Development and Characterization of an Avirulent Leishmania major Strain Mukesh Kumar Jha, Aditya Y. Sarode, Neelam Bodhale, Debasri Mukherjee, Surya Prakash Pandey, Neetu This information is current as Srivastava, Abdur Rub, Ricardo Silvestre, Arup Sarkar and of September 29, 2021. Bhaskar Saha J Immunol 2020; 204:2734-2753; Prepublished online 3 April 2020;
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Supplementary http://www.jimmunol.org/content/suppl/2020/04/02/jimmunol.190136 Material 2.DCSupplemental http://www.jimmunol.org/ References This article cites 48 articles, 21 of which you can access for free at: http://www.jimmunol.org/content/204/10/2734.full#ref-list-1
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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2020 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology
Development and Characterization of an Avirulent Leishmania major Strain
Mukesh Kumar Jha,*,1 Aditya Y. Sarode,*,1 Neelam Bodhale,† Debasri Mukherjee,* Surya Prakash Pandey,* Neetu Srivastava,* Abdur Rub,* Ricardo Silvestre,‡ Arup Sarkar,x and Bhaskar Saha*,x
Leishmania major causes cutaneous leishmaniasis. An antileishmanial vaccine for humans is unavailable. In this study, we report development of two attenuated L. major strains—5ASKH-HP and LV39-HP—by continuous culture (high passage) of the corre- sponding virulent strains (low passage). Both avirulent strains showed similar changes in proteome profiles when analyzed by surface-enhanced laser desorption ionization mass spectrometry. Liquid chromatography–mass spectrometry and microarray characterization of 5ASKH strains revealed substantially altered gene and protein expression profiles, respectively. Both virulent and avirulent L. major strains grew comparably in culture, but the avirulent strain survived significantly less in BALB/c-derived Downloaded from peritoneal macrophages. Both attenuated strains failed to infect BALB/c mice and elicited IFN-g, but not IL-4 and IL-10, responses. 5ASKH-HP parasites failed to induce significant infection even in severely immunocompromised- SCID or inducible NO synthase–, CD40-, or IL-12–deficient mice, indicating attenuation. The avirulent strain induced less IL-10, but higher IL-12, in macrophages. The avirulent strain failed to reduce CD40 relocation to the detergent-resistant membrane domain and to inhibit CD40-induced phosphorylation of the kinases Lyn and protein kinase C-b and MAPKs MKK-3/6 and p38MAPK or to upregulate MEK-1/2 and ERK-1/2 in BALB/c-derived peritoneal macrophages. The virulent and the avirulent strains reciprocally modulated http://www.jimmunol.org/ CD40-induced Ras-mediated signaling through PI-3K and Raf-1. Avirulent 5ASKH-primed BALB/c mice were protected against virulent L. major challenge infection. The loss of virulence accompanied by substantially altered proteome profiles and the elicitation of host-protective immune responses indicate plausibly irreversible attenuation of the L. major strain and its potential use as a vaccine strain. The Journal of Immunology, 2020, 204: 2734–2753.
he protozoan parasite Leishmania causes a complex of that more than 12 million people are currently infected with diseases called Leishmaniases that range from self-healing Leishmania and 350 million people are at risk for infection in T cutaneous to potentially fatal visceral leishmaniasis. The tropical and subtropical regions of the world (5). Despite being by guest on September 29, 2021 parasite Leishmania is dimorphic: elongated, motile, flagellated, a major parasitic disease, its chemotherapy is severely limited extracellular promastigotes and ovoid, sessile, aflagellate, and by the restricted repertoire of chemotherapeutic drugs, the toxicity intraphagosomal amastigotes in the mammalian macrophages (1). of the available drugs, and emerging drug-resistant parasites. As the phagosomes fuse with the lysosomes rich in hydrolytic en- Therefore, a prophylactic vaccine is an imperative choice for zymes, the amastigotes are killed. In contrast, virulent parasites preventing the disease. However, no antileishmanial vaccine subvert the macrophage’s antileishmanial surveillance system (2–4) for human use is available till date. and cause the disease. Leishmania major causes the disease cuta- To date, three basic prototypes of antileishmanial vaccines have neous leishmaniasis. The World Health Organization has estimated been tested: the first generation vaccine was with live virulent parasite (Leishmanization), the second generation vaccines were *National Centre for Cell Science, Savitribai Phule Pune University, Pune, Maharashtra recombinant proteins and peptides, and the third generation vaccine 411007, India; †Jagadis Bose National Science Talent Search, Kolkata, West Bengal was the genetic construct encoding multiple peptides from different 700107, India; ‡Life and Health Sciences Research Institute, School of Health x proteins (reviewed in Ref. 6). However, all these candidates have so Sciences, University