC class I (HDAC1-3 and 8), while DAC3 and 4 could not be definitively assigned to a specific family but exhibit greater similarity to human HDAC class IIa (HDAC4, 5, 7, and 9) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). In contrast to human class IIa HDACs, neither DAC3 nor DAC4 possesses N- or C-terminal extensions. However, like \u003cem\u003eT. cruzi\u003c/em\u003e DAC3 \u003csup\u003e19\u003c/sup\u003e, \u003cem\u003eL. mexicana\u003c/em\u003e DAC3 contains insertions within its catalytic domain (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eComparative analysis of the catalytic domain amino acid sequences between \u003cem\u003eL. mexicana\u003c/em\u003e and human HDACs revealed low identity (\u0026lt;\u0026thinsp;49%) across all \u003cem\u003eLeishmania\u003c/em\u003e DACs, with DAC4 and DAC5 exhibiting the highest divergence (~\u0026thinsp;41%) (Supplementary Data 1A). Further sequence analysis of \u003cem\u003eL. mexicana\u003c/em\u003e HDAC domains in comparison to their \u003cem\u003eT. brucei\u003c/em\u003e and \u003cem\u003eT. cruzi\u003c/em\u003e orthologs demonstrated high identity for DAC1, DAC33, and DAC4 (53\u0026ndash;73%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). In contrast, \u003cem\u003eL. mexicana\u003c/em\u003e DAC2 shared very low identity (\u0026lt;\u0026thinsp;26%) with its trypanosome orthologs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), leading us to rename this enzyme as DAC5. This significant divergence was also evident in phylogenetic analysis of \u003cem\u003eLeishmania\u003c/em\u003e and trypanosome DACs (Supplementary Data 1B).\u003c/p\u003e\u003cp\u003eMoreover, multiple sequence alignments of \u003cem\u003eL. mexicana\u003c/em\u003e DACs with human HDAC classes I and II revealed conservation of residues critical for zinc binding and the catalytic tyrosine residue (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD and Supplementary Data 1C). Despite the phylogenetic proximity of DAC3 and 4 to human HDAC class IIa, the characteristic substitution of the catalytic tyrosine (Y) to histidine (H) is absent in these parasite enzymes, which retain a tyrosine residue (Supplementary Data 1C). To gain insights in \u003cem\u003eL. mexicana\u003c/em\u003e DACs, we compared their predicted protein structures to those of human HDAC1 and \u003cem\u003eT. cruzi\u003c/em\u003e DAC2. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD illustrates a high degree of structural conservation among these enzymes, including the catalytic site region.\u003c/p\u003e\u003cp\u003e\u003cb\u003eL. mexicana\u003c/b\u003e \u003cb\u003eDACs subcellular localization and expression in different parasite stages\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo functionally characterize \u003cem\u003eL. mexicana\u003c/em\u003e DACs, we generated parasite cell lines expressing DAC1, DAC3, DAC4, or DAC5, each endogenous fused to mNeonGreen and a 3xMyc tags. To confirm successful transfection, we performed flow cytometry analysis to measure mNeonGreen fluorescence in procyclic forms of parental (Cas9) and transfected (DAC-tag) parasites. All transfected parasites exhibited higher fluorescence levels compared to their counterpart Cas9 control, confirming mNeonGreen expression (Supplementary Data 2A). Next, to verify the correct expression of DAC-tagged fusion proteins, Western blot analysis was performed using anti-c-Myc antibody. As expected, we detected the DAC-tagged proteins exclusively in the transfected parasites, with the expected molecular weights of 75, 93, 91, and 84 kDa for DAC1, DAC3, DAC4, and DAC5, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003eUsing the endogenous DAC-tagged cell lines, we determined the subcellular localization of these enzymes in the major parasite stages: procyclic, metacyclic, and amastigote forms. We found that DAC1 and DAC5 are primarily localized to specific regions of the cytoplasm, while DAC3 and DAC4 are nuclear proteins in all parasite stages (Fig.\u0026nbsp;1FC and Supplementary Data 2B). Interestingly, although both DAC3 and DAC4 are nuclear, DAC3 is predominantly found in euchromatin regions, while DAC4 is primarily localized to the nucleolar region (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG).\u003c/p\u003e\u003cp\u003eTo investigate the indirect expression of DACs in procyclic, metacyclic, and axenic amastigote stages of \u003cem\u003eL. mexicana\u003c/em\u003e, we evaluated mNeonGreen fluorescence levels by flow cytometry. We observed a decreasing expression for DAC3 and DAC4 from promastigote to metacyclic and axenic amastigote stages, while DAC1 and DAC5 showed a slight increase (Supplementary Data 2C).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eDAC1 and DAC3 are probably essential in\u003c/b\u003e \u003cb\u003eL. mexicana\u003c/b\u003e \u003cb\u003epromastigotes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo further characterize \u003cem\u003eL. mexicana\u003c/em\u003e DACs, we employed a CRISPR/Cas9-based loss-of-function approach to generate knockout mutant parasites. Following selection of transfected parasites, diagnostic PCR reactions were performed using total genomic DNA and oligonucleotide primers flanking the specific gene locus of each DAC (Supplementary Data 3A). PCR amplification products confirmed the generation of hemizygous knockout parasites (one disrupted allele) for DAC1 (DAC1\u003csup\u003e\u0026minus;/+\u003c/sup\u003e) and DAC3 (DAC3\u003csup\u003e\u0026minus;/+\u003c/sup\u003e), and homozygous knockout parasites (both alleles disrupted) for DAC4 (DAC4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e) and DAC5 (DAC5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e) (Supplementary Data 3B), suggesting that DAC1 and DAC3 may be essential for the promastigote stage of \u003cem\u003eL. mexicana\u003c/em\u003e. To further validate these findings, we quantified gene copy number and mRNA expression levels of each DAC in the knockout mutants by qPCR, which confirmed the results obtained by PCR (Supplementary Data 3C and D). At least four rounds of transfection were performed for each DAC, yielding consistent results. In addition, we confirmed the re-expression of each DAC in add-back transfectants (DAC1-AB, DAC3-AB, DAC4-AB, and DAC5-AB) by Western blot, RT-qPCR, and immunofluorescence (Supplementary Data 4).