Unexpected Role of Sterol Synthesis in RNA Stability and Translation in Leishmania

biomedicines

Article Unexpected Role of Sterol Synthesis in RNA Stability and Translation in Leishmania

Zemﬁra N. Karamysheva 1,*, Samrat Moitra 1, Andrea Perez 1,2, Sumit Mukherjee 1,3, Elena B. Tikhonova 4, Andrey L. Karamyshev 4 and Kai Zhang 1,*

1 Department of Biological Sciences, Texas Tech University, Lubbock, TX 79409, USA; [email protected] (S.M.); [email protected] (A.P.); [email protected] (S.M.) 2 Honors College, Texas Tech University, Lubbock, TX 79409, USA 3 Division of Infectious Diseases, Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA 4 Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA; [email protected] (E.B.T.); [email protected] (A.L.K.) * Correspondence: zemﬁ[email protected] (Z.N.K.); [email protected] (K.Z.); Tel.: +1-806-834-5075 (Z.N.K.); +1-806-834-0550 (K.Z.)

Abstract: Leishmania parasites are trypanosomatid protozoans that cause leishmaniasis affecting millions of people worldwide. Sterols are important components of the plasma and organellar membranes. They also serve as precursors for the synthesis of signaling molecules. Unlike ani- mals, Leishmania does not synthesize cholesterol but makes ergostane-based sterols instead. C-14- demethylase is a key enzyme involved in the biosynthesis of sterols and an important drug target.

Citation: Karamysheva, Z.N.; In Leishmania parasites, the inactivation of C-14-demethylase leads to multiple defects, including Moitra, S.; Perez, A.; Mukherjee, S.; increased plasma membrane ﬂuidity, mitochondrion dysfunction, hypersensitivity to stress and Tikhonova, E.B.; Karamyshev, A.L.; reduced virulence. In this study, we revealed a novel role for sterol synthesis in the maintenance Zhang, K. Unexpected Role of Sterol of RNA stability and translation. Sterol alteration in C-14-demethylase knockout mutant leads to Synthesis in RNA Stability and increased RNA degradation, reduced translation and impaired heat shock response. Thus, sterol Translation in Leishmania. Biomedicines biosynthesis in Leishmania plays an unexpected role in global gene regulation. 2021, 9, 696. https://doi.org/ 10.3390/biomedicines9060696 Keywords: Leishmania; sterol; C-14-demethylase; stress tolerance; RNA degradation; polysome; endoplasmic reticulum; translation; regulation of gene expression Academic Editors: Gabriela Santos-Gomes and Maria Pereira

Received: 18 May 2021 1. Introduction Accepted: 15 June 2021 Protozoan parasites of the genus Leishmania cause leishmaniasis infecting 10–12 million Published: 19 June 2021 people worldwide [1]. There are three major forms of leishmaniasis. Visceral leishmaniasis is the most severe form, with a mortality rate of almost 100% if left untreated [2,3]. Publisher’s Note: MDPI stays neutral Mucocutaneous leishmaniasis can produce disfiguring lesions of the nose, mouth and with regard to jurisdictional claims in throat cavities. The cutaneous form of leishmaniasis is the most common type representing published maps and institutional affil- 50–75% of all new cases [4]. iations. All three forms of leishmaniasis are transmitted through the bite of sand fly vectors (Phlebotomus spp. and Lutzomyia spp.). During their life cycle, these dixenic protozoans alternate between flagellated, extracellular promastigotes, which live in the midgut of sand flies, and non-flagellated amastigotes residing in the phagolysosomal compartment Copyright: © 2021 by the authors. of mammalian macrophages [5]. Promastigotes are transmitted with sand fly saliva into Licensee MDPI, Basel, Switzerland. the mammalian host during blood feeding, where they are rapidly engulfed by phagocytic This article is an open access article cells and differentiate into amastigotes. The changes in temperature, pH and nutrients distributed under the terms and that Leishmania parasites encounter in the mammalian host appear to be essential for the conditions of the Creative Commons promastigote to amastigote differentiation [6]. Options for leishmaniasis control are very Attribution (CC BY) license (https:// limited due to the lack of a vaccine, toxic side effects of drugs and rapid emergence of creativecommons.org/licenses/by/ drug-resistant strains [7,8]. Therefore, there is an urgent need to understand the molecular 4.0/).

Biomedicines 2021, 9, 696. https://doi.org/10.3390/biomedicines9060696 https://www.mdpi.com/journal/biomedicines Biomedicines 2021, 9, 696 2 of 16

mechanisms utilized by Leishmania parasites to survive in different hosts, as discoveries in basic biology can lead to new medicine. Lipid metabolism is a very important yet understudied area in protozoan parasites. Besides serving as an energy source and building blocks of the membrane, lipids are im- plicated in parasite-host interaction and pathogenesis [9–11]. Drugs such as miltefosine and antimony induce lipid perturbations which contribute to the development of drug resistance in Leishmania parasites [12,13]. Notably, Leishmania parasites produce different types of sterols from humans, making the sterol synthesis pathway an attractive pharma- cological target [14,15]. Specifically, Leishmania produces ergostane-based sterols such as ergosterol and 5-dehydroepisterol while human cells synthesize cholesterol [16]. Sterols are important constituents of the plasma membrane (PM), endoplasmic reticulum (ER) and organellar membranes. Because of their rigid and hydrophobic structure, sterols reduce the flexibility of acyl chains of neighboring phospholipids and increase membrane rigidity and tightness [17]. They are also involved in vesicular transport [17]. Sterols defects are known to affect not only membrane permeability and fluidity but also the localization of membrane-bound proteins and transport of proteins [18]. In mammalian cells, the distribution of sterols in specific organellar membranes is strictly regulated, and its impairment leads to many diseases [19]. In yeasts, defects in sterol synthesis lead to disruption in ER organization and changes in lipid organization of the PM [20]. C-14-demethylase (C14DM) catalyzes the removal of a methyl group from the carbon- 14 position of lanosterol, a key step in the synthesis of ergostane-based sterols [21]. The C14DM-null mutant (c14dm−) has been characterized in Leishmania major LV39 strain [22]. This mutant cannot remove the C-14-methyl group from lanosterol or other sterol intermediates. The defect leads to increased membrane fluidity, mitochondrion dysfunction, superoxide accumulation, hypersensitivity to heat and severely reduced virulence in mice [22,23]. In this study, we revealed an unexpected role of C14DM in the regulation of RNA levels in Leishmania parasites. We uncovered that defects in sterol synthesis lead to reduced RNA stability and protein synthesis, which likely contribute to their impaired stress response. Our study is the first of its kind that links sterol synthesis to the global regulation of gene expression at the level of RNA stability.

2. Materials and Methods 2.1. Reagents Actinomycin D and MitoSox Red were purchased from Sigma (St. Louis, MO, USA) and Thermo Fisher Scientiﬁc (Waltham, MA, USA), respectively. Trizol and Trizol LS were purchased from Life Technologies (Carlsbad, CA, USA). L-Glutathione (reduced form) and antimycin A were purchased from ENZO Life Sciences (Farmingdale, NY, USA). All other chemicals were purchased from VWR International (Radnor, PA, USA) unless otherwise speciﬁed.

2.2. Leishmania Culturing and Treatments Leishmania major LV39 (Rho/SU/59/P), c14dm−(C14DM-null mutant) and c14dm−/ + C14DM (episomal add-back) promastigotes were cultivated at 27 ◦C in complete M199 media containing 10% fetal bovine serum and additional supplements as previously described [24]. Culture densities over time were determined by direct cell counting using a hemacytometer. The BCA protein assay kit (Thermo Fisher Scientiﬁc) was used to determine protein concentration in cell lysates according to the manufacturer’s recommendation. In order to block transcription and monitor RNA degradation, actinomycin D was added at 10 µg/mL to mid-log phase cultures (3–6 × 106 cells/mL). Equal aliquots of cultures were taken at the indicated times for further analysis. Some experiments required antioxidant treatment of cells. Brieﬂy, Leishmania promastigotes were seeded at 4 × 105 cells/mL and treated with different concentrations of L-glutathione (1, 2 or 4 mM) for 48 h prior to cell collection for further analysis. In Biomedicines 2021, 9, 696 3 of 16

order to induce mitochondrial oxidative stress, LV39 wild-type (WT) cells prepared at a concentration of 1.0 × 107 cells/mL were treated with 5 µM of antimycin A for 3 h at 27 ◦C. Then parasites were stained with 10 µM of MitoSox Red. Mean fluorescence intensities (MFI) were determined by flow cytometry using an Attune NxT Acoustic Flow Cytometer (Thermo Fisher Scientific) to confirm the induction of mitochondrial stress prior to cell collection for downstream analysis. Cell viability was determined by measuring the incorporation of propidium iodide (PI, 5.5 µg/mL) via flow cytometry as described [23]. Neither antimycin A nor L-glutathione treatments significantly affected cell viability based on PI staining (<5% PI positive).