of Minho, 4710-057 Braga, Portugal; and Trident Academy of Creative Technology, Bhubaneswar, Odisha 751024, India far failed because of high infectivity in the first generation vaccine 1M.K.J. and A.Y.S. contributed equally. whereas various immunological shortcomings including priming of ORCIDs: 0000-0002-0685-4942 (A.Y.S.); 0000-0003-1040-5506 (N.B.); 0000-0003- limited T cell repertoire with the second and the third generation 3935-2655 (S.P.P.); 0000-0003-1301-0761 (A.R.); 0000-0002-9270-2717 (R.S.); vaccines (reviewed in Ref. 6). Therefore, we first generated the 0000-0002-5833-7912 (B.S.). avirulent parasites by continuous culture of two virulent L. major Received for publication November 14, 2019. Accepted for publication March 5, strains. All assays were therefore direct comparison of the same 2020. virulent and avirulent parasites. We characterized these strains by This work was supported by Infect-eRA funding through the Department of Biotech- nology, New Delhi, Government of India. microarray and mass spectrometry. Both avirulent parasites expressed comparably altered proteome profiles as shown by surface-enhanced Address correspondence and reprint requests to Dr. Bhaskar Saha, National Centre for Cell Science, Ganeshkhinnd Road, Savitribai Phule Pune University, Pune, laser desorption ionization (SELDI) analyses. We then tested whether Maharashtra 411007, India. E-mail address: [email protected] priming of a susceptible host with an avirulent L. major strain The online version of this article contains supplemental material. would elicit a host-protective immune response that would protect Abbreviations used in this article: CSA, crude soluble Ag; HP, high-passage; LN, the host against the challenge infection with corresponding lymph node; LP, low-passage; SELDI, surface-enhanced laser desorption ionization; virulent parasites. Treg, regulatory T. In this study, we report that the promastigotes of these two Copyright Ó 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50 avirulent and the respective virulent strains comparably replicated www.jimmunol.org/cgi/doi/10.4049/jimmunol.1901362 The Journal of Immunology 2735 in cultures, but although both virulent and avirulent parasites in- p–SHP-1, p–MEK-1/2, MEK-1/2, p–MKK-3/6, MKK-3/6, Ras GTPase, fected macrophages, the avirulent strains’ survival within mac- CD40, b-actin, and Luminol reagent were from Santa Cruz Biotechnology rophages was compromised. In fact, the avirulent parasites failed (Santa Cruz, CA). Anti-cytokine Abs (IL-10, IL-12, IFN-g, and IL-4), cytokine standard for ELISA, anti-CD40 Ab (no azide/low endotoxin; to establish sustained infections in susceptible BALB/c mice. The clone 3/23), and isotype control (rat IgG2ak) were purchased from BD attenuation was complete, as these parasites could not establish Biosciences (San Diego, CA). infection even in severely immunocompromised mice. As mac- Animals and parasites culture rophages express CD40, a cell membrane-located costimulatory 2 2 receptor, and as CD40–CD40L interaction plays significant anti- BALB/c, C57BL/6, SWISS, NOD/SCID on Swiss, CD402/2 and IL-12 / 2/2 2/2 leishmanial functions (7–9) through modulation of signaling in on a BALB/c background, and IL-10 and iNOS mice on C57BL/6 background (Jackson Laboratory, Bar Harbor, ME) were bred in the macrophages (10–17), we compared how these avirulent parasites experimental animal facility at the National Centre for Cell Science in differed from their virulent counterparts in terms of intracellular Thoren caging units (Thoren Caging System, Hazleton, PA). The signaling and immune response profiles. These avirulent parasites progress of infection was monitored weekly by measuring footpad had impaired ability to selectively modulate CD40-mediated thickness and parasite load was assessed following euthanization 5 wk phosphorylation of phosphatases and kinases. The virulent and postinfection. All experiments were in accordance with the animal use protocol approved by the Institutional Animal Care and Use Com- avirulent strains differ in their ability to induce IL-10 or IL-12 from mittee and the Committee for the Purpose of Control and Supervision macrophages and to induce IL-4 or IFN-g from the lymph node of Experiments on Animals, the regulatory authorities for animal (LN) cells cocultured with virulent or avirulent L. major–infected experimentation. macrophages. The LN cells from the avirulent L. major–infected L. major (strain MHOM/Su73/5ASKH and MRHO/SU/59/P/LV39) was maintained in vitro in complete RPMI 1640 medium (Life Technologies-BRL), BALB/c mice produced low IL-4 but higher IFN-g in response to containing penicillin (70 mg/ml), streptomycin (100 mg/ml), 2-ME (50 mM), Downloaded from leishmanial Ags. Therefore, developing avirulent strains that fail sodium pyruvate (1 mM), and FCS (10%; Life Technologies-BRL). Virulence to establish an infection but elicit a host-protective T cell re- wasmaintainedbypassagethroughBALB/c mice. Avirulent or high-passage sponse is the prerequisite for generating an effective antileishmanial (HP) L. major promastigotes were derived from continuous in vitro culture for vaccine. 