\u003c/p\u003e\u003cp\u003e\u003cb\u003eDAC1, DAC3, and DAC5 influence\u003c/b\u003e \u003cb\u003eL. mexicana\u003c/b\u003e \u003cb\u003epromastigote multiplication\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCompared to Cas9 parental cells, DAC1\u003csup\u003e\u0026minus;/+\u003c/sup\u003e, DAC3\u003csup\u003e\u0026minus;/+\u003c/sup\u003e, and DAC5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mutants exhibited a reduction in promastigote multiplication varying from 25 to 40% (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). In contrast, DAC4 null mutants showed no significant effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). This phenotype was partially restored in DAC5-AB but not in DAC1-AB or DAC3-AB parasites (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eDAC1 and DAC5 affect\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e \u003cb\u003emetacyclogenesis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eNext, we investigated the impact of DACs on \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e differentiation into metacyclics. To assess \u003cem\u003ein vitro\u003c/em\u003e metacyclogenesis, we compared the number of non-metacyclic forms before differentiation to the number of metacyclic forms after the differentiation protocol. We found that DAC5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e and DAC1\u003csup\u003e\u0026minus;/+\u003c/sup\u003e parasites exhibited a nearly 80% and 40% reduction in metacyclogenesis, compared to the Cas9 parental cell line (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, left panel). These phenotypes were rescued by re-expressing DAC1 and 5 in add-back parasites (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, right panel). We did not observe any significant differences in metacyclogenesis of DAC3\u003csup\u003e\u0026minus;/+\u003c/sup\u003e and DAC4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mutants.\u003c/p\u003e\u003cp\u003eGiven the observed effects on \u003cem\u003ein vitro\u003c/em\u003e metacyclogenesis, we investigated the impact of DACs on metacyclogenesis during \u003cem\u003ein vivo\u003c/em\u003e infection with \u003cem\u003eL. longipalpis\u003c/em\u003e. Female \u003cem\u003eL. longipalpis\u003c/em\u003e were fed with procyclic forms of parental and DAC mutant parasites. Metacyclogenesis was assessed by counting parasite stages (procyclic, nectomonads, and metacyclics) at 1, 4, and 8-days post-infection. As observed \u003cem\u003ein vitro\u003c/em\u003e, DAC5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e parasites exhibited impaired differentiation to metacyclics compared to the parental cell line (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Interestingly, DAC3\u003csup\u003e\u0026minus;/+\u003c/sup\u003e showed a decreased differentiation rate compared to parental cells, while DAC1\u003csup\u003e\u0026minus;/+\u003c/sup\u003e had no significant effect on \u003cem\u003ein vivo\u003c/em\u003e differentiation, contrasting with the \u003cem\u003ein vitro\u003c/em\u003e observations (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-B). Unfortunately, we did not observe a reversion of the DAC3\u003csup\u003e\u0026minus;/+\u003c/sup\u003e and DAC5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e phenotypes in the DAC3-AB and DAC5-AB cells (Supplementary Data 5). Detailed metacyclogenesis data for 1-, 4-, and 8-days post-infection of all DACs mutants are provided in Supplementary Data 5.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eDAC3 and DAC5 impair axenic amastigote multiplication but not differentiation\u003c/h2\u003e\u003cp\u003eTo investigate the role of DACs in promastigote-to-axenic amastigote differentiation and multiplication, we performed differentiation kinetics of promastigote to axenic amastigotes using DAC mutants (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). We found no significant effect of DACs on the differentiation process, as the frequency of promastigotes, intermediate axenic amastigotes, and axenic amastigote (axAMA) forms remained unchanged in the first 24 h comparing DACs mutant cells and Cas9 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). However, we observed a major deleterious effect on axAMA multiplication in DAC5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e, about a\u0026thinsp;~\u0026thinsp;70% decrease, and DAC3\u003csup\u003e\u0026minus;/+\u003c/sup\u003e (about 50% reduction) compared to Cas9 cells after 24 h, while DAC1\u003csup\u003e\u0026minus;/+\u003c/sup\u003e and DAC4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mutants showed no significant effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). The observed deleterious phenotype in axAMA multiplication observed in DAC3\u003csup\u003e\u0026minus;/+\u003c/sup\u003e and DAC5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e was restored to similar levels of Cas9 in the DAC3-AB and DAC5-AB parasites (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003e\u003cb\u003eEffect of DACs on\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e \u003cb\u003emacrophage infection\u003c/b\u003e\u003c/p\u003e\u003cp\u003eUsing BMDMs from BALB/c mice, we performed \u003cem\u003ein vitro\u003c/em\u003e infection assays with axenic amastigotes from parental and DAC knockout mutant parasites. After infection, we monitored intracellular amastigote multiplication at 12-, 24-, and 48h post-infection. A significant reduction in the infection index was observed for all mutants, as measured by the number of intracellular parasites and infected cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The most pronounced reduction (~\u0026thinsp;90%) was observed for DAC5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e after 48h of infection, while DAC1\u003csup\u003e\u0026minus;/+\u003c/sup\u003e, DAC3\u003csup\u003e\u0026minus;/+\u003c/sup\u003e, and DAC4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e exhibited reductions of approximately 50%, 70%, and 60%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Consistent with these results, microscopy images revealed more intracellular amastigotes in macrophages infected with Cas9 than those infected with DAC mutants, particularly for DAC5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). The infection index was restored in DAC5-AB parasites (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC and Supplementary Data 6A).\u003c/p\u003e\u003cp\u003e\u003cb\u003eDAC5 impairs\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e \u003cb\u003einfection of\u003c/b\u003e \u003cb\u003eL. mexicana\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFemale BALB/c mice were infected with stationary-phase procyclic forms of parental and DACs knockout mutant parasites in the left hind footpad. Footpad thickness was measured daily for 48 days to monitor parasite-induced lesion development. We observed no significant lesion development in mice infected with DAC5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e parasites compared to the control group, indicating that disruption of the DAC5 gene attenuates parasite virulence (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). No significant differences in lesion development were observed for DAC1\u003csup\u003e\u0026minus;/+\u003c/sup\u003e, DAC3\u003csup\u003e\u0026minus;/+\u003c/sup\u003e, and DAC4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e parasites compared to the parental strain (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). These findings were further supported by parasite load quantification and representative photographs of BALB/c mice at day 48 post-infection (Supplementary Date 6B-C). No phenotype reversion was observed when we performed the same experiments comparing DAC5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e and DAC5-AB parasites (data not shown).