2.3. Polysome Profiling Polysome profiling experiments were performed as published earlier [25]. Briefly, Leishmania promastigotes of WT, c14dm− and c14dm−/ + C14DM were grown in flasks until cell density reached 5 × 106 cells/mL. An equal number of cells (1.5 × 108 promastigotes) were used for polysome profiling for each experimental condition. Before lysis, cells were treated with 100 µg/mL of cycloheximide for 15 min at 27 ◦C to stabilize ribosomes on mRNAs. Cells were lysed on ice in a buffer containing 20 mM Hepes–KOH (pH 7.5), 10 mM MgCl2, 100 mM KCl, 2 mM DTT, 1% NP-40, 1 × protease inhibitor cocktail (EDTA- free) from Sigma, 200 units/mL RNasin (Thermo Fisher Scientific) and 100 µg/mL of cycloheximide. Details of cell lysate preparation were described previously [25]. The lysate was clarified by centrifugation at 11,200 g for 10 min at 4 ◦C. In order to separate polysomes, 500 µL of the clarified lysate was loaded on top of a 10%–50% sucrose gradient containing 20 mM Hepes–KOH (pH 7.5), 100 mM KCl, 10 mM MgCl2, 1 mM DTT and 200 units/mL RNasin, and subjected to ultracentrifugation using an SW41 rotor for 2 h at 260,000× g and 4 ◦C. After centrifugation, 500 µL of fractions were collected using a Piston Gradient Fractionator from BioComp Instruments (Fredericton, NB, Canada). Trizol LS was added to fractions immediately, and samples were stored at −80 ◦C until RNA extraction.

2.4. RNA Extraction, cDNA Preparation and Real-Time Reverse-Transcription Quantitative PCR (RT-qPCR) Total RNA was isolated using Trizol or Trizol LS (for polysomal fractions) and quantified spectrophotometrically using a NanoDrop device (Thermo Fisher Scientific) as described [25]. cDNA samples were prepared using a High-Capacity cDNA Reverse Transcrip- tion Kit from Applied Biosystems (Waltham, MA, USA) according to the manufacturer’s recommendation. Real-time quantitative polymerase chain reactions (RT-qPCR) were performed on a Quant Studio 12 K Flex Real-Time PCR System using Power SYBR Green PCR Master Mix (Applied Biosystems), according to the manufacturer’s protocol. The comparative ∆∆CT method was used to quantify the qPCR results [26]. For the analysis of gene expression, synthetic outer membrane protein A (OmpA) mRNA was added to all samples prior to RNA extraction and used for normalization as described [25]. The RNA levels were analyzed by RT-qPCR for the following genes: tubulin, heat shock protein 70 (HSP70), heat shock protein 83 (HSP83), sterol-24-C- methyltransferase (SMT), 18S ribosomal RNA (18S rRNA) and 28S ribosomal RNA (28S rRNA). The primers used in RT-qPCR reactions and corresponding gene identification numbers are presented in Supplementary File S1.

2.5. ER Labelling and Confocal Microscopy ER staining was performed using an anti-Trypanosoma brucei BiP antibody (kind gift from Dr. Jay Bangs, University at Buffalo, SUNY). Log phase WT, c14dm− and c14dm−/ + C14DM promastigotes were incubated at 27 ◦C (control) or 37 ◦C (experimental) for 2 h. Afterwards, parasites were washed in phosphate-buffered saline (PBS), attached to poly-L- lysine coated coverslips, ﬁxed with 3.7% formaldehyde, and then permeabilized on ice with ethanol. Incubation with the rabbit anti-T. brucei BiP antiserum (1:1000) was performed at room temperature for 40 minutes. After washing with PBS, coverslips were incubated with a goat anti-rabbit-Alexa Fluor 488 (1:2000) antiserum for 40 min. An Olympus (Center Valley, PA, USA) Fluoview FV3000 Laser Scanning Confocal Microscope was used to visualize the Biomedicines 2021, 9, x FOR PEER REVIEW 4 of 16

C14DM promastigotes were incubated at 27 °C (control) or 37 °C (experimental) for 2 h. Afterwards, parasites were washed in phosphate-buffered saline (PBS), attached to poly- L-lysine coated coverslips, fixed with 3.7% formaldehyde, and then permeabilized on ice with ethanol. Incubation with the rabbit anti-T. brucei BiP antiserum (1:1000) was per-

Biomedicines 2021, 9, 696 formed at room temperature for 40 minutes. After washing with PBS, coverslips were4 of in- 16 cubated with a goat anti-rabbit-Alexa Fluor 488 (1:2000) antiserum for 40 min. An Olym- pus (Center Valley, PA, USA) Fluoview FV3000 Laser Scanning Confocal Microscope was used to visualize the intensity and localization of BiP from randomly selected cells using intensitythe cellSens and Imaging localization Software of BiP (Olympus). from randomly The number selected of cells cells using analyzed the cellSens at 27 °C Imaging was 63 ◦ − Software(WT), 131 (Olympus). (c14dm ̅ ) and The 95 number (c14dm of ̅ / cells+ C14DM analyzed). The at number 27 C was of cells 63 (WT), analyzed 131 (c14dm at 37 °C) andwas − ◦ − 95152 (c14dm (WT), 106/ + C14DM).(c14dm ̅ ) Theand number65 (c14dm of ̅ cells/ + C14DM analyzed). at 37 C was 152 (WT), 106 (c14dm ) and 65 (c14dm−/ + C14DM). 2.6. Statistical analysis 2.6. Statistical Analysis Unless specified otherwise, assay values in all figures are averaged from three inde- pendentUnless biological specified repeats, otherwise, and error assay bars values represent in all figures standard are deviations. averaged from The Student’s three inde- t- pendent biological repeats, and error bars represent standard deviations. The Student’s test was used in pairwise comparisons. Differences among multiple groups were assessed t-test was used in pairwise comparisons. Differences among multiple groups were assessed by One-way Anova followed by Tukey’s or Dunnett’s test. P-values indicating statistical by One-way Anova followed by Tukey’s or Dunnett’s test. p-values indicating statistical significance were grouped in figures as ns: not significant; *: p < 0.05; **: p < 0.01; and ***: significance were grouped in figures as ns: not significant; *: p < 0.05; **: p < 0.01; and p < 0.001. ***: p < 0.001.

3.3. ResultsResults 3.1.3.1. DefectsDefects inin SterolSterol SynthesisSynthesis LeadLead toto GlobalGlobal ReductionReduction inin RNARNA andand ProteinProtein LevelsLevels inin c14dmc14dm− ̅ C14DMC14DM isis an an important important enzyme enzyme in ergosterolin ergosterol biosynthesis biosynthesis and theand target the target of azole of drugs. azole Indrugs.L. major In ,L. this major enzyme, this enzyme is mostly is localized mostly localized at the ER, at and the onlyER, and a minor only amount a minor is amount found in is thefound mitochondrion in the mitochondrion [22]. Genetic [22]. or Genetic chemical or inactivation chemical inactivation of C14DM ledof C14DM to a complete led to lossa com- of ergostane-basedplete loss of ergostane-based sterols and accumulation sterols and of ac C-14-methylatedcumulation of C-14-met sterols [22hylated]. c14dm sterols− mutants [22]. alsoc14dm displayed ̅ mutants increased also displayed membrane increased ﬂuidity, membra reducedne fluidity, virulence reduced and extreme virulence sensitivity and ex- totreme stress. sensitivity to stress. InIn orderorder toto fullyfully understandunderstand thethe consequencesconsequences causedcaused byby defectivedefective sterolsterol synthesis,synthesis, wewe examinedexamined the total RNA and and protein protein levels levels in in c14dmc14dm .̅ − Remarkably,. Remarkably, we wefound found an ap- an approximatelyproximately 40% 40% reduction reduction in intotal total RNA RNA levels levels in c14dm in c14dm ̅ in− comparisonin comparison to WT to and WT add- and add-backback parasites parasites (Figure (Figure 1A).1 TheA). Themutant mutant also alsohad ~20% had ~20% less total less protein total protein (Figure (Figure 1B). These1B). Theseresults results suggest suggest a global a global dysregulation dysregulation of gene of expression gene expression in the incells the defective cells defective in sterol in sterolproduction. production. Adding Adding C14DMC14DM back completelyback completely rescues rescues the defects the defects at both atRNA both and RNA protein and proteinlevels. levels.