10 y. Avirulent L. major parasites were maintained in vitro in complete RPMI 1640 medium. Stationary-phase L. major promastigotes (2 3 106 per mouse, s.c.) were used for infecting the indicated mice. 8 Typically, 2 3 10 stationary phase parasites were harvested for SELDI http://www.jimmunol.org/ Materials and Methods experiment. Reagents Isolation of virulent and avirulent amastigotes Western blot Abs for p-PKCd and p-PKCz/l (Cell Signaling Technology, Danvers, MA); pan-Ras (Pierce, Rockford, IL); p-PKCb1, PKCb1, p-PKCd, BALB/c-derived peritoneal macrophages (5 3 107/100 mm petri dish) PKCd,p-PKCz,p-PKCz/l, p-p38MAPK, p38MAPK, p–ERK-1/2, ERK-1/2, were infected with virulent and avirulent L. major promastigotes for 72 h. p-Lyn, Lyn, p-Syk, Syk, p-PI3k, PI3k, p-Raf, Raf, p–MKP-1, p–MKP-3, After 72 h, cells were scraped with 13 PBS and passed through insulin by guest on September 29, 2021
FIGURE 1. HP and LP have many differences as observed by proteomic analyses by SELDI. Parasites were harvested at stationary phase of culture by centrifugation at 2500 rpm for 10 min, washed thrice with 13 PBS, and finally resuspended in 300 ml of lysis buffer (20 mM Tris [pH 7.4], 150 mM NaCl, 0.1% Triton X-100, 1 mM EDTA, and protease inhibitor mixture from Roche). After 1 h of lysis at 4˚C, samples were centrifuged at 12,000 rpm at 4˚C for 1 h. Cleared supernatant was collected, protein was estimated using BCA kit (Pierce), and SELDI analysis was performed as described in Materials and Methods.(A) 5ASKH (B) LV39 avirulent and virulent parasite; spectrum and gel view (left panel and right panel, intensity and fold change). The ex- periments were performed thrice, and representative data from one experiment are shown. 2736 CD40 SIGNALING AND LEISHMANIA VIRULENCE syringe 10 times. Cells were centrifuged at 1400 rpm for 5 min at 22˚C. Active Ras Pull-Down and Detection Kit (Thermo Fisher Scientific, Supernatants were collected and again centrifuged at 5000 rpm for 5 min Rockford, IL), as previously described (14). at 22˚C. Pellet was collected and added TRIzol for further RNA isolation (12–14). Isolation of detergent-resistant and soluble membrane fractions Peritoneal macrophage collection Based on their resistance to Triton X-100 (1%), detergent-resistant and Mice were injected with 2 ml thioglycolate (3% i.p.). Four days later, soluble fractions were isolated. Macrophages were infected with virulent or 3 peritoneal exudate cells were harvested in sterile HBSS, centrifuged (210 g, avirulent promastigotes parasites and treated with indicated doses of anti- 8 min at 4˚C), and the pellet was resuspended in RPMI 1640–10% FCS. The CD40 Ab at 37˚C for 7 min and immunoblot for CD40 (13). cells were seeded in 96-well, 24-well, or 6-well culture plates according to experimental requirements and were cultured at 37˚C in a humidified CO2 L. major infection of macrophages incubator for 12 h. Nonadherent cells were removed by replacing the medium with fresh RPMI 1640–10% FCS. The culture was maintained as Thioglycolate-elicited BALB/c-derived peritoneal macrophages were per the requirements of the experiments. infected with stationary phase Leishmania promastigotes, as indicated, at a 1:10 ratio for 6 h (10). Extracellular parasites were washed, and Western blotting macrophages were cultured for 72 h. The cells were fixed with chilled methanol for 5 min and stained with Giemsa, followed by counting the Cells were washed twice with chilled PBS and lysed in cell lysis buffer number of amastigotes per 100 infected macrophages and percentage (20 mM Tris [pH 7.4], 150 mM NaCl, 1% Nonidet P-40, 10% glycerol, of infected macrophages under a Nikon Eclipse E600 microscope. 2 mM EDTA, protease inhibitor mixture [Roche Applied Science, Mannheim, Germany], and phosphatase inhibitor mixture [Pierce]). Protein was Assaying LN cells’ antileishmanial functions quantified by a BCA kit (Pierce) or Bradford reagent, and an equal amount of protein was run on SDS-PAGE. Resolved proteins were BALB/c-derived peritoneal macrophages were infected with L. major Downloaded from blotted onto polyvinylidene difluoride membrane (MilliporeSigma, promastigotes at a 1:10 ratio for 6 h. The extracellular parasites were Bedford, MA) and blocked with 5% nonfat dried milk in TBST (25 mM washed out and cultured for 36 h. The popliteal lymph node was col- Tris [pH 7.6], 137 mM NaCl, and 0.2% Tween 20). Membranes were lected from the fifth week L. major–infected BALB/c or naive mice and incubated with primary Ab at 4˚C overnight, washed with TBST, and the L. major–infected macrophages were cocultured with lymph node incubated with HRP-conjugated secondary Ab. Immunoreactive bands cells isolated from the L. major–infected BALB/c mice at 1:3 ratios for were visualized with the chemiluminescent Luminol reagent (Santa 72 h, followed by enumeration of parasites in macrophages. Cruz Biotechnology) (14). Cytokine and Ab ELISA http://www.jimmunol.org/ Active Ras pull-down assay The supernatants obtained from the 48-h crude soluble Ag (CSA; 25 mg/ml)– A total of 107 macrophages were treated with anti-CD40 Ab (3 mg/ml) or stimulated cultures of macrophages and lymph node cells were assayed isotype control (3 mg/ml) for 7 min. Ras activation was assessed using for IL-10/IL-12 and IL-4/IFN-g secretion, respectively, by ELISA (7). by guest on September 29, 2021