\u003c/p\u003e\u003cp\u003eGiven the attenuated phenotype observed in DAC5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e parasites, we investigated their potential to confer protection against subsequent infection. To address this, BALB/c mice previously infected with DAC5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e parasites were re-challenged with Cas9 parasites in the contralateral paw, and footpad thickness was monitored for additional 60 days. No significant difference in footpad thickness was observed between mice previously infected with DAC5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e and re-challenged with Cas9 parasites compared to PBS-treated control mice up to 44 days post-infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE), suggesting a protective phenotype.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003eDAC5 delays axenic amastigote-to-procyclic differentiation\u003c/h2\u003e\u003cp\u003eAxenic amastigotes from parental and DACs knockout mutants were inoculated into M199 medium at 28\u0026deg;C to induce differentiation into procyclic forms. After differentiation, we monitored parasite multiplication for four days and observed a similar growth phenotype (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA) to that seen in our previous procyclic multiplication experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Markedly, we observed a significant delay in procyclic differentiation in DAC5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e compared to parental cells at 24h post-differentiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), which prompted us to evaluate DAC5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e differentiation at a time series from 2 to 24h. We observed that compared to parental cells, most of DAC5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e still resemble as axenic amastigotes with a rounded morphology after 24h (Supplementary Data 7), indicating a delay in parasite differentiation.\u003c/p\u003e\u003cp\u003eTo link this phenotype to DAC5 function, we repeated the experiments using DAC5-AB parasites, and as expected, the axenic amastigote-procyclic differentiation phenotype was rescued, as detected in the number and morphology of procyclic cells after 24h (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB and C).\u003c/p\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003eDAC mutants express the procyclic, metacyclic and amastigote markers\u003c/h2\u003e\u003cp\u003eTo confirm the expression of stage-specific markers in the parasite stages quantified in our phenotype differentiation experiments, we collected total RNA samples from each stage of DAC mutants and measured the expression of \u003cem\u003ehistone h4\u003c/em\u003e (procyclic), \u003cem\u003esherp\u003c/em\u003e (metacyclic), and \u003cem\u003eamastin\u003c/em\u003e (axenic amastigote) genes by RT-qPCR \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. We successfully validated the stages of \u003cem\u003eL. mexicana\u003c/em\u003e in our analysis, and no significant differences in the expression of these markers were observed between DAC mutants and the parental cell line (Supplementary Data 8).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003eDACs effect on the transcriptome of procyclic stages\u003c/h2\u003e\u003cp\u003eTo investigate the mechanisms regulated by DACs that may influence \u003cem\u003eL. mexicana\u003c/em\u003e stage differentiation, we first performed RNA-Seq analyses comparing procyclic DAC knockout mutants with parental cells. We identified 516, 165, 641, and 381 upregulated genes in the DAC1, DAC3, DAC4, and DAC5 mutants, respectively, and 763, 437, 941, and 1935 downregulated genes in the same mutants (Supplementary Data 9A and Supplementary Table\u0026nbsp;4). Assessment of all differentially expressed genes across the DAC mutants revealed only 21 commonly upregulated and 40 commonly downregulated genes (Supplementary Data 9B and Supplementary Table\u0026nbsp;4). Comparing only cytosolic (DAC1 and DAC5) or nuclear (DAC3 and DAC4) DAC mutants we identified 36 commonly upregulated and 111 commonly downregulated genes between DAC1 and DAC5, and 159 commonly upregulated and 393 commonly downregulated genes between DAC3 and DAC4 (Supplementary Data 9C).\u003c/p\u003e\u003cp\u003eDifferentially expressed genes (DEGs) from the DAC mutant RNA-Seq data were subjected to functional enrichment analysis using the Gene Ontology (GO) terms \"Cellular Compartment (CC)\" and \"Biological Process (BP)\". A stringent \u003cem\u003ep\u003c/em\u003e-value cutoff of 0.05 was applied to identify significantly enriched GO terms. Consequently, not all mutants yielded significant enrichment results. Analysis of the \"Cellular Compartment (CC)\" GO category revealed distinct enrichment patterns for each DAC mutant (Supplementary Data 10). In DAC1\u003csup\u003e\u0026minus;/+\u003c/sup\u003e, upregulated genes were enriched for membrane-related terms (membrane, intrinsic component of membrane, and integral component of membrane), while downregulated genes were enriched for general cellular component terms (cellular component, cellular anatomical entity, and intracellular anatomical structure). In DAC3\u003csup\u003e\u0026minus;/+\u003c/sup\u003e, upregulated genes were enriched for membrane, ribosome, and chromosome terms, whereas downregulated genes were enriched for cellular component and intracellular anatomical structure terms, as well as terms related to the nucleus and chromatin. Both upregulated and downregulated genes in DAC4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e were enriched for nucleus- and chromatin-related terms. In DAC5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e, upregulated genes were enriched for cell projection, cilium, plasma membrane-bounded cell projection, and cytoskeleton terms, while downregulated genes were enriched for ribonucleoprotein complex, ribosomal subunit, and mitochondrial matrix terms.