A B ns ns 120 *** 120 *

** % **

% 100 100 level,

80 80 level,

60 60 RNA protein

40 40 Relative Relative 20 20

0 0 WT c14dm ̅ c14dm ̅/+C14DM WTWT cc14dm14dm ̅̅ c14c14dmdm ̅/+C ̅/+C14DM14DM

FigureFigure 1.1.The Thetotal total RNA RNA and and protein protein levels levels are are reduced reduced in inc14dm c14dm– mutant. ̅ mutant. ( A(A)) The The total total RNA RNA was was extracted extracted from from 1 1× ×10 1077 cellscells ofof WT,WT,c14dm c14dm− ̅ and andc14dm c14dm− / ̅ /+ + C14DMC14DM cellscells andand quantiﬁedquantified usingusing NanoDropNanoDrop inin relationrelation toto WT.WT. (B(B)) The The total total protein protein 7 levelslevels from from 1 1× × 107 cells of WT, c14dmc14dm− ̅ andandc14dm c14dm− ̅// + C14DM were measured using BCA assay,assay, andand theirtheir relativerelative levelslevels werewere calculated calculated in in relation relation to to WT. WT. ns: ns: not not signiﬁcant; significant; *: *:p p< < 0.05; 0.05; **: **:p p< < 0.01;0.01; andand ***:***: pp << 0.001.

In eukaryotic cells, most RNA is rRNA, while the protein-encoding mRNA constitutes about 5% of total RNA [27,28]. Global reduction in RNA level suggests that rRNA is likely reduced. In the next experiment, we examined what type of RNA is affected by defects in sterol synthesis. Total RNA was extracted, and RT-qPCR was performed to measure levels of selected RNAs as described [25]. We found that both 28S and 18S rRNA, as well as Biomedicines 2021, 9, x FOR PEER REVIEW 5 of 16

In eukaryotic cells, most RNA is rRNA, while the protein-encoding mRNA constitutes about 5% of total RNA [27,28]. Global reduction in RNA level suggests that rRNA is Biomedicines 2021, 9, 696 likely reduced. In the next experiment, we examined what type of RNA is affected5of by 16 defects in sterol synthesis. Total RNA was extracted, and RT-qPCR was performed to measure levels of selected RNAs as described [25]. We found that both 28S and 18S rRNA, as well as individual mRNAs for tubulin and HSP83, displayed substantially lower levels individual mRNAs for tubulin and HSP83, displayed substantially lower levels in c14dm− in c14dm ̅ and c14dm ̅ / + C14DM rescued the defects (Figure 2). and c14dm−/ + C14DM rescued the defects (Figure2).

28S rRNA 18S rRNA A 1.2 ns 1.4 ns ** 1.2 1 **

level 1 level 0.8 0.8

RNA 0.6 RNA 0.6 0.4 0.4 0.2

Relative 0.2 Relative 0 0 B WTwt c14dm C14- ̅ c14dm AB̅/+C14DM WTwt c14dm C14- ̅ c14dm AB̅/+C14DM Tubulin mRNA Hsp83 mRNA 1.2 ns 1.2 ns *** 1 *** 1 level level

0.8 0.8

RNA 0.6 RNA

0.6

0.4 0.4 0.2 0.2 Relative Relative 0 0 WTwt c14dm C14DM- ̅ c14dm C14-/AB ̅/+C14DM WTwt c14dm C14DM- ̅ c14dm C14-/AB ̅/+C14DM

− FigureFigure 2. 2. LevelsLevels of of ribosomal ribosomal RNA RNA and and individual individual mRNAs mRNAs are are reduced reduced inin c14dmc14dm ̅ . .The The total total RNA RNA was was extracted extracted from from 1 7 − − ×1 10×710 cellscells of WT,of WT, c14dmc14dm ̅ andand c14dmc14dm ̅ / + C14DM/ + C14DM cells. cells.The artificial The artiﬁcial outer membrane outer membrane protein protein A (OmpA) A (OmpA) mRNA mRNAwas added was prioradded to priorRNA toextraction RNA extraction for further for normalizatio further normalization.n. RNA levels RNA were levels measured were measured by RT-qPCR. by RT-qPCR. (A) 28S (left (A) panel) 28S (left and panel) 18S (rightand 18S panel) (right rRNA panel) levels. rRNA (B levels.) Tubulin (B) Tubulin(left) and (left) HSP83 and (right) HSP83 mRNA (right) levels. mRNA ns: levels. not significant; ns: not signiﬁcant; **: p < 0.01; **: p and< 0.01; ***: andp < 0.001.***: p < 0.001.

TheseThese results results suggest suggest that impairment in sterolsterol biosynthesis causescauses globalglobal downregu-downreg- ulationlation of of rRNA rRNA and and mRNAs mRNAs in inLeishmania Leishmania major majorparasites. parasites.

− 3.2.3.2. RNA RNA Stability Stability is Is Compromised Compromised in in c14dm c14dm ̅ TheThe reduction reduction in in RNA RNA levels levels can can be be caused caused either either by by increased increased RNA RNA degradation degradation or defectsor defects in transcription. in transcription. In order In order to examine to examine whether whether defects defects in sterol in sterolsynthesis synthesis affect affectRNA − − stability,RNA stability, we treated we treated WT, c14dm WT, c14dm ̅ and c14dmand c14dm ̅ / + C14DM/ + C14DM cells with cells actinomycin with actinomycin D to block D to transcriptionblock transcription and monitored and monitored the rates the ratesof RN ofA RNA degradation. degradation. Actinomycin Actinomycin D inhibits D inhibits all transcriptionall transcription independent independent of the of the types types of ofRNA RNA polymerases polymerases [29]. [29]. Actinomycin Actinomycin D D was was addedadded to to mid-log phase cells,cells, andand aa time-coursetime-course experiment experiment was was performed performed as as described described in inFigure Figure3. The3. The RNA RNA levels levels were were quantiﬁed quantified relative relative to theto the starting starting point point of actinomycinof actinomycin D treatment for each strain. The absolute amount of total RNA was lower in c14dm− at the D treatment for each strain. The absolute amount of total RNA was lower in c14dm ̅ at the− startingstarting point point (Figure (Figure 11B).B). OurOur resultsresults demonstratedemonstrate that that total total RNA RNA decays decays faster faster in inc14dm c14dm ̅ afterafter blocking blocking transcription transcription with with actinomycin actinomycin D, D,providing providing strong strong support support to the to idea the ideathat that RNA stability is substantially compromised in the mutant (Figure3A). We checked RNA stability is substantially compromised in the mutant (Figure 3A). We checked the the rate of degradation of individual mRNAs as well (Figure3B). Tubulin mRNA decays rate of degradation of individual mRNAs as well (Figure 3B). Tubulin mRNA decays much faster in the mutant; after one hour of treatment, only about 57% of RNA remains in much faster in the mutant; after one hour of treatment, only about 57% of RNA remains c14dm− versus 94% in WT parasites. HSP83 mRNA exhibits even faster degradation. The in c14dm ̅ versus 94% in WT parasites. HSP83 mRNA exhibits even faster degradation. The half-life of HSP83 mRNA is 48.9 min in c14dm−, 137.7 min in WT and 110.5 min in c14dm−/ half-life of HSP83 mRNA is 48.9 min in c14dm ,̅ 137.7 min in WT and 110.5 min in c14dm ̅ + C14DM parasites, respectively (Figure3B). / + C14DM parasites, respectively (Figure 3B). BiomedicinesBiomedicines2021 2021,,9 9,, 696x FOR PEER REVIEW 66 ofof 1616