FIGURE 2. Proteomic analysis of virulent and avirulent L. major parasites. Parasites were harvested at stationary phase of culture by centrifugation at 2500 rpm for 10 min and washed thrice with 13 PBS. Trypsin was digested and cleaned up by C18 columns, as described in Materials and Methods. (A) The Venn diagram shows a total of 283 differentially expressed proteins in 5ASKH LP and 503 proteins in 5ASKH HP. (B) Samples abundances normalized values virulent versus avirulent L. major parasites. (C) Expression of 26 significant proteins in avirulent or virulent strains of L. major by heat map. (D) Volcano plot shows differentially abundant proteins in virulent and avirulent parasites. The plot constructed was using log10 (p value) against the log2 (fold change). The nonaxial vertical lines represent 61-fold change. The experiments were performed thrice, and representative data from one experiment are shown. The Journal of Immunology 2737
Table I. Proteins uniquely expressed either in 5ASKH-LP or in 5ASKH-HP
Molecular Serial No. Accession No. Gene mass, kDa Calc. pI L. major LP L. major HP Cytoskeleton and cellular transport 1. Q66VD0 a-Tubulin (fragment) 27.9 4.77 High Not found 2. Q4FX37 Actin-like protein 53.4 6.98 Not found High 3. Q4QB82 Actin interacting protein-like protein 56.9 6.74 Not found High 4. E9BM84 Calmodulin-related protein, putative 81.1 4.79 High Not found 5. A0A3Q8IFZ5 Microtubule-associated protein, putative 286.8 4.67 High Not found 6. E9BIK6 Microtubule-associated protein Gb4, putative 286.9 4.65 High Not found 7. E9AF06 Putative ER–Golgi transport protein gp25L 24.5 9.14 High Peak found Heat shock protein 1. Q4Q740 Putative heat shock 70-related protein 1, mitochondrial 70.6 5.91 High Not found 2. A0A165DJZ9 Heat shock protein 70 (fragment) 20.7 6.8 Not found High 3. E9BM26 Chaperonin HSP60/CNP60, putative 58.1 5.47 Not found High 4. E9BK14 Heat-shock protein hsp70, putative (fragment) 13.4 6.86 High Peak found 5. Q4Q711 Putative chaperonin HSP60/CNP60 58 5.54 Not found High 6. A0A3S5H7Z8 Chaperone protein DNAj, putative 50.9 8.65 High Not found 7. E9BSH3 Chaperone protein DNAj, putative 51 8.56 High Not found 8. E9AFC2 Putative chaperone protein DNAj 51 8.82 High Not found
9. A0A3Q8ICB1 MORN repeat, putative 43.5 5.58 Not found High Downloaded from 10. A0A3S5H808 Hsp70 protein, putative 78.9 6.51 Not found High Metabolic proteins: glycolytic 1. Q4QD34 Phosphoglycerate kinase 51.5 8.81 High Not found 2. Q2PDE6 Glucose-6-phosphate isomerase 67 6.7 Not found High 3. P50312 Phosphoglycerate kinase, glycosomal 51.5 8.9 High Not found Amino acid metabolism 1. A0A0R6YBP0 Putative argininosuccinate synthase (fragment) 12.1 5.26 High Not found 2. Q6QQZ0 Glutamate oxaloacetate (fragment) 41.9 6.62 Not found High http://www.jimmunol.org/ 3. E9ABT3 Aspartate transaminase (fragment) 35.7 7.24 Not found High 4. Q6QQY0 Glutamate oxaloacetate (fragment) 41.9 6.8 Not found High 5. Q6QQY3 Glutamate oxaloacetate (fragment) 41.8 6.8 Not found High 6. E9BEX2 Aminoacylase, putative (fragment) 30.5 8.12 Not found High 7. E9BEX0 Aminoacylase, putative (Fragment) 11.5 4.96 Not found High 8. E9BMC8 Peptidyl-prolyl cis–trans isomerase 24.6 8.35 High Not found 9. Q4Q6Q9 Peptidyl-prolyl cis–trans isomerase 24.6 8.29 High Not found 10. A0A3Q8IG44 S-adenosylmethionine synthetase 43.1 5.76 High Not found 11. E9BK65 Propionyl-coa carboxylase b-chain, putative 57 8.95 High Not found
12. E9ACK5 Putative arginine N-methyltransferase 44.4 6.39 High Not found by guest on September 29, 2021 13. Q4Q9I8 Glycine cleavage system P protein 106.4 7.53 Not found High 14. E9ACF2 2-Aminoethylphosphonate/pyruvateaminotransferase-like 48.1 9.1 High Not found protein 15. E9BJC5 Branched-chain amino acid aminotransferase, putative 44.1 7.97 High Not found (fragment) 16. A0A3Q8IGG4 Branched-chain amino acid aminotransferase, putative 44.1 7.97 High Not found 17. A0A3Q8IWI0 A distinct subfamily of CDD/CDA-like deaminases, putative 68.8 8.22 High Not found 18. E9BI16 Glycine cleavage system P protein 106.4 7.75 High Not found 19. A0A3Q8IEG8 Aspartyl aminopeptidase, putative 49.4 6.57 Not found High 20. E9ACD7 g-Glutamyl phosphate reductase-like protein 68.5 7.24 High Not found 21. G1C2I2 O-acetyl serine sulfhydrylase (fragment) 34.5 8.05 High Not found 22. Q4Q5Z6 Acetoin dehydrogenase e3 component-like protein 60.8 8.85 High Not found Mitochondrial 1. Q9U0W1 Acetyl-CoA synthetase 77.4 6.57 Not found High 2. E9BN78 Acetyl-CoA carboxylase, putative 241 6.43 Not found High 3. A0A3Q8IIV7 