\u003c/p\u003e\u003cp\u003eAnalyses of the \"Biological Process (BP)\" GO category revealed that in DAC1\u003csup\u003e\u0026minus;/+\u003c/sup\u003e, upregulated genes were enriched for autophagy-related terms (autophagy and process utilizing autophagy mechanisms), while downregulated genes were enriched for metabolic process terms (cellular nitrogen compound metabolic process, organic cyclic compound metabolic process, and heterocycle metabolic process) (Supplementary Data 10). In DAC3\u003csup\u003e\u0026minus;/+\u003c/sup\u003e, upregulated genes were enriched for biosynthetic process terms (biosynthetic process, organic substance biosynthetic process, and cellular biological process), and downregulated genes were enriched for catabolic process terms (catabolic process, organic substance catabolic process, and proteolysis). No significantly enriched terms were identified among upregulated genes in DAC4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e; however, downregulated genes were enriched for metabolic processes related to nitrogen compounds, nucleobases, and RNA (cellular nitrogen compound metabolic process, nucleobase-containing compound metabolic process, and RNA metabolic process). In DAC5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e, upregulated genes were enriched for terms related to cellular movement and microtubules (movement of cell or subcellular component, microtubule-based process, and microtubule-based movement). Among the downregulated genes we found cellular component, cellular anatomical entity and intracellular anatomical structure as the most enriched terms in DAC5 mutants ((Supplementary Data 10).\u003c/p\u003e\u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\u003ch2\u003eDAC mutants have altered protein acetylation levels\u003c/h2\u003e\u003cp\u003eTo further investigate the mechanisms associated with DAC regulatory function, we performed proteomic analyses of lysine-acetylated enriched (Kac) and non-modified procyclic protein fractions, comparing DAC mutants to Cas9 control cells. We identified differentially expressed proteins (DEPs) with a log\u003csub\u003e2\u003c/sub\u003e fold change\u0026thinsp;\u0026gt;\u0026thinsp;1.5: 210 upregulated and 262 downregulated in DAC1\u003csup\u003e\u0026minus;/+\u003c/sup\u003e, 230 upregulated and 151 downregulated in DAC3\u003csup\u003e\u0026minus;/+\u003c/sup\u003e, 120 upregulated and 183 downregulated in DAC4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e, and 630 upregulated and 624 downregulated in DAC5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e compared to Cas9 (Supplementary Data 11A-C and Supplementary Table\u0026nbsp;5). Comparison of DEPs across all mutants revealed only 19 commonly downregulated and 36 commonly upregulated proteins (Supplementary Data 11D). However, comparison of cytoplasmic DAC mutants (DAC1\u003csup\u003e\u0026minus;/+\u003c/sup\u003e vs. DAC5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e) identified 119 commonly upregulated and 104 commonly downregulated proteins, while comparison of nuclear DAC mutants (DAC3\u003csup\u003e\u0026minus;/+\u003c/sup\u003e vs. DAC4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e) identified 54 commonly downregulated and 130 commonly upregulated proteins (Supplementary Fig.\u0026nbsp;11D). We observed no preferential distribution of DEPs according to subcellular localization, except for DAC3\u003csup\u003e\u0026minus;/+\u003c/sup\u003e and DAC4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e, with a tendency of DEPs to be nuclear (Supplementary Fig.\u0026nbsp;11E).\u003c/p\u003e\u003cp\u003eAnalysis of the top 30 differentially expressed proteins (DEPs) revealed the five most downregulated proteins for each DAC mutant. In the DAC1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e, there were: protein of unknown function (DUF1077, putative), RNA recognition motif (putative), GP63 (leishmanolysin), cytochrome c oxidase VIII (COX VIII, putative), and hypothetical protein (LmxM.34.0620). For DAC3\u003csup\u003e\u0026minus;/+\u003c/sup\u003e, the five most downregulated proteins were: ring-box protein 1, protein of unknown function (DUF1077), galactokinase-like protein, GP63, leishmanolysin and xylulokinase. For DAC4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e, we found DnaJ domain containing protein, hypothetical predicted multi-pass transmembrane protein, aldehyde dehydrogenase, mitochondrial precursor, GP63, leishmanolysin and hypothetical protein (\u003cem\u003eLmxM.11.0750\u003c/em\u003e) as the most downregulated proteins. Finally, in the DAC5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e, the most downregulated proteins were ascorbate peroxidase, Kinesin-13 3, thymine-7-hydroxylase, 3-hydroxy-3-methylglutaryl-CoA synthase, metallo-peptidase, Clan MH, Family M18 and aminopeptidase-like protein, metallo-peptidase, Clan MA(E), Family M1 (Supplementary Data 11F and Supplementary Table\u0026nbsp;6).\u003c/p\u003e\u003cp\u003eConversely, the five most upregulated proteins varied across the DAC mutants. In the DAC1\u003csup\u003e\u0026minus;/+\u003c/sup\u003e mutant, these were: hypothetical proteins (\u003cem\u003eLmxM.31.0470;\u003c/em\u003e and \u003cem\u003eLmxM.31.3650\u003c/em\u003e), rab-like GTPase activating protein, conserved hypothetical protein (\u003cem\u003eLmxM.17.0990\u003c/em\u003e), and surface antigen-like protein (Supplementary Data 11F and Supplementary Table\u0026nbsp;6). For the DAC3\u003csup\u003e\u0026minus;/+\u003c/sup\u003e mutant, the five most upregulated proteins were: zinc transporter 3, ferrous iron transport protein, rab-like GTPase activating protein, cytochrome b5-like heme/steroid binding domain-containing protein, and peptidylprolyl isomerase-like protein (Supplementary Data 11F and Supplementary Table\u0026nbsp;6). In the DAC4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mutant, the five most upregulated proteins were: putative ribosomal protein L29, hypothetical protein (\u003cem\u003eLmxM.31.3650\u003c/em\u003e), acyl-CoA binding protein, rab-like GTPase activating protein, and polyketide cyclase/dehydrase and lipid transport protein (Supplementary Data 11F and Supplementary Table\u0026nbsp;6). Finally, in the DAC5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mutant, the five most upregulated proteins were: hypothetical protein (\u003cem\u003eLmxM.23.0840\u003c/em\u003e), acyl-CoA binding protein, microtubule-binding stalk of dynein motor, protein of unknown function (DUF3437), and 1,2-dihydroxy-3-keto-5-methylthiopentene dioxygenase (Supplementary Data 11F and Supplementary Table\u0026nbsp;6).