AB 1.2 1.2 1 Tubulin mRNA Total RNA 0.8 1 0.6 ** 0.4 0.8 ** 0.2 Actinomycin D

Fraction of remaining mRNA mRNA remaining of Fraction 0 0.6 Actinomycin D 01368 hours wt C14DM- C14-/ABc14dm ̅ /+C14DM 0.4 WT c14dm ̅

Fraction of remaining RNA RNA remaining of Fraction 1.2 0.2 1 HSP83 mRNA 0.8 0 01368 0.6 hours ** 0.4 WT c14dmc14dm ̅̅ c14dm ̅/+C14DM ̅ /+C14DM 0.2 Actinomycin D

Fraction of remaining mRNA mRNA remaining of Fraction 0 01368 hours wtWT C14DM-c14dm ̅ C14-/ABc14dm ̅ /+C14DM

− FigureFigure 3.3. RNA in in c14dmc14dm ̅ degradesdegrades faster faster in in comparison comparison to toWT WT and and add- add-backback parasites. parasites. Actinomycin Actinomycin D was D wasadded added to mid- to mid-loglog cultures, cultures, and andequal equal aliquots aliquots of cultures of cultures were were taken taken at the at indicated the indicated times. times. RNA RNA was wasextracted extracted and andanalyzed analyzed by RT- by RT-qPCRqPCR relative relative to the to values the values at the at starting the starting point. point. (A) Total (A) TotalRNA RNAwas quantified was quantiﬁed using NanoDrop using NanoDrop over time. over (B time.) The (relativeB) The relativeabundance abundance of tubulin of tubulinand HSP83 and mRNA HSP83 mRNAwas quantified was quantiﬁed by RT-qPCR by RT-qPCR over time. over **: time. p < 0.01. **: p < 0.01.

We alsoalso examinedexamined the the degradation degradation of of 18S 18S and and 28S 28S rRNA. rRNA. While While rRNA rRNA is consideredis considered to beto morebe more stable, stable, it is it also is also subject subject to quality to quality control control in eukaryotes in eukaryotes [30]. It [30]. also It degrades also degrades under conditionsunder conditions of stress of orstress starvation or starvation [31]. We [31]. have We found have found that 18S that and 18S 28S and rRNA 28S rRNA degrades de- signiﬁcantlygrades significantly faster in faster the mutant, in the mutant, although alth theyough are they more are stable more than stable tubulin than tubulin and HSP83 and mRNAsHSP83 mRNAs (Figure (Figure S1). S1).

3.3. RNA Degrades Faster during Heat Shock andand thethe InductionInduction ofof HeatHeat ShockShock ResponseResponse IsIs Com- − Compromisedpromised in c14dm in c14dm ̅ When cellscells faceface stress, stress, such such as as heat heat shock, shock, a globala global downregulation downregulation of geneof gene expression expres- occurs, but genes involved in stress response to increase survival are selectively upregu- sion occurs, but genes involved in stress response to increase survival are selectively up-− latedregulated [32]. HSP83[32]. HSP83 is known is known to be upregulatedto be upregulated in response in response to heat shockto heat [32 shock]. The [32].c14dm The mutantc14dm ̅ mutant displays displays extreme extreme sensitivity sensitivity to stress to conditions stress conditions such as such heat as shock heat and shock starva- and tion [22,23]. The ability to cope with stress is essential for Leishmania survival in mammals starvation [22,23]. The ability to cope with stress is essential for Leishmania survival in where they encounter dramatic changes in temperature, nutrient availability and pH [33]. mammals where they encounter dramatic changes in temperature, nutrient availability To examine if the heat shock response is altered in c14dm−, we subjected promastigotes to and pH [33]. To examine if the heat shock response is altered in c14dm ̅ , we subjected 37 ◦C treatment and measured HSP70 and HSP83 mRNA levels by RT-qPCR (Figure4A,B ). promastigotes to 37 °C treatment and measured HSP70 and HSP83 mRNA levels by RT- Sterol-24-C-methyltransferase (SMT) mRNA and 18S rRNA were included as negative qPCR (Figure 4A–B). Sterol-24-C-methyltransferase (SMT) mRNA and 18S rRNA were controls, which should not be induced under heat shock (Figure4C,D). As expected, HSP70 included as negative controls, which should not be induced under heat shock (Figure 4C– and HSP83 mRNAs showed a 2–4-fold increase in WT parasites after four hours at 37 ◦C. D). As expected, HSP70 and HSP83 mRNAs showed a 2–4-fold increase in WT parasites In contrast, c14dm− mutant had a substantially diminished response (1.2–1.5-fold increase after four hours at 37 °C. In contrast, c14dm ̅ mutant had a substantially diminished re- after two hours) to the heat shock. This initial increase was fast and signiﬁcantly dropped sponse (1.2–1.5-fold increase after two hours) to the heat shock. This initial increase was down with longer incubation. The defects were completely reversed in c14dm−/ + C14DM fast and significantly dropped down with longer incubation. The defects were completely cells (Figure4A,B). We also observed accelerated degradation of SMT mRNA and 18S ̅ rRNAreversed in c14dmin c14dm− (Figure / + C14DM4C,D). cells After (Figure 8 h of 4A–B). incubation We also at 37observed◦C, SMT accelerated mRNA level degrada- was ̅ dramaticallytion of SMT mRNA diminished and to18S ~5% rRNA in the in c14dm mutant, (Figure whereas 4C–D). only slightAfter changes8 h of incubation were detected at 37 in°C, WT SMT and mRNAc14dm level−/ + was C14DM dramatically parasites. diminished While 18S to rRNA ~5% in remained the mutant, stable whereas in WT only and slight changes were detected in WT and c14dm ̅ / + C14DM parasites. While 18S rRNA Biomedicines 2021, 9, x FOR PEER REVIEW 7 of 16

Biomedicines 2021, 9, 696 7 of 16

remained stable in WT and add-back parasites even after 8 h of heat shock, its level was add-backstrikingly parasites reduced even to 18% after in 8sterol h of heatdefect shock,ive mutant. its level As was previously strikingly described reduced [22], to 18% c14dm in ̅ sterolparasites defective were mutant. mostly dead As previously after 24 h describedof heat shock, [22], whilec14dm both− parasites WT and were c14dm mostly ̅ / + C14DM dead afterparasites 24 h of were heat mostly shock, whilealive bothby that WT time and andc14dm maintained−/ + C14DM 18S parasites rRNA level were at mostly around alive 80% by(Figure that time 4D, and E). maintained 18S rRNA level at around 80% (Figure4D,E).

A B E 3.5 4.5 HSP70 mRNA HSP83 mRNA 3 4 100 3.5 90 *** 2.5 80 ** 3 70 2 *** *** 60 ns 2.5 ns ns ***ns 50 1.5 2 40 ** 1.5 30 1 1 death cell of % 20 10 Relative mRNARelative levels 0.5 0.5 Relative mRNA levels mRNA Relative 0 0 0 0 2 4 6 8 24 0246824 0246824 Hours at 37°C Hours at 37°C Hours at 37 °C WT c14dmc14dm ̅ ̅ c14dmc14dm ̅ / +C14DM ̅/+C14DM WT c14dmc14dm ̅ ̅ c14dmc14dm ̅/+C14DM̅ /+C14DM C D WT c14dm ̅ c14dmc14dm ̅ /̅ /+C14DM+C14DM 1.2 SMT mRNA *** 1.4 18S rRNA *** ns *** ns 1 *** 1.2 ns ns 1 0.8 0.8 0.6 0.6 0.4 0.4

0.2 levelsRNA Relative 0.2 Relative mRNA levels mRNA Relative 0 0 0246824 0246824 Hours at 37°C Hours at 37°C WT c14dmc14dm ̅ ̅ c14dmc14dm ̅/+C14DM ̅ /+C14DM WT c14dmc14dm ̅ ̅ c14dmc14dm ̅/+C14DM ̅ /+C14DM