Acetyl-CoA carboxylase, putative 240.9 6.46 Not found High 4. E5LCR1 Acetyl-CoA synthetase 77.2 6.57 Not found High 5. E9B982 Flavoprotein subunit-like protein 61 8.63 High Not found 6. E9BGU0 Malic enzyme 62.8 6.19 Not found High 7. E9B7U0 Cytochrome C oxidase copper chaperone, putative 9.4 6.49 High Not Found 8. E9BFJ4 Cytochrome C oxidase subunit VI, putative 19.2 7.93 Not found High 9. E9ACL9 Putative cytochrome C oxidase copper chaperone 9.4 6.49 High Not Found 10. Q4QC12 Putative cytochrome C oxidase subunit VI 19.2 7.27 High Not Found 11. F2QKI7 NADH–cytochrome b5 reductase 32 8 Not found High 12. F2QKM3 NADH–cytochrome b5 reductase 32 8 Not found High 13. F2QKI8 NADH–cytochrome b5 reductase 32 8 Not found High 14. Q4Q5Z7 2-Oxoglutarate dehydrogenase, e3 component, 66.8 7.87 High Not found lipoamidedehydrogenase-like protein 15. E9BJK6 p450 Reductase, putative 72.3 6.43 High Not found 16. E9B7D1 Mitochondrial processing peptide b subunit, putative 55 6.58 Not found High 17. E9AC56 Putative mitochondrial processing peptide b subunit 55 6.58 Not found High 18. E9BFN8 Carnitine palmitoyltransferase-like protein 74.6 8.31 High Not found 19. E9B7L1 Mitochondrial carrier protein, putative 34.4 9.67 High Not found 20. Q4Q8E2 Putative p450 reductase 72.2 6.64 High Not found 21. E9BA42 Cytochrome b5-like, putative 12.9 4.91 High Not found 22. E9ACE1 Putative mitochondrial carrier protein 34.3 9.64 High Not found (Table continues) 2738 CD40 SIGNALING AND LEISHMANIA VIRULENCE
Table I. (Continued)
Molecular Serial No. Accession No. Gene mass, kDa Calc. pI L. major LP L. major HP 23. A0A3S5H6T5 Cytochrome-b5 reductase, putative 31.5 9.19 High Not found 24. E9B959 Electron transfer flavoprotein–ubiquinone oxidoreductase, 62.8 7.37 High Not found putative 25. Q4QIN2 Putative electron transfer flavoprotein–ubiquinone 62.8 7.36 High Not found oxidoreductase 26. E9BGB2 Mitochondrial RNA binding protein, putative 39.8 9.32 High Not found 27. A0A3Q8IF71 Mitochondrial RNA binding complex 1 subunit, putative 108.8 6.98 High Peak found 28. Q4QBS0 Putative NADH–cytochrome b5 reductase 31.8 7.5 Not found High 29. E9BSW9 Mitochondrial phosphate transporter, putative (fragment) 19.9 8.72 High Not found Purine and pyrimidine metabolism 1. Q27679 Adenine phosphoribosyltransferase 26.2 6.4 High Not found 2. E9BI25 Adenine phosphoribosyltransferase 26.2 6.4 High Not found 3. A0A3Q8IFK1 Adenine phosphoribosyltransferase 26.2 6.4 High Not found 4. E9BF84 Hypoxanthine phosphoribosyltransferase 23.6 7.3 High Not found 5. Q4QCC3 Hypoxanthine phosphoribosyltransferase 23.6 7.77 High Not found 6. Q4QCC2 Xanthine phosphoribosyltransferase 26.7 7.81 High Not found 7. Q4Q6T6 S-adenosylmethionine synthase 43 5.88 High Not found
8. O96439 Adenosine kinase 37.1 5.97 Not found High Downloaded from 9. Q4QHB3 Nucleoside phosphorylase-like protein 36.8 6.35 High Not found 10. Q4QEF8 Cytidine deaminase-like protein 19.3 6.86 Not found High 11. H9ABU0 Uracil phosphoribosyltransferase 27.2 5.72 High Not found 12. Q4Q3A1 Putative uracil phosphoribosyltransferase 27.2 5.52 High Not found 13. A0A3S5H592 AMP deaminase, putative 212.9 6.77 Not found High 14. E9B7Z3 AMP deaminase, putative 200.8 6.62 Not found High Fatty acid metabolism 1. E9BQ16 2,4-Dienoyl-coa reductase fadh1, putative 78.4 8.59 High Peak found http://www.jimmunol.org/ 2. E9AC43 Putative long-chain-fatty-acid–CoA ligase 77.6 6.61 Not found Not found 3. Q4QBL1 Farnesyl pyrophosphate synthase 40.9 5.81 High Not found 4. E9BPM4 b-Ketoacyl synthase family protein, putative 47.2 6.46 High Not found 5. Q4Q3E6 Putative enoyl-[acyl-carrier-protein] reductase 33.9 8.59 Not found High 6. E9BIW5 Methylmalonyl-Co a mutase, putative 79.4 6.71 High Peak found 7. Q4Q8M1 Putative propionyl-coa carboxylase b-chain 56.7 8.57 High Not found 8. Q4Q2V8 Putative dolichyl-P-Man:GDP-Man1GlcNAc2-PP-dolichyl 60.1 8.34 Not found High a-1,3-mannosyltransferase 9. E9BI15 Methylmalonyl-coa epimerase-like protein 16.5 7.5 High Not found
OS = Leishmania donovani (strain BPK282A1) by guest on September 29, 2021 10. Q4Q9I9 Methylmalonyl-coa epimerase-like protein 15.3 6.51 High Not found OS = Leishmania major 11. Q4Q3R4 Putative b-ketoacyl synthase family protein 47.1 7.06 High Not found 12. E9BSI3 Glycerol kinase, glycosomal, putative 55.9 7.4 Not found High 13. E9AD07 Putative methylmalonyl-Co a mutase 79.3 6.61 High Peak found 14. D0AB09 Glycylpeptide N-tetradecanoyltransferase 48.6 6.76 High Not found 15. Q8T4P8 Glycylpeptide N-tetradecanoyltransferase (fragment) 20.9 9.82 High Not found 16. A0A3S5H7R0 N-myristoyl transferase, putative 48.6 6.92 High Not found 17. Q4Q758 Sphingosine-1-phosphate lyase 59.3 8.48 Not found High 18. Q4Q4Q4 3-Hydroxyisobutyryl-CoA hydrolase, mitochondrial 40.1 6.19 High Not found 19. Q9BIF7 Dihydrolipoamide dehydrogenase-like protein (fragment) 19.8 8.05 High Not found Sugar metabolism 1. Q4QES5 Sucrose-phosphate synthase-like protein 52.4 6.43 High Not found 2. Q4QBD1 Aldose 1-epimerase-like protein 44.2 5.59 High Not found 