\u003c/p\u003e\u003cp\u003eOur analysis of the lysine-acetylated enriched fraction identified 7,515 Kac sites on 2,895 proteins. The replicates of the same cell line cluster closely in the principal component analysis (PCA) (Supplementary Data 12A and B). We defined a high confidence \"common dataset\" shared between the DAC mutants and the Cas9 control, which required the detection of a given peptide in at least three biological replicates. This dataset consisted of 2,390 Kac sites on 1,061 proteins. Considering all these findings, we could define the \u003cem\u003eL. mexicana\u003c/em\u003e procyclic acetylome containing 5,125 Kac sites in 1,834 proteins (22.5% of the total proteome), which is like \u003cem\u003eT. brucei\u003c/em\u003e, \u003cem\u003eT. evansi\u003c/em\u003e and \u003cem\u003eT. cruzi\u003c/em\u003e published acetylomes \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e (Supplementary Data 12C).\u003c/p\u003e\u003cp\u003eSubsequent analysis of individual mutants showed that protein acetylation was predominantly upregulated in DAC1\u003csup\u003e\u0026minus;/+\u003c/sup\u003e (1,502 Kac sites/763 proteins) and DAC3\u003csup\u003e\u0026minus;/+\u003c/sup\u003e (1,365 Kac sites/708 proteins). In contrast, the DAC4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e (150 sites/134 proteins) and DAC5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e (32 sites/25 proteins) displayed far fewer upregulated sites (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, Supplementary Table\u0026nbsp;7). Downregulation of protein acetylation was more pronounced in DAC5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e (505 sites/344 proteins) and DAC4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e (105 sites/134 proteins) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, Supplementary Table\u0026nbsp;7). Notably, the majority of these upregulated or downregulated proteins in any given mutant contained only one modified lysine site (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB-C).\u003c/p\u003e\u003cp\u003eComparing the differentially acetylated proteins (DAPs) across all mutants showed limited overlap, with only 8 commonly downregulated and 18 commonly upregulated proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). A more focused comparison between the cytoplasmic mutants (DAC1\u003csup\u003e\u0026minus;/+\u003c/sup\u003e vs. DAC5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e) yielded 22 upregulated and 18 downregulated proteins. A similar analysis of the nuclear mutants (DAC3\u003csup\u003e\u0026minus;/+\u003c/sup\u003e vs. DAC4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e) identified 18 commonly downregulated and 122 commonly upregulated proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003eWe validated our acetylome data by targeting histones, successfully identifying Kac-sites in all four canonical histones (H3, H4, H2A, and H2B) as well as the variants H2Az and H2Bv (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). Also, in parallel we analyzed the effect of DACs on protein implicated in glycolysis, protein kinases, cell cycle progression and microtubule organization, which might be regulated by these enzymes impacting parasite stage differentiation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\u003ch2\u003eGlycolysis\u003c/h2\u003e\u003cp\u003eIn the glycolytic pathway, we found that nine of the ten core enzymes were acetylated, with pyruvate kinase (PK) being the only exception (Supplementary Data 13A). Notably, acetylated phosphoglycerate kinase (PGK), phosphoglycerate mutase (PGM), and enolase (ENO) were not detected in DAC4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e and DAC5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe number of Kac sites on commonly acetylated enzymes also differed among the mutants. For instance, three enzymes \u0026ndash; glucose phosphate isomerase (PGI), phosphofructokinase (PFK), and aldolase (ALD) \u0026ndash; exhibited a greater number of Kac sites in DAC5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e compared to the other mutants (Supplementary Data 13B). Furthermore, analysis of specific residues revealed significant differences in acetylation levels (Supplementary Data 13C). The ALD catalytic sites K51 and K239, which are conserved and known to play a role regulating the enzymatic activity in other organisms \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, were hyperacetylated in the DAC5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mutant (Supplementary Data 13C-D). This finding suggests that lysine acetylation may exert a negative regulatory effect on ALD activity in \u003cem\u003eL. mexicana\u003c/em\u003e and might influence parasite differentiation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eProtein kinases\u003c/p\u003e\u003cp\u003eIn eukaryotes, signal transduction via phosphorylation is fundamental to the regulation of protein activity. In \u003cem\u003eLeishmania\u003c/em\u003e, this process is strongly implicated in driving differentiation, as indicated by stage-specific phosphorylation patterns and recent comprehensive analyses of the \u003cem\u003eL. mexicana\u003c/em\u003e kinome \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Among the whole kinome, which comprises 204 proteins kinases, 15 and 29 were identified as required for colonization of the sand fly and for survival as amastigotes \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e, respectively \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eWe hypothesized that acetylation could regulate the activity of protein kinases involved in \u003cem\u003eL. mexicana\u003c/em\u003e differentiation. We assessed our DACs acetylome and identified 15 differentially acetylated protein kinases, where six are among those identified as required for parasite survival (AEK1, PKAC1, CK1.2, CRK1, GSK3 and TOR2) and two (CK2A2 and PKAC3) as crucial for amastigote \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF).\u003c/p\u003e\u003cp\u003eTarget of rapamycin (TOR) is a serine/threonine kinase that acts as a master regulator of multiple signaling pathways in eukaryotes \u003csup\u003e\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. In trypanosomatids, four TOR paralogs \u0026mdash; TOR1, TOR2, TOR3 and TOR4 \u0026mdash; have been identified. TOR1 is associated with cell cycle progression and protein synthesis \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e, whereas TbTOR2 has been linked to the regulation of cell polarity and cytokinesis \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. In our study, TOR2 was found to be hypoacetylated specifically in the DAC5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mutant (K1299), but not in the other mutants, suggesting a potential regulatory mechanism that may contribute to the altered parasite proliferation and differentiation phenotypes observed in DAC5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section3\"\u003e\u003ch2\u003eMicrotubule organization and cell cycle progression\u003c/h2\u003e\u003cp\u003eMicrotubule