FigureFigure 4. 4.C14dm C14dm– mutants ̅ mutants show show a dramatic a dramatic decrease decrease in RNAin RNA levels levels during during heat heat shock. shock. Promastigotes Promastigotes were were incubated incubated at at 3737◦C °Cfor for 0–24 0–24 h, h, and and cells cells were were collected collected and and analyzed analyzed at at the the indicated indicated times. times. Artificial Artificial OmpA OmpA mRNA mRNA was was added added prior prior to to RNARNA extraction extraction for for normalization. normalization. HSP70 HSP70 (A (A),), HSP83 HSP83 (B (),B), SMT SMT (C ()C mRNA) mRNA and and 18S 18S rRNA rRNA (D (D) levels) levels were were measured measured by by RT-qPCR.RT-qPCR. (E )( CellE) Cell viability viability was was monitored monitored by PI by staining PI staining during during heat shock. heat shock. ns: not ns: significant; not significant; **: p < 0.01;**: p and< 0.01;***: andp < 0.001***: p. < 0.001. These results suggest that in addition to increased membrane fluidity and accumulation ofThese superoxide, results asuggest defective that heat in addition shock response to increased (failure membrane to properly fluidity induce and HSP accumula- gene expressiontion of superoxide, and accelerated a defective degradation heat shock of otherresponse RNAs) (failure contributes to properly to the induce extreme HSP heat gene sensitivityexpression displayed and accelerated by c14dm degradation− (Figure4E) of [22 ot,23her]. RNAs) contributes to the extreme heat sensitivity displayed by c14dm ̅ (Figure 4E) [22,23]. 3.4. Reduced RNA Levels in c14dm− Is Not Caused by the Accumulation of Oxidants ̅ 3.4.C14dm Reduced– cells RNA have Levels elevated in c14dm levels Is Not of reactive Caused oxygenby the Accumulation species (ROS), of Oxidants mostly in the form of mitochondrialC14dm ̅ cells superoxide have elevated [23]. levels In order of reactive to examine oxygen if oxidative species (ROS), stress contributes mostly in the to theform RNAof mitochondrial reduction, we superoxide cultivated c14dm [23]. In− cellsorder in to the examine presence if oxidative of L-glutathione stress contributes and performed to the totalRNA RNA reduction, extractions. we cultivated Cell growth c14dm rates ̅ cells and in survival the presence (by PI staining)of L-glutathione were similar and performed between controltotal RNA and L-glutathione-treatedextractions. Cell growth cells rates (Figure and5A). survival Importantly, (by PI L-glutathionestaining) were treatment similar be- didtween not restorecontrol the and RNA L-glutathione-treated levels in c14dm− (Figure cells 5(FigureB), although 5A). itImportantly, could partially L-glutathione alleviate thetreatment ROS accumulation did not restore [23]. the RNA levels in c14dm ̅ (Figure 5B), although it could partially alleviateIn addition, the ROSL. accumulation major WT cells [23]. were treated with antimycin A to induce oxidative stress in the mitochondria, and total RNA was extracted from an equal number of cells. While mitochondrial superoxide was induced as expected based on MitoSox staining (Figure6A )[23], it did not affect the RNA levels (Figure6B). Cell death was negligible in control and antimycin-treated cells (Figure6C). Biomedicines 2021, 9, x FOR PEER REVIEW 8 of 16

AB RNA

ml 3.5 140 Growth ns Biomedicines 2021, 9, 696 % 8 of 16 Biomedicines 2021, 9, x FOR PEER REVIEW 8 of 16 3 120

per 7 2.5 100 levels,

x10 2 80 1.5 60 RNA

AB1 40 number

RNA

ml 3.5 140 0.5 Growth 20 ns %

Cell 03 Relative 1200 per 7 2.5 Control 1 mM 2 mM 4 mM 100 Control 1 mM 2 mM 4 mM levels,

x10 2 L-glutathione80 L-glutathione

1.5 60 RNA

Figure1 5. Antioxidant treatment of c14dm ̅ mutant40 does not restore RNA levels. (A) C14dm ̅ cells number 5 were0.5 inoculated at 4 × 10 cells/mL and treated with20 1–4 mM of L-glutathione, and culture densities

Cell 7 were determined0 after 48 h. (B) Total RNA was extractedRelative 0 from 1 × 10 c14dm ̅ cells after L-glutathione treatmentControl and 1quantifi mM 2ed mM by NanoDrop. 4 mM PercentagesControl represen 1 mMt RNA 2 mMlevels relative 4 mM to the untreated control. ns: notL-glutathione significant. L-glutathione

Figure 5. Antioxidant treatment of c14dm− mutant does not restore RNA levels. (A) C14dm– cells FigureIn 5.addition, Antioxidant L. majortreatment WT of cells c14dm were ̅ mutant treated does with not antimycinrestore RNA A levels. to induce (A) C14dm oxidative ̅ cells were inoculated at 4 × 1055 cells/mL and treated with 1–4 mM of L-glutathione, and culture densities stresswere inoculated in the mitochondria, at 4 × 10 cells/mL and andtotal treated RNA withwas 1–4extracted mM of L-glutathione,from an equal and number culture ofdensities cells. were determined afterafter 4848 h.h. ((BB)) TotalTotal RNA RNA was was extracted extracted from from 1 ×1 ×10 107 c14dm7 c14dm− cells ̅ cells after after L-glutathione L-glutathi- While mitochondrial superoxide was induced as expected based on MitoSox staining (Fig- onetreatment treatment and and quantiﬁed quantifi byed NanoDrop. by NanoDrop. Percentages Percentages represent represen RNAt RNA levels levels relative relative to the to untreated the un- ure 6A) [23], it did not affect the RNA levels (Figure 6B). Cell death was negligible in treatedcontrol. control. ns: not ns: signiﬁcant. not significant. control and antimycin-treated cells (Figure 6C). In addition, L. major WT cells were treated with antimycin A to induce oxidative ABCstress in the mitochondria, and total RNA was extracted from an equal number of cells. 140 4.5 While mitochondrial superoxide was induced as expected based on MitoSox staining (Fig- 4 ure 6A) *[23], it did not120 affect the RNA levels (Figure 6B). Cell death was negligible in intensity 3.5 % control and antimycin-treated 100 cells (Figure 6C). 3 Treatment PI staining, % 2.5 80 levels,

ABC2 60 Control 0.16 fluorescence RNA

1.5 140

in 4.5 40 Antimycin A 0.17 14 * 120 intensity

20 0.53.5 Relative % change

100 03 0 Treatment PI staining, % Fold Control Antimycin A 2.5 80 Control Antimycin A levels,

2 60 Control 0.16 fluorescence RNA

FigureFigure 6. 6. MitochondrialMitochondrial 1.5 oxidative oxidative stress stress induced induced by by antimycin antimycin A A does does not not reduce reduce RNA RNA levels levels in in WT WT parasites parasites.. (A (A) )WT WT in 40 Antimycin A 0.17 parasitesparasites were were treated treated1 with with antimycin antimycin A A for for 3 3 h h at at 27 27 °C◦C and and stained stained with with 10 10 μµM of MitoSox Red. Mean Mean fluorescence fluorescence 20 intensitiesintensities were were0.5 determined byby flowflow cytometry. cytometry. Relative RelativeRelative mitochondrial mitochondrial ROS ROS levels levels were were plotted plotted in comparison in comparison to untreated to un- change 0 7 treatedWT control WT cells.control (B )cells. Total (B RNA) Total was RNA extracted was extracted from 1 ×0 10from7 WT 1 × cells 10 afterWT cells antimycin after antimycin A treatment A andtreatment quantified. and quantified. Percentages