3. E9BJT1 Ribose 5–phosphate isomerase, putative 18.7 5.77 High Not found 4. A0A3Q8ID81 Ribose 5–phosphate isomerase, putative 18.7 5.77 High Not found 5. Q4Q869 Putative ribose 5–phosphate isomerase 18.6 6.52 High Not found 6. Q4Q2G5 Phosphomannomutase-like protein 65.2 7.47 Not found High 7. E9BH77 Transketolase 71.8 6.64 High Not found 8. Q4Q882 Probable methylthioribulose-1-phosphate dehydratase 27.2 6.34 High Not found 9. Q4QIK7 Phosphoacetylglucosamine mutase 65.3 5.87 High Not found 10. Q4QBG5 Mannose-1-phosphate guanyltransferase 41.7 5.99 High Not found 11. Q4Q3Z4 Putative phosphoribosyl transferase 39 7.2 High Peak found 12. E9BNB3 Glycylpeptide N-tetradecanoyltransferase 48.6 6.92 High Not found 13. E9BT64 N-acetylglucosamine-6-phosphate deacetylase-like protein 46.8 7.21 Peak found High 14. A0A3Q8IGB4 Glycerophosphoryl diester phosphodiesterase family, 38.3 8.46 High Peak found putative 15. E9BDJ0 a-Glucosidase II subunit, putative 90.8 6.35 High Not found 16. Q4QE33 Putative a-glucosidase II subunit 90.5 6.06 High Not found NADP and ATP metabolism 1. A1Y2C8 z-Crystallin/NADPH-oxidoreductase-like protein 33 6.89 High Not found (fragment) 2. E9BGK5 V-type proton ATPase subunit a 87.6 5.11 High Not found 3. E9ADR4 Putative ATP-binding cassette protein subfamily A, 206.9 7.37 Not found High member 10 4. Q4QH08 ATPase ASNA1 homolog 43.9 5.44 High Not found (Table continues) The Journal of Immunology 2739
Table I. (Continued)
Molecular Serial No. Accession No. Gene mass, kDa Calc. pI L. major LP L. major HP 5. P11718 Probable proton ATPase 1A 107.4 5.44 High Not found 6. P12522 Probable proton ATPase 1B 107.2 5.73 High Not found 7. E9BDY2 Plasma membrane ATPase 107.3 5.78 High Not found 8. Q5VLR7 ADP ribosylation factor 1 20.6 7.12 High Not found 9. Q4Q8N2 Sacchrp_dh_NADP domain–containing protein 41.8 8.16 Not found High 10. A1Y2C7 z-Crystallin/NADPH-oxidoreductase-like protein (fragment) 33.1 6.52 High Not found 11. Q4QDN7 Plasma membrane ATPase 106.9 5.57 High Not found 12. Q4QDN8 Plasma membrane ATPase 107 5.52 High Not found 13. E9BNS8 NAD+ synthase, putative 32.5 6 Not found High 14. A0A3Q8II62 Methylthioribulose-1-phosphate dehydratase, putative 25.5 6.06 High Not found 15. E9BG63 ATP-binding cassette protein subfamily G, member 5, 135 6.39 High Not found putative 16. Q4QBD6 Putative ATP-binding cassette protein subfamily 134.8 6.38 High Not found 17. A0A3Q8IGN1 Vacuolar proton-ATPase-like protein, putative 101 5.82 Not found High Other metabolic genes 1. Q572M7 6-Phosphogluconate dehydrogenase (fragment) 29.5 6.57 High Peak found 2. Q572M9 6-Phosphogluconate dehydrogenase (fragment) 29.6 5.78 High Peak found
3. Q4Q8S1 6-Phosphogluconolactonase 28.4 5.78 Not found High Downloaded from 4. Q5XQR1 Glyoxalase I 16.3 5.11 Peak found High 5. E9BSH6 Glyoxalase I 15.7 4.83 Peak found High 6. Q4Q0Z7 Putative oxidoreductase 36.1 7.23 High Not found 7. Q4Q2M5 Epimerase domain-containing protein 55.4 8.18 High Not found 8. A0A3Q8ICX4 Thioesterase-like superfamily/Thioesterase superfamily, 37.2 9.94 Not found High putative 9. A0A3Q8IC15 Glutathione synthetase, putative 67.1 6.54 Not found High 10. Q4Q8A8 Putative hydrolase, a/b fold family 38.3 8.78 High Not found http://www.jimmunol.org/ 11. E9BP84 Pyrroline-5-carboxylate synthetase-like protein 50.6 6.13 High Not found 12. Q4QC52 2-Oxoisovalerate dehydrogenase subunit a 53.3 6.16 High Not found 13. Q4QG40 AB hydrolase-1 domain-containing protein 43.6 7.23 High Not found 14. A0A3Q8I9M0 a/b Hydrolase family, putative 43.6 8.37 High Not found 15. Q4QC34 Choline dehydrogenase, like protein 58.4 8.38 High Not found 16. A0A3Q8IRG0 Oxidoreductase, putative 36.2 6.92 High Not found 17. Q4QC30 PEROXIDASE_4 domain-containing protein 38.3 7.87 High Not found 18. A0A3S5H6W5 Phosphotransferase enzyme family, putative 94.6 8.48 Not found High 19. E9BDK1 UDP-galactopyranose mutase 54.9 6.54 High Not found
20. E9AFC6 1-Alkyl-2-acetylglycerophosphocholine esterase 50 8.28 High Not found by guest on September 29, 2021 21. A0A3S5H601 N-acetylglucosamine-phosphate mutase, putative 65.4 6.02 High Not found 22. Q4Q0N8 Putative glycosyl hydrolase 128.6 6.8 Not found High 23. E9B7M2 2-Aminoethylphosphonate:pyruvateaminotransferas 41.8 8.79 High Not found e-like protein 24. E9BUR6 2-Methoxy-6-polyprenyl-1,4-benzoquinol methylase, 32 5.92 High Not found mitochondrial 25. Q4QE87 Hydrolase-like protein 46.7 9.66 Not found High 26. Q4Q735 Formate–tetrahydrofolate ligase 66.5 6.8 Not found High 27. A0A3S5H5Y9 Vacuolar-type Ca2+-ATPase, putative 122.1 6.32 High Not found 28. E9BT49 Isopentenyl-diphosphate d-isomerase, putative 39.6 8.02 Not found High 29. Q5QQ43 Isopentenyl-pyrophosphate isomerase 39.5 7.66 Not found High 30. E9BK68 Carbonic anhydrase-like protein 67.6 7.66 High Not found 31. A0A3Q8IDR5 Carbonic anhydrase-like protein 67.6 7.74 High Not found 32. E9BJP1 Hydrolase, a/b fold family, putative 38.3 8.38 High Not found 33. Q4Q8M3 Carbonic anhydrase-like protein 67.2 9.11 High Not found 34. E9BM02 