organization, together with the coordinated action of associated proteins, plays a critical role in regulating the cell cycle and maintaining cellular morphology in \u003cem\u003eLeishmania\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Protein acetylation has emerged as an important regulatory mechanism influencing microtubule dynamics, cell cycle progression, and morphological transitions \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. In this context, analysis of the DAC mutant acetylomes revealed differential acetylation of several key proteins, including α-tubulin, actin, cyclins, and Kharon (Supplementary Table\u0026nbsp;7). Notably, α-tubulin, actin, and Kharon were hyperacetylated in the DAC5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mutant compared to other lines, while no acetylated cyclins were detected (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\u003ch2\u003eDepletion of DAC1, DAC3, and DAC5 alters G1 and S phase lengths\u003c/h2\u003e\u003cp\u003eGiven the altered acetylation of proteins involved in cell cycle and cytokinesis in our DAC mutant acetylomes, we evaluated cell cycle phase durations. Doubling times were 6.25 h (Cas9), 6.8 h (DAC1\u003csup\u003e\u0026minus;/+\u003c/sup\u003e), 6 h (DAC3\u003csup\u003e\u0026minus;/+\u003c/sup\u003e), 6.5 h (DAC4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e), and 8.5 h (DAC5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e), indicating impaired proliferation in DAC5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Next, based on the doubling time, we estimated the duration of each cell cycle phase.\u003c/p\u003e\u003cp\u003eCytokinesis duration was similar across all lines \u0026mdash; 0.45 h (Cas9), 0.4 h (DAC1\u003csup\u003e\u0026minus;/+\u003c/sup\u003e), 0.41 h (DAC3\u003csup\u003e\u0026minus;/+\u003c/sup\u003e), 0.43 h (DAC4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e), and 0.36 h (DAC5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e) \u0026mdash; with a slight, non-significant reduction in DAC5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e (Supplementary Data 14). Mitosis also showed no significant variation, lasting\u0026thinsp;~\u0026thinsp;0.2 h in most lines and 0.34 h in DAC5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e (Supplementary Data 14). G2 phase, estimated by subtracting M from G2\u0026thinsp;+\u0026thinsp;M duration, was comparable across mutants: 0.55 h (Cas9), 0.54 h (DAC1\u003csup\u003e\u0026minus;/+\u003c/sup\u003e), 0.53 h (DAC3\u003csup\u003e\u0026minus;/+\u003c/sup\u003e), 0.58 h (DAC4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e), and 0.66 h (DAC5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e). S-phase, however, showed distinct changes: 0.92 h (Cas9), 0.48 h (DAC1\u003csup\u003e\u0026minus;/+\u003c/sup\u003e), 0.36 h (DAC3\u003csup\u003e\u0026minus;/+\u003c/sup\u003e), 1 h (DAC4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e), and 2.2 h (DAC5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e). DAC1-/+ and DAC3\u003csup\u003e\u0026minus;/+\u003c/sup\u003e showed shortened S phase, while DAC5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e displayed a marked increase. As S-phase length largely reflects replication fork progression, our data suggest that DAC1 and DAC3 may act as negative regulators, and DAC5 as a positive regulator of replication fork progression. Finally, G1-phase durations were 4.13 h (Cas9), 5.17 h (DAC1\u003csup\u003e\u0026minus;/+\u003c/sup\u003e), 4.48 h (DAC3\u003csup\u003e\u0026minus;/+\u003c/sup\u003e), 4.32 h (DAC4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e), 4.94 h (DAC5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e), with DAC1\u003csup\u003e\u0026minus;/+\u003c/sup\u003e and DAC3\u003csup\u003e\u0026minus;/+\u003c/sup\u003e showing increases, and DAC5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e a modest reduction (Supplementary Data 14). These shifts are consistent with compensatory changes in response to altered S-phase duration.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAdapting to diverse host environments requires \u003cem\u003eLeishmania\u003c/em\u003e to rapidly modulate cellular processes. Post-translational modifications, such as acetylation, offer a rapid and efficient mechanism for regulating the proteins involved in these processes. Here, we characterized the DACs of \u003cem\u003eL. mexicana\u003c/em\u003e and their roles in parasite life cycle progression and differentiation. Our findings reveal that specific DACs are crucial for distinct life stages of \u003cem\u003eL. mexicana\u003c/em\u003e, strengthening the importance of protein acetylation in regulating key cellular processes and pointing out DACs as potencial drug targets.\u003c/p\u003e\u003cp\u003eConsistent with observations in trypanosomatids \u003cem\u003eT. brucei\u003c/em\u003e and \u003cem\u003eT. cruzi\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, bioinformatic analyses identified four DAC-encoding genes (DAC1, DAC3, DAC4, and DAC5) in the \u003cem\u003eL. mexicana\u003c/em\u003e genome. However, unlike those species, \u003cem\u003eL. mexicana\u003c/em\u003e possesses a divergent DAC2 ortholog, designated here as DAC5. Functional analysis revealed that \u003cem\u003eL. mexicana\u003c/em\u003e DAC1 and DAC3 are essential for procyclic forms, whereas DAC4 and DAC5 are not, indicating a lack of redundance in the deacetylation machinery. DAC1 and DAC5 are cytosolic, while DAC3 and 4 are nuclear, mirroring the general localization and essentiality patterns in \u003cem\u003eT. brucei\u003c/em\u003e and \u003cem\u003eT. cruzi\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, except for TbDAC1, which is nuclear in bloodstream forms and shifts to the cytoplasm in procyclic forms, and TbDAC4, which is cytosolic \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Subcellular localization data showed that TcDAC1 is nuclear/cytosolic (Sielecki et al., 2024, personal communication).\u003c/p\u003e\u003cp\u003eTo elucidate the role of DACs in parasite adaptation throughout the life cycle, we conducted a detailed phenotypic analysis, which revealed that each enzyme contributes uniquely to parasite stage differentiation. Assessment of procyclic multiplication showed that disruption of DAC1, DAC3, and DAC5 resulted in reduced growth compared to parental cells, with DAC5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e exhibiting the most pronounced effect. These mutants did not exhibit significant cell-cycle alterations (Supplementary Data 9). This contrasts with \u003cem\u003eT. brucei\u003c/em\u003e, where disruption of DAC1, DAC3, or DAC4 impairs parasite multiplication and TbDAC4 affects mitotic entry \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, and with \u003cem\u003eTc\u003c/em\u003eDAC1 and \u003cem\u003eTc\u003c/em\u003eDAC2 single-knockout epimastigotes, which show reduced proliferation and abnormal cell-cycle progression \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Like the lack of effect observed in TcDAC4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e epimastigotes \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, we found that disruption of \u003cem\u003eL. mexicana\u003c/em\u003e DAC4 did not affect procyclic growth.