Fold Control Antimycin A Percentages represent RNA levels relative to the untreated control.Control (C Antimycin) Cell death A was measured by PI staining. *: p < 0.05;. represent RNA levels relative to the untreated control. (C) Cell death was measured by PI staining. *: p < 0.05. Figure 6. Mitochondrial oxidativeThus,Thus, stress our our induced data data demonstrate demonstrateby antimycin thatA that does mitochondrial mitochondrial not reduce RNA ROS ROS levels stress stress in is WT is not not parasites responsible responsible. (A) WT for for the the parasites were treated with antimycin A for 3 h at 27 °C and stained with 10 μM of MitoSox Red. Mean fluorescence reductionreduction of of RNA RNA levels levels in in c14dmc14dm ̅−mutant.mutant. intensities were determined by flow cytometry. Relative mitochondrial ROS levels were plotted in comparison to untreated WT control cells. (B) Total RNA was extracted from 1 × 107 WT cells after antimycin A treatment and quantified. 3.5. Polysome Profiling Reveals Defects in Translation in c14dm−̅ Percentages represent RNA3.5. levels Polysome relative Profiling to the untreated Reveals control. Defects ( Cin) TranslationCell death was in measuredc14dm by PI staining. *: p < 0.05;. ReducedReduced mRNA andand rRNArRNA levels levels support support the the hypothesis hypothesis that that mRNA mRNA translation translation could − couldbe compromisedThus, be compromised our data in thedemonstratec14dm in the c14dmmutant. that ̅ mitochondrialmutant. To test thisTo notion,test ROS this astress polysomenotion, is nota profilingpolysome responsible experiment profiling for the experimentreductionwas performed, of was RNA performed, which levels isin basedc14dm which on̅ mutant.is based monosome on monosome and polysome and polysome separation separation in a sucrose in a sucrosegradient. gradient. mRNA mRNA can be can translated be translated by more by thanmore one than ribosome one ribosome leading leading to the to formation the for- mation3.5.of light Polysome of and light Profiling heavy and polysomes.heavy Reveal polysomes.s Defects A higher in TranslationA higher association association in c14dm of mRNAs ̅ of mRNAs with lightwith andlight heavy and polysomes supports more efficient translation, while a higher association with monosomes heavyReduced polysomes mRNA supports and rRNAmore levelsefficient support translation, the hypothesis while a higher that mRNA association translation with indicates poor translation or translational arrest, as we demonstrated earlier [25]. An equal monosomescould be compromised indicates poor in translationthe c14dm ̅ ormutant. translational To test arrest, this notion, as we demonstrateda polysome profiling earlier number of cells from WT, c14dm− and c14dm−/ + C14DM were used for polysome profiling. [25].experiment An equal was number performed, of cells which from is WT,based c14dm on monosome ̅ and c14dm and ̅ / polysome+ C14DM separationwere used infor a Transcripts were stalled on ribosomes with cycloheximide [34]. Then mRNAs containing polysomesucrose gradient. profiling. mRNA Transcripts can be were translated stalled by on more ribosomes than one with ribosome cycloheximide leading [34]. to the Then for- a different number of ribosomes were separated by sucrose gradient [35]. Although an mRNAsmation ofcontaining light and a heavydifferent polysomes. number of A ribosomes higher association were separated of mRNAs by sucrose with lightgradient and equal number of cells were used in each polysome profiling, c14dm− mutant displays heavy polysomes supports more efficient translation, while a higher association with substantially reduced ribosomal peaks (Figure7A). The heights of both monosome and monosomespolysome peaks indicates were 30%–40%poor translation lower than or translational WT and c14dm arrest,−/ + C14DMas we demonstrated parasites, indicating earlier ̅ ̅ [25].overall An reduced equal number engagement of cells of ribosomesfrom WT, inc14dm translation and c14dm (Figure / 7+B). C14DM were used for polysome profiling. Transcripts were stalled on ribosomes with cycloheximide [34]. Then mRNAs containing a different number of ribosomes were separated by sucrose gradient Biomedicines 2021, 9, x FOR PEER REVIEW 9 of 16

[35]. Although an equal number of cells were used in each polysome profiling, c14dm ̅ mutant displays substantially reduced ribosomal peaks (Figure 7A). The heights of both Biomedicines 2021, 9, 696 monosome and polysome peaks were 30%–40% lower than WT and c14dm ̅ / + C14DM9 of 16 parasites, indicating overall reduced engagement of ribosomes in translation (Figure 7B).

A B 1.2 ns WT c14dm-c14dm ̅ c14dm-/+C14DMc14dm ̅ /+C14DM ns * ns 0.8 1 80S * WT * 0.7 WT * * c14dm-c14dm ̅ 0.8 0.6 * 0.5 c14dm-/+C14DMc14dm ̅ /+C14DM 0.6 0.4 0.3 0.4

ABS at 260 nm at ABS 0.2 n=8+ 40S 60S 0.2 0.1

0 Peak area relatively WT monosomes 0 024681012141618202224 monosomesmonosome lightlight polysomes polysomes heavyheavy polysomes polysomes fractions FigureFigure 7.7. C14dmC14dm– ̅ mutant mutant displays displays reduced reduced monosomal monosomal and and polysomal polysomal peaks. peaks. (A (A) Polysomal) Polysomal profile profile of of WT WT (blue), (blue),c14dm c14dm− ̅ (orange)(orange) andandc14dm c14dm− /̅/ ++ C14DM C14DM (grey). (grey). Ribosomal Ribosomal subunits subunits and and polysomes polysomes are are indicated based on absorbance at 260 nm. TheThe experimentexperiment was was conducted conducted in in four four biological biological repeats, repeats, and and one one representative representative profile profile is shownis shown here. here. (B )(B Quantification) Quantification of of the results shown in (A). Areas for peaks of monosomal fractions (80S), the light polysomes (n = 2-4) and heavy poly- the results shown in (A). Areas for peaks of monosomal fractions (80S), the light polysomes (n = 2–4) and heavy polysomes somes (n = 5-8+) were calculated using trapezoidal sum (https://bit.ly/36YJVo1 (accessed on 12/07/2020)). Peak areas were (n = 5–8+) were calculated using trapezoidal sum (https://bit.ly/36YJVo1 (accessed on 12 July 2020)). Peak areas were then then normalized to the WT monosomes. ns: not significant; *: p < 0.05. normalized to the WT monosomes. ns: not significant; *: p < 0.05. Next, we examined if the association of individual mRNAs with monosomes and Next, we examined if the association of individual mRNAs with monosomes and light light and heavy polysomes were affected in the c14dm ̅ mutant. We expected two scenarios and heavy polysomes were affected in the c14dm− mutant. We expected two scenarios here: here: first, the association of all mRNAs with ribosomes was consistently reduced similar first, the association of all mRNAs with ribosomes was consistently reduced similar to the to the reduction in steady-state mRNA levels; alternatively, the association with ribo- reduction in steady-state mRNA levels; alternatively, the association with ribosomes for somes for some mRNAs could be affected more than others, leading to a global gene some mRNAs could be affected more than others, leading to a global gene dysregulation insteaddysregulation of proportional instead of reductionproportional in protein reduction production. in protein Lowerproducti associationon. Lower ofassociation mRNAs withof mRNAs light and with heavy light polysomes and heavy is polysomes indicative ofis in lessdicative efficient of translation,less efficient and translation, reductions and in polysomereductions peak in polysome heights suggested peak heights that suggested individual that RNAs individual could be RNAs translated could less be efficientlytranslated asless well. efficiently Our RT-qPCR as well. results,Our RT-qPCR based on results, four mRNAs,based on demonstrate four mRNAs, the demonstrate degree of individ- the de- ualgree mRNA’ of individual association mRNA’ with association monosomes, with and monosomes, light and and heavy light polysomes and heavy were polysomes largely unaffectedwere largely by unaffectedC14DM-deletion, by C14DM supporting-deletion, asupporting mostly proportional a mostly proportional reduction in reduction protein productionin protein production (Figure8). We(Figure observed 8). We a observed minor increase a minor in associationincrease in association with monosomes with mon- for HSP70osomes mRNA for HSP70 in c14dm mRNA−, butin c14dm the difference ̅, but the wasdifference not statistically was not statistically significant. significant.