Formate–tetrahydrofolate ligase 66.6 6.62 Not found High Transporters 1. Q4QBE9 ABC-thiol transporter 173.7 6.32 High Not found 2. A0A3Q8IR35 Transportin2-like protein 102.2 4.96 High Not found 3. Q4Q1E8 Transportin2-like protein 102.2 5.06 High Not found 4. E9BLL1 Importin subunit a 58 5.01 Not found High 5. Q4Q7J1 Importin subunit a 58.1 5.01 Not found High 6. E9BQK5 Importin b-1 subunit, putative 95.5 4.67 Not found High 7. A0A3Q8IUD8 Importin b-1 subunit, putative 95.6 4.67 Not found High Protein synthesis 1. Q4Q1V1 Putative 40S ribosomal protein S9 22.1 10.65 High Not found 2. E9ADB9 60S acidic ribosomal protein P0 34.8 5.1 High Not found 3. Q4Q6S9 40S ribosomal protein S14 22.9 9.73 High Not found 4. E9B7A7 40S ribosomal protein S7 (fragment) 11.5 10.67 Not found High 5. Q4QIB4 Translation initiation factor-like protein 38 5.2 Not found High 6. E9B9Z9 Eukaryotic translation initiation factor 2 subunit, 31.1 6.32 Not found High putative (fragment) 7. Q7YWB3 Translation initiation factor 2 a subunit 35.9 5.82 High Not found 8. E9B9G2 Translation initiation factor-like protein 37.9 5.26 Not found High 9. E9BAE7 Translation initiation factor eif-2b b subunit, putative 57.6 5.55 High Not found 10. A0A3Q8IJA9 Eukaryotic translation initiation factor 4 g, putative 83.9 6 High Not found (Table continues) 2740 CD40 SIGNALING AND LEISHMANIA VIRULENCE
Table I. (Continued)
Molecular Serial No. Accession No. Gene mass, kDa Calc. pI L. major LP L. major HP 11. Q4FXY8 Elongation factor Ts, mitochondrial 30.1 7.8 High Not found 12. E9BKM1 Elongation factor Ts, mitochondrial 29.9 7.8 High Not found 13. M1LZZ9 Putative elongation initiation factor 2 a subunit (fragment) 25.9 8.21 High Not found 14. Q4QDS1 Putative prolyl-tRNA synthetase 81.5 6.37 High Not found 15. Q4QDS0 Putative prolyl-tRNA synthetase 81.5 6.54 High Not found 16. Q4QBJ3 Alanine–tRNA ligase 106.3 5.87 High Not found 17. E9BKF3 Tryptophanyl-tRNA synthetase, putative 45.4 8.1 High Not found 18. Q4QGR8 Putative cysteinyl-tRNA synthetase 88.5 6.05 High Not found 19. Q4QEQ4 Tyrosyl or methionyl-tRNA synthetase-like protein 19.6 5.44 Not found High 20. A0A3Q8IHR2 Histidyl-tRNA synthetase, putative 52.8 6.09 High Not found 21. E9AE98 Aspartyl putative aminopeptidase 49.6 6.61 High Not found 22. Q4Q7P3 Putative histidyl-tRNA synthetase 52.9 5.92 High Not found 23. E9BCX1 Tyrosyl or methionyl-tRNA synthetase-like protein 25.2 6.55 Not found High 24. A0A3S5H6Y3 Tyrosyl or methionyl-tRNA synthetase-like protein 19.6 5.43 Not found High 25. A0A3Q8IA00 Lysyl-tRNA synthetase, putative 67.1 6.28 Not found High 26. E9BC68 Lysine–tRNA ligase 67.1 6.28 Not found High 27. Q4Q6 3 7 Putative valyl-tRNA synthetase 109.6 6.64 High Not found
28. E9BM60 Valyl-tRNA synthetase, putative 109.8 6.74 High Not found Downloaded from 29. A0A3Q8IJP4 Valyl-tRNA synthetase, putative 109.7 6.79 High Not found 30. Q4Q648 tRNA_lig_CPD domain-containing protein 103.2 7.88 Not found High Proteases and peptidases 1. E9B9W9 Oligopeptidase b 83.1 6.16 High Not found 2. C9EF60 Oligopeptidase B 83.1 6.16 High Not found 3. A0A2I6J0 3 3 Cysteine proteinase b (fragment) 38 6.96 High Peak found 4. Q4QI66 Cathepsin L-like protease 48 7.8 High Peak found http://www.jimmunol.org/ 5. K7P522 Cathepsin L-like protease 37.8 6.71 High Peak found 6. Q4QI64 Cathepsin L-like protease 37.8 6.77 High Peak found 7. K7P582 Cathepsin L-like protease 37.8 7.18 High Peak found 8. A0A2I6J0 3 1 Cysteine proteinase b (fragment) 37.8 6.77 High Peak found 9. K7PN70 Cysteine protease 37.7 6.6 High Peak found 10. K7PN53 Cysteine protease 37.8 7.18 High Peak found 11. K7PNP9 Cysteine protease 37.8 6.61 High Peak found 12. Q4QI68 Cathepsin L-like protease 37.9 7.4 High Peak found 13. A0A2I6J0W9 Cysteine proteinase b 37.9 7.4 High Peak found 14. A0A3Q8IBQ6 Mitochondrial processing peptidase a subunit, putative 57.8 7.99 High Not found
15. Q4Q3T0 Putative aminopeptidase 55.7 7.08 High Not found by guest on September 29, 2021 16. E9BMG5 Calpain-like cysteine peptidase, putative 89.2 5.12 Not found High 17. A0A3Q8IKU0 Mitochondrial intermediate peptidase, putative 75.9 6.46 Not found High 18. E9B9J8 Cathepsin L-like protease (fragment) 37.3 7.14 High Not found 19. M9SY92 Cysteine protease b 48 7.61 High Not found 20. Q5EF92 Cathepsin L-like cysteine protease (fragment) 26.4 6.07 High Not found 21. D9YIV5 Cysteine protease F (fragment) 25.7 5.77 High Not found 22. Q95WR6 Cysteine protease 42.6 7.56 High Not found 23. Q5EF90 Cathepsin L-like cysteine protease (fragment) 22.6 4.84 High Not found 24. A0A3S5H682 Cysteine peptidase B (CPB) 42.7 7.78 High Not found 25. Q599P0 Cysteine proteinase type B (fragment) 13 8.29 High Not found 26. Q5EF94 Cathepsin L-like cysteine protease (fragment) 26.4 6.07 High Not found 27. E9LZ04 Cysteine proteinase B (fragment) 35.6 6.83 High Not found 28. A0A3S5H683 Cysteine peptidase B (CPB) 48 7.44 High Not found 29. Q5EF91 Cathepsin L-like cysteine protease (fragment) 24.4 6.07 High Not found 30. D1MG61 Cysteine proteinase B (fragment) 19.2 4.91 High