\u003c/p\u003e\u003cp\u003eTransmission of \u003cem\u003eLeishmania\u003c/em\u003e parasites by the insect vector depends on the metacyclic stage, which evolves through a developmental process called metacyclogenesis \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Metacyclogenesis involves a significant reduction in RNA, protein, and lipid turnover compared to highly replicative procyclics \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e, reflecting the decreased cell volume and non-replicative state of metacyclics. \u003cem\u003eIn vitro\u003c/em\u003e metacyclogenesis assays revealed that DAC1\u003csup\u003e\u0026minus;/+\u003c/sup\u003e and DAC5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e parasites exhibited 40% and 80% reductions in metacyclic formation compared to parental cells. This phenotype was rescued using DAC1-AB and DAC5-AB cells and was also observed in the \u003cem\u003ein vivo L. longipalpis\u003c/em\u003e infections with DAC5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e parasites.\u003c/p\u003e\u003cp\u003eGiven the cytosolic localization of DAC1 and DAC5 and the established role of alpha-tubulin acetylation in maintaining microtubule organization \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e, we hypothesize that the reduction of both enzymes might affect parasite tubulin acetylation, thereby impairing the procyclic-to-metacyclic transition. In \u003cem\u003eT. cruzi\u003c/em\u003e, overexpression of \u003cem\u003eTc\u003c/em\u003eATAT, the a-tubulin acetyltransferase, induced morphological changes that are associated with cell division impairment and ultrastructural alterations in the mitochondrial branches and kDNA topology \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Moreover, TcDAC4 null mutants display morphological alterations in metacyclics, including changes in nucleus/kinetoplast distance and a thinner cell body compared to wild-type parasites \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Also, \u003cem\u003eTc\u003c/em\u003eSir2rp1 sirtuin overexpression impaired epimastigote to metacyclic transition \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. The involvement of DAC1 and DAC5 in a-tubulin acetylation and microtubule organization is yet to be described.\u003c/p\u003e\u003cp\u003eIntracellular replication of \u003cem\u003eL. mexicana\u003c/em\u003e amastigotes is crucial for parasite propagation and infection of new host cells. To investigate the role of DACs in amastigote differentiation, we first evaluated procyclic-to-axenic amastigote differentiation and found no significant differences in the DAC mutants. However, we observed a reduction in axenic amastigote multiplication in DAC3\u003csup\u003e\u0026minus;/+\u003c/sup\u003e and DAC5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e parasites after initial differentiation, a phenotype that was rescued upon re-expression of the corresponding genes. This growth defect in DAC5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mutants was also observed in \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e infection assays.\u003c/p\u003e\u003cp\u003eThe precise mechanisms underlying the reduction of amastigote multiplication observed in DAC3\u003csup\u003e\u0026minus;/+\u003c/sup\u003e and DAC5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e parasites remain unclear and warrant additional exploitation. In \u003cem\u003eT. cruzi\u003c/em\u003e, TcDAC1 overexpression reduces host cell infection by approximately 40% (Sielecki et al., personal communication), while overexpression of the sirtuin TcSir2rp3 enhances intracellular amastigote multiplication \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Furthermore, pan-HDAC inhibitors targeting TcDAC2 (quisinostat and in-house TB compounds) exhibit only a modest anti-\u003cem\u003eT. cruzi\u003c/em\u003e effect \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e mouse infection assays \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. A finding consistent with studies using other FDA-approved inhibitors (vorinostat [SAHA], romidepsin, belinostat, and panobinostat) against intracellular amastigotes of \u003cem\u003eL. amazonensis\u003c/em\u003e and \u003cem\u003eL. donovani\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Despite these findings, exploring DACs as potential drug targets deserves further consideration.\u003c/p\u003e\u003cp\u003eAs observed during the promastigote-to-axenic amastigote differentiation, the reverse transition (axenic amastigote to procyclic) was also impaired in DAC5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mutants compared to parental cells, potentially due to defects in microtubule reorganization. Transcriptomic analysis of DAC5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e revealed enrichment of genes related to cell projection, cytoskeleton, microtubule-associated complexes, and dynein complexes, which may underlie this phenotype.\u003c/p\u003e\u003cp\u003eAlterations in global protein acetylation levels have been linked to parasite stage transitions and physiological adaptations, as previously shown by us in \u003cem\u003eT. brucei\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Proteomic and acetylome profiling of DAC mutants confirmed extensive remodeling, with DAC5 deficiency causing the most pronounced changes. Notably, DAC5 loss led to hyperacetylation of cytoskeletal and metabolic proteins, including aldolase, α-tubulin, and actin, potentially contributing to defects in cell cycle progression and differentiation.\u003c/p\u003e\u003cp\u003eFurthermore, the differential acetylation of kinases essential for \u003cem\u003eL. mexicana\u003c/em\u003e development, such as TOR2, supports a role for acetylation in modulating signaling pathways critical for stage progression. The acetylation of cytoskeletal components and TOR2 in DAC5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e is particularly intriguing, given the known involvement of TOR2 in cytokinesis in \u003cem\u003eT. brucei\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. This, along with the altered cell cycle phase durations and differentially acetylation of Kharon, a well-known player involved in trypanosomatids cell cycle, observed in DAC5\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e parasites \u003csup\u003e\u003cspan additionalcitationids=\"CR50 CR51\" citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e, suggests that DAC5 regulates both morphological transitions and cell cycle progression in promastigote forms through acetylation-mediated control of cytoskeletal organization and signaling.