3.6. Intensity of ER Staining Is Compromised in c14dm− under Heat Shock Sterol is present in both PM and organellar membranes [36]. Rough ER carries ribosomes and is where the majority of translation takes place [37]. Perturbations in sterol synthesis may alter the structure of ER and affect the recruitment of ribosomes for translation. To examine the ER in promastigotes, WT, c14dm− and c14dm−/ + C14DM parasites were grown at 27 ◦C (insect vector temperature) and exposed to 37 ◦C for two hours (mammalian temperature) followed by staining with the antibody against BiP, an ER protein (Figure9). Our results indicate that BiP staining at 27 ◦C was comparable between WT and c14dm−. However, the 2-h treatment at 37 ◦C led to a dramatic reduction (~30%) in c14dm−, suggesting an altered ER at higher temperatures. BiomedicinesBiomedicines 20212021,, 99,, 696x FOR PEER REVIEW 1010 ofof 1616

AB HSP70 mRNA HSP83 mRNA % 60 % 50

50 40 mRNA, mRNA,

40 of of 30

30 20 20 10 10 Distribution Distribution 0 0 monosomes light polysomes heavy polysomes monosomes light polysomes heavy polysomes

C WT c14dm ̅ c14c14dmdm ̅/+C ̅ /+C14DM14DM D WT c14dm ̅ c14c14dmdm ̅/+C ̅ /+C14DM14DM % 60 SMT mRNA% 60 Tubulin mRNA

50 50 mRNA, mRNA,

40 40 of of

30 30 20 20 10 10 Distribution Distribution 0 0 monosomes light polysomes heavy polysomes monosomes light polysomes heavy polysomes

WT c14dm ̅ c14c14dmdm ̅/+C ̅ /+C14DM14DM WT c14dm ̅ c14c14dmdm ̅/+C ̅ /+C14DM14DM

FigureFigure 8.8. The association of of individual individual mRNAs mRNAs with with monosomes monosomes and and light light and and heavy heavy polysomes polysomes is islargely largely unaffected unaffected in Biomedicines 2021−, 9, x FOR PEER REVIEW 11 of 16 inc14dmc14dm ̅ mutant.mutant. Distribution Distribution of HSP70 of HSP70 (A), ( AHSP83), HSP83 (B), (SMTB), SMT (C) and (C) andtubulin tubulin (D) mRNAs (D) mRNAs among among monosomes, monosomes, light poly- light polysomessomes and andheavy heavy polysomes polysomes was wasdetermined determined by RT-qPCR by RT-qPCR after after RNA RNA extraction extraction from from polysomal polysomal fractions. fractions.

3.6. Intensity of ER Staining Is Compromised in c14dm ̅ under Heat Shock A WT c14dm ̅ c14dm ̅/+C14DM B Sterol is present in both PM and organellar membranes [36]. Rough*** ER carries ribosomes and is where the majority of translation takes place [37].*** Perturbations in sterol synthesis may alter the structure of ER and af1200fect the recruitment of ribosomes*** for trans- 5 µm 5 µm 5 µm *** 27°C 27°Clation. To examine the27°C ER in promastigotes, WT, c14dm ̅ and c14dm ̅ / + C14DM parasites WT c14dmwere ̅ grown at 27 °Cc14dm (insect ̅/+C14DM vector temperature)1000 and exposed to 37 °C for two hours (mammalian temperature) followed by staining with the antibody against BiP, an ER protein 800 (Figure 9). Our results indicate that BiP staining at 27 °C was comparable between WT 27°C 5 µm 27°Cand c14dm ̅ . 5However, µm 27°C the 2-h treatment5 µm at 37600 °C led to a dramatic reduction (~30%) in intensity c14dm ,̅ suggesting an altered ER at higher temperatures.

WT c14dm ̅ c14dm ̅/+C14DM ER 400

200

5 µm 5 µm 37°C 37°C 37°C 5 µm 0 27°C; 2 hours 1 37°C; 2 hours WT c14dm ̅ c14dm ̅/+C14DM WT c14dm ̅ cc14dm14dm ̅ ̅ /+C/+C14DM14DM

37°C 5 µm 37°C 5 µm 37°C 5 µm

− − FigureFigure 9. 9. HeatHeat treatment treatment leads to a significant signiﬁcant reduction inin ERER stainingstaining ininc14dm c14dm ̅ .(. (A) Representative imagesimages ofof WT,WT,c14dm c14dm ̅ andand c14dmc14dm − ̅ // ++ C14DM C14DM cells cells are are shown. shown. BiP BiP staining staining was was perfor performedmed after after 2 2 h h of of incubation incubation at at 27 27 or or 37°C. 37◦C. ( (BB)) Quantitative Quantitative analysisanalysis of of BiP-staining BiP-staining intensity. ***: p < 0.001. 0.001.

4.4. Discussion Discussion LipidLipid homeostasis homeostasis is iscritical critical for for the the proliferation proliferation and and pathogenesis pathogenesis of Leishmania of Leishmania parasites,parasites, and changes and changes in lipid in composition lipid composition can lead can to leadmultiple to multiple defects [22,23,38–43]. defects [22,23 Despite,38–43]. the progress in understanding many lipid pathways, the role of sterol synthesis is not fully defined in protozoan parasites. In contrast to mammalian cells that synthesize cholesterol as the main sterol, Leishmania parasites primarily make ergosterol and 5-dehydroepisterol [44–46]. C14DM is responsible for the removal of a C-14-methyl group from sterol intermediates. Genetic deletion of this key enzyme leads to the loss of ergostane-based sterols and the accumulation of C-14-methylated sterol intermediates [22]. These defects cause increased PM fluidity and permeability. The presence of sterols is required not only in the PM but also in the membranes of intracellular organelles and lipid droplets [47]. The lack of proper sterols in c14dm ̅ leads to altered mitochondrial morphology, accumulation of ROS and impairment in respiration [23]. In this study, we revealed an unexpected role of sterol synthesis in RNA stability and translation. First, we found that both total RNA and protein levels are substantially reduced in c14dm ̅ mutants (Figure 1). Then, we demonstrated that all tested RNAs, including 18S rRNA, 28S rRNA, tubulin and HSP83 mRNAs, display 50%–60% reduction in steady-state RNA levels in comparison to WT and add-back parasites when extracted from an equal number of cells (Figure 2). Next, actinomycin D treatment experiments demonstrated that both mRNA and rRNA are much less stable in the mutant and adding the C14DM gene back rescues this defect (Figure 3). In c14dm ,̅ RNA instability becomes exacerbated under heat shock stress. Moreover, their response to heat shock is severely compromised, as evident from the insufficient upregulation of HSP83 and HSP70 mRNAs in c14dm ̅ (Figure 4A–B). Heat shock proteins are known to be induced in response to heat and other stress to increase cell survival under adverse conditions [48,49]. They act as molecular chaperones that facilitate the correct folding or degradation of misfolded proteins [50]. In addition, heat shock proteins in trypanosomatids are functionally adapted to a parasitic lifestyle, including resistance to the temperature changes and essential for the differentiation of parasites [51–53]. Our results suggest that the inability of c14dm ̅ to cope with stress is, at least in part, due to dysregulation of gene expression. Biomedicines 2021, 9, 696 11 of 16

Despite the progress in understanding many lipid pathways, the role of sterol synthesis is not fully defined in protozoan parasites. In contrast to mammalian cells that synthesize cholesterol as the main sterol, Leishmania parasites primarily make ergosterol and 5-dehydroepisterol [44–46]. C14DM is responsible for the removal of a C-14-methyl group from sterol intermediates. Genetic deletion of this key enzyme leads to the loss of ergostane- based sterols and the accumulation of C-14-methylated sterol intermediates [22]. These defects cause increased PM fluidity and permeability. The presence of sterols is required not only in the PM but also in the membranes of intracellular organelles and lipid droplets [47]. The lack of proper sterols in c14dm− leads to altered mitochondrial morphology, accumulation of ROS and impairment in respiration [23]. In this study, we revealed an unexpected role of sterol synthesis in RNA stability and translation. First, we found that both total RNA and protein levels are substantially reduced in c14dm− mutants (Figure1). Then, we demonstrated that all tested RNAs, including 18S rRNA, 28S rRNA, tubulin and HSP83 mRNAs, display 50%–60% reduction in steady-state RNA levels in comparison to WT and add-back parasites when extracted from an equal number of cells (Figure2). Next, actinomycin D treatment experiments demonstrated that both mRNA and rRNA are much less stable in the mutant and adding the C14DM gene back rescues this defect (Figure3). In c14dm−, RNA instability becomes exacerbated under heat shock stress. Moreover, their response to heat shock is severely compromised, as evident from the insufficient upregulation of HSP83 and HSP70 mRNAs in c14dm− (Figure 4A–B). Heat shock proteins are known to be induced in response to heat and other stress to increase cell survival under adverse conditions [48,49]. They act as molecular chaperones that facilitate the correct folding or degradation of misfolded proteins [50]. In addition, heat shock proteins in trypanosomatids are functionally adapted to a parasitic lifestyle, including resistance to the temperature changes and essential for the differentiation of parasites [51–53]. Our results suggest that the inability of c14dm− to cope with stress is, at least in part, due to dysregulation of gene expression. It is known that a high level of oxidative stress can contribute to increased RNA degradation and reduced translation [54–56]. We have demonstrated earlier that c14dm− mutant displays mitochondrial dysfunction and elevated levels of oxidative stress [23]. However, the antioxidant treatment of the mutant with L-glutathione did not restore RNA levels (Figure5). Moreover, the induction of oxidative stress in the mitochondrion of WT cells did not lead to RNA reduction. Thus, our data indicate that oxidative stress is not the main cause of RNA instability in c14dm−. C14DM is primarily localized in the ER [22]. The lack of endogenous sterols and accumulation of abnormal, C-14-methylated intermediates may lead to defects not only in the PM but also in the ER. Rough ER contains ribosomes actively involved in translation [57,58]. We hypothesize that impairment in sterol production leads to significant defects in the ER (Figure 10) and reduced recruitment of ribosomes to ER where the majority of translation occurs [37]. As a result, rRNA and mRNA not involved in translation are subjected to degradation. Our polysome profiling data demonstrate that monosome and light and heavy polysome peaks are lower in c14dm− mutant, supporting an overall reduced engagement in translation (Figure7). Moreover, ER staining with BiP, an ER marker, is substantially reduced (by ~30%) during heat shock, suggesting defective ER in the mutant (Figure9). It is possible that ribosomes cannot attach sufficiently due to the alteration of sterol content in the ER. It leads to reduced translation and RNA degradation, as shown in our proposed model (Figure 10A). Biomedicines 2021, 9, x FOR PEER REVIEW 12 of 16

It is known that a high level of oxidative stress can contribute to increased RNA degradation and reduced translation [54–56]. We have demonstrated earlier that c14dm ̅ mutant displays mitochondrial dysfunction and elevated levels of oxidative stress [23]. How- ever, the antioxidant treatment of the mutant with L-glutathione did not restore RNA levels (Figure 5). Moreover, the induction of oxidative stress in the mitochondrion of WT cells did not lead to RNA reduction. Thus, our data indicate that oxidative stress is not the main cause of RNA instability in c14dm .̅ C14DM is primarily localized in the ER [22]. The lack of endogenous sterols and accumulation of abnormal, C-14-methylated intermediates may lead to defects not only in the PM but also in the ER. Rough ER contains ribosomes actively involved in translation [57,58]. We hypothesize that impairment in sterol production leads to significant defects in the ER (Figure 10) and reduced recruitment of ribosomes to ER where the majority of translation occurs [37]. As a result, rRNA and mRNA not involved in translation are subjected to degradation. Our polysome profiling data demonstrate that monosome and light and heavy polysome peaks are lower in c14dm ̅ mutant, supporting an overall reduced engagement in translation (Figure 7). Moreover, ER staining with BiP, an ER marker, is substantially reduced (by ~30%) during heat shock, suggesting defective ER in the mutant Biomedicines 2021, 9, 696 (Figure 9). It is possible that ribosomes cannot attach sufficiently due to the alteration12 of of 16 sterol content in the ER. It leads to reduced translation and RNA degradation, as shown in our proposed model (Figure 10A).

A Endoplasmic reticulum Endoplasmic reticulum WT c14dm ̅ Defects in sterol

Normal turnover of RNA

Reduced translation mRNA and ribosomal RNA not involved in translation are degraded B WT RNA is protected by c14dm ̅ RNA is de-protected due to the expression/traffic defects of RNA binding proteins proteins involved in RNA protection

cap X cap AAAAA AAAAA

Figure 10. A model depicting the putative mechanism of RNA reduction in c14dm .̅ (A) Sterol synthesis defects may alter Figure 10. A model depicting the putative mechanism of RNA reduction in c14dm−.(A) Sterol synthesis defects may alter the lipid content of the ER and reduce ribosome recruitment, causing degradation of rRNA and mRNA not involved in the lipid content of the ER and reduce ribosome recruitment, causing degradation of rRNA and mRNA not involved in translation. (B) Alternatively, defects in sterol synthesis may cause traffic defects preventing proteins involved in RNA translation.protection from (B) Alternatively,reaching and defectsprotecting in sterol RNA. synthesis Lightning may bolts cause in red traffic depict defects defe preventingcts in sterol. proteins Small (yellow) involved and in RNAlarge protection(orange) ovals from represent reaching small and protecting and large RNA.subunits Lightning of ribosomes, bolts in respectively. red depict defects mRNA in is sterol.shown Small as a blue (yellow) line. andEnzymes large (orange)involved ovalsin RNA represent degradation small are and shown large as subunits partial ofred ribosomes, circles. RNA-binding respectively. proteins mRNA are is shownshown asin blue a blue and line. purple. Enzymes involved in RNA degradation are shown as partial red circles. RNA-binding proteins are shown in blue and purple. However, we cannot rule out other possibilities. Sterol defects in other organisms are knownHowever, to affect we not cannot only membrane rule out other permeabilit possibilities.y and Sterol fluidity defects but also in other the localization organisms are of membrane-boundknown to affect not proteins only membrane and protein permeability transport [5 and9]. Therefore, fluidity but it alsois possible the localization that defects of inmembrane-bound sterol synthesis proteinsmay compromise and protein the transport expression/trafficking [59]. Therefore, of it isRNA-stabilizing possible that defects proteins,in sterol thus synthesis reducing may their compromise ability to reach the expression/trafficking and protect RNA (Figure of RNA-stabilizing 10B). proteins, thus reducing their ability to reach and protect RNA (Figure 10B).

5. Conclusions The role of lipids in controlling gene expression is not sufficiently understood not only in trypanosomatids but also in other eukaryotic organisms, including mammals. In the past, lipids have been mostly viewed as components of membranes and energy storage molecules. However, during the last few decades, a substantial amount of research revealed the importance of lipids as signaling molecules regulating many processes and gene expression pathways in the cell. Sphingolipids and their metabolites, such as ceramides, sphingosine and sphingosine-1-phosphate (S1P), regulate cellular processes, including proliferation, differentiation and cell death [60–62]. The S1P acts extracellularly through cell surface receptors. It also has important intracellular targets involved in inflammation, cancer and neurodegenerative diseases [63,64]. Phospholipids are exceptionally important for cellular signaling, and they can modulate the activity of nuclear hormone transcription factors responsible for the tuning of genes involved in the metabolism [65]. Sterols are known to be involved in signaling as well. For example, cholesterol activates cytokine production and regulates inflammation in mammalian cells [66]. While sterols are known to serve as structural components of eukaryotic membranes and signaling molecules regulating certain pathways in the cell, they appear to play an additional unexpected role. Our study is the first of its kind and supports a novel role of sterol in RNA stability, mRNA translation and global regulation of gene expression. As a result, it shifts our Biomedicines 2021, 9, 696 13 of 16

traditional understanding of the role of lipids. For future studies, it will be very important to determine what mechanism is responsible for RNA instability caused by sterol defects. Do defects in ER and PM structures contribute to reduced RNA stability? Is the recruitment of RNA-binding proteins affected? Is ribosome recruitment to ER affected? More studies are needed to determine the role of sterols in the regulation of RNA levels and reveal the mechanism of global RNA destabilization caused by lipid imbalance. Finally, the identiﬁcation of components of RNA degradation and protection machinery may provide us new drug targets, and their inhibition combined with sterol inhibition may provide a better strategy to treat leishmaniasis.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/biomedicines9060696/s1, Figure S1: Ribosomal RNA in c14dm− degrades faster than in WT and add-back parasites, File S1: Primers used in RT-qPCR. Author Contributions: Z.N.K. and K.Z. contributed to the conceptualization and experimental design; Z.N.K. wrote the manuscript, analyzed the data and prepared the figures; Z.N.K., S.M. (Samrat Moitra), A.P., S.M. (Sumit Mukherjee), E.B.T. and A.L.K. performed the experiments. All authors discussed and edited the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the US National Institutes of Health grant AI099380 (KZ). The funder had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available on request from the corresponding authors. Acknowledgments: We thank Igor Ponomarev for providing advice on statistical analysis. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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