\u003c/p\u003e\u003cp\u003eIn conclusion, our study highlights the critical role of lysine acetylation and the regulatory functions of zinc-dependent lysine deacetylases (DACs) in \u003cem\u003eLeishmania mexicana\u003c/em\u003e stage differentiation and virulence. Through functional, phenotypic, transcriptomic and proteomic analyses, we demonstrate that specific DACs are essential for parasite development, proliferation, and morphological transitions. These findings not only emphasize the importance of acetylation dynamics in the regulation of key cellular processes but also open new avenues of research into the broader role of acetylation in trypanosomatid biology. In this sense, the attenuation driven by DAC5 disruption together with the \u003cem\u003ein vivo\u003c/em\u003e protection against a new infection can be further explored to develop live-attenuated parasite vaccines against leishmaniases. Moreover, our results suggest that targeting DACs may represent a promising strategy for therapeutic intervention in leishmaniases.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Dr. Carolina Catta-Preta for scientific support during the generation of DACs mutant parasites. The authors also acknowledge the Fiocruz Core Facilities (Next Generation Sequencing Facility-RPT 01I at Aggeu Magalh\u0026atilde;es Institute). The authors thank Fiocruz\u0026rsquo;s Network of Technological Platforms (https://plataformas.fiocruz.br/) and the animal facility at Ren\u0026eacute; Rachou Institute, Oswaldo Cruz Founation \u0026ndash; IRR/Fiocruz Minas, Belo Horizonte, Minas Gerais, Brazil, for providing technical assistance and research support services. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was financed, in part, by the S\u0026atilde;o Paulo Research Foundation (FAPESP), Brasil. Process Number 2018/09948-0, 2020/07870-4 and 2022/03075-0 to N.M.; 2019/13765-1 and 2021/13477-6 to S.R.M; 2023/16672-0 to ABL; 2025/03898-5 to MANG; 2019/10753-2 and 2020/10277-3 to MSdS). NM and RLMN are CNPq research fellows (grant numbers CNPq 312353/2023-5 and 306191/2024-5). A.M.R. was supported by Instituto Aggeu Magalh\u0026atilde;es/FIOCRUZ - CNPq grant 400739/2019-4. Research coordinated by RLMN is supported by \u0026ldquo;Funda\u0026ccedil;\u0026atilde;o de Amparo \u0026agrave; Pesquisa do Estado de Minas Gerais \u0026ndash; Fapemig, from \u0026ldquo;Rede Mineira de Imunobiol\u0026oacute;gicos Aplicada \u0026agrave; Vacinas, Biof\u0026aacute;rmacos e Diagn\u0026oacute;stico para Leishmaniose Visceral\u0026rdquo; grant #RED-0003222 and \u0026ldquo;ReMinD - Rede Mineira de Diagn\u0026oacute;stico de Doen\u0026ccedil;as Infecciosas\u0026rdquo; #RED-00196\u0026ndash;23; and \u0026ldquo;Programa Inova Fiocruz\u0026rdquo; from Oswaldo Cruz Foundation. National Natural Science Foundation of China (32402916 to N.Z. and 32072880 to Q.C.). This research was supported in part by the Intramural Research Program of the National Institutes of Health (NIH). The contributions of the NIH authors were made as part of their official duties as NIH federal employees, are in compliance with agency policy requirements, and are considered Works of the United States Government. However, the findings and conclusions presented in this paper are those of the author(s) and do not necessarily reflect the views of the NIH or the U.S. Department of Health and Human Services.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBurza, S., Croft, S. L. \u0026amp; Boelaert, M. Leishmaniasis. \u003cem\u003eLancet\u003c/em\u003e \u003cstrong\u003e392\u003c/strong\u003e, 951\u0026ndash;970 (2018).\u003c/li\u003e\n\u003cli\u003eDe Pablos, L. M., Ferreira, T. R. \u0026amp; Walrad, P. B. 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A. \u003cem\u003eet al.\u003c/em\u003e KHARON Is an Essential Cytoskeletal Protein Involved in the Trafficking of Flagellar Membrane Proteins and Cell Division in African Trypanosomes. \u003cem\u003eJ Biol Chem\u003c/em\u003e \u003cstrong\u003e291\u003c/strong\u003e, 19760\u0026ndash;73 (2016).\u003c/li\u003e\n\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Leishmania, acetylation, acetylome, lysine deacetylases, parasite differentiation","lastPublishedDoi":"10.21203/rs.3.rs-7520632/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7520632/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eProtein acetylation regulates essential processes across eukaryotes. In trypanosomatids, stage-specific acetylation suggests roles in parasite differentiation. Here, we functionally characterized zinc-dependent lysine deacetylases (DAC1, DAC3, DAC4, and DAC5) in \u003cem\u003eLeishmania mexicana\u003c/em\u003e. CRISPR-Cas9-mediated disruption revealed that DAC1 and DAC3 are essential for procyclics, while DAC4 and DAC5 are dispensable. DAC1 and DAC5 are localized in the cytoplasm, and DAC3 and DAC4 in the nucleus. Functional analysis implicates DAC1, DAC3, and DAC5 in procyclic proliferation, whereas DAC1 and DAC5 drive promastigote-to-metacyclic differentiation. DAC5 was required for metacyclogenesis in the sand flies, the promastigote\u0026ndash;amastigote transition, and amastigote intracellular replication. Notably, DAC5-null parasites failed to induce lesions in mice, displaying an attenuated phenotype. Proteomic profiling uncovered altered acetylation patterns in DAC mutants, linking DAC5 to cytoskeleton regulation and cell cycle control. These findings identify acetylation as a central regulator of \u003cem\u003eLeishmania\u003c/em\u003e stage differentiation and highlight DAC5 as a key factor in parasite virulence.\u003c/p\u003e","manuscriptTitle":"AcetyLeish: acetylation-driven control of stage differentiation and virulence in Leishmania mexicana","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-17 06:18:52","doi":"10.21203/rs.3.rs-7520632/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"224e8b02-0fc4-47c4-abbb-a6dd5d420355","owner":[],"postedDate":"September 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":54826090,"name":"Biological sciences/Microbiology/Parasitology/Parasite host response"},{"id":54826091,"name":"Biological sciences/Microbiology/Parasitology/Parasite biology"}],"tags":[],"updatedAt":"2025-11-13T15:20:25+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-17 06:18:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7520632","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7520632","identity":"rs-7520632","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}