Not found 31. A0A3S5H685 Cysteine peptidase B (CPB) 48.1 7.61 High Not found 32. A0A346M343 Cysteine protease (fragment) 20.2 4.84 High Not found 33. A0A3S5H684 Cysteine peptidase B (CPB) 47.9 7.09 High Not found 34. Q95WR7 Cysteine protease 47.9 7.61 High Not found 35. Q5EF93 Cathepsin L-like cysteine protease (fragment) 26.3 6.65 High Not found 36. Q599N8 Cysteine proteinase type B (fragment) 6.9 5.1 High Not found 37. D9YIG2 Cysteine protease F (fragment) 25.6 5.76 High Not found 38. A0A346M344 Cysteine protease (fragment) 20.2 4.93 High Not found 39. Q4Q0W9 Putative mitochondrial intermediate peptidase 76 6.54 Not found High 40. Q4Q990 Peptidase_M28 domain–containing protein 59.6 7.12 High Not found 41. E9BIT9 CAAX prenyl protease 49.1 8.5 High Not found 42. Q4QCC5 Methionine aminopeptidase 2 51.2 6.99 High Not found 43. E9BGD0 Cytosolic leucyl aminopeptidase 60 9.17 High Not found 44. A0A3Q8ID62 Calpain-like cysteine peptidase, putative 716.8 5.48 Not found Not found 45. E9ACY1 CAAX prenyl protease 49.2 8.48 High Not found 46. Q4Q1H9 Serine peptidase, Clan SC, Family S9B 94.2 5.92 Not found High Virulence factors 1. Q4QDY0 Putative ATP-dependent zinc metallopeptidase 64.9 6.38 High Peak found 2. A0A3Q8IFG3 Metalloprotease M41 FtsH, putative 77.8 8.35 High Not found 3. Q4Q5D1 Putative ATP-dependent zinc metallopeptidase 78 8.35 High Not found 4. Q4QIT4 Pitrilysin-like metalloprotease 115.6 6.24 High Not found (Table continues) The Journal of Immunology 2741
Table I. (Continued)
Molecular Serial No. Accession No. Gene mass, kDa Calc. pI L. major LP L. major HP 5. E9BQB6 Macrophage migration inhibitory factor-like protein 12.7 7.77 High Not found 6. Q4QD50 Putative ATP-dependent zinc metallopeptidase 64.5 7.14 Not found High DNA synthesis 1. E9B9I5 DNA polymerase OS = Leishmania donovani 42.6 8.95 High Not found (strain BPK282A1) 2. Q4QI80 DNA polymerase 42.7 8.95 High Not found 3. A0A3Q8IFC3 Replication factor A protein 3, putative 13.2 5.39 Not found High 4. O15922 DNA-(apurinic or apyrimidinic site) lyase 48.6 8.54 Not found High 5. E9BJR5 Replication protein A subunit 52.3 6.43 Not found High Flagellar proteins 1. A0A3Q8IGZ2 Paraflagellar Rod Proteome Component 9, putative 15.7 5.14 High Not found 2. Q9NJT6 Major paraflagellar rod protein (fragment) 3 10.46 High Not found 3. A0A3Q8IG75 Radial spoke protein 3, putative 42.1 6.2 Not found High 4. E9AD29 Putative radial spoke protein 3 42 5.81 Not found High 5. A0A3S5H752 Intraflagellar transport protein 52, putative 73.5 5.2 Not found High 6. E9BE28 Intraflagellar transport protein component, putative 73.5 5.2 Not found High 7. Q4QDI9 Putative intraflagellar transport protein component 73.2 5.19 Not found High
8. E9B894 Paraflagellar rod component par4, putative 68.5 5.49 Not found High Downloaded from 9. A0A3Q8IJX2 Intraflagellar transport protein 80, putative 66.5 7.88 Not found High 10. Q4QJJ9 Putative paraflagellar rod component par4 68.2 5.36 High Not found 11. A0A3Q8IH47 Flagellar Member 8 241.7 5.07 High Not found 12. E9AD91 Putative intraflagellar transport protein IFT88 91.2 6.62 High Not found Signaling proteins 1. E7E1K5 PKA regulatory subunit 1 56.2 4.97 High Peak found 2. E9BB91 Protein kinase A regulatory subunit, putative 56.2 4.91 High Peak found 3. A0A3Q8IGR4 Rab-GTPase-TBC domain containing protein, putative 91.2 5.62 High Not found http://www.jimmunol.org/ 4. R9VXI6 Activated protein kinase C receptor (fragment) 24.9 6.79 High Not found 5. A0A3Q8I8T8 Ras-related protein RabX1, putative 24.5 8.24 High Not found 6. Q4QH98 Putative rab1 small GTP-binding protein 24.6 8.24 High Not found 7. A0A3Q8I9L6 Ran-binding protein 1, putative 17.8 5.72 High Not found 8. Q4QEZ7 Protein tyrosine phosphatase PRL-1 19.4 8.37 High Not found 9. E9BCM8 Protein tyrosine phosphatase-like protein 19.4 8.38 High Not found 10. Q9GNR4 Protein kinase A catalytic subunit isoform 1 42.2 8.94 High Not found 11. A0A109NYM0 Rab5a 25 7.85 High Not found 12. E9BDU4 Ras-related protein rab-5, putative 25.3 7.11 High Not found
13. Q4QG06 Putative Ran-binding protein 1 17.8 5.72 High Not found by guest on September 29, 2021 14. Q95ZB3 Possible rac-family ser/thr kinase homolog (fragment) 17.9 9.22 High Not found 15. E9BTF6 Nonspecific serine/threonine protein kinase 87.4 8.76 High Not found 16. E9BGI0 Ran-binding protein, putative 23.2 4.61 High Not found 17. Q4Q1Y6 Nonspecific serine/threonine protein kinase 88 8.92 High Not found 18. E9BCN0 Protein tyrosine phosphatase-like protein 19.5 7.81 High Not found 19. Q4QB12 Putative Ran-binding protein 23 4.78 High Not found 20. E9BQB2 Protein kinase, putative 72.2 6.43 Not found High 21. Q4QCV0 Protein kinase domain-containing protein 360 7.31 High Not found 22. Q4QJ04 Putative serine-threonine dehydratase 37 8.16 High Not found 23. A0A3S5H5S2 Serine-threonine dehydratase, putative 37 8.16 High Not found 24. Q4Q5N4 Putative ras-related rab-4 22.2 5.38 Not found Not found 25. E9BDJ6 Serine/threonine protein phosphatase type 5, putative 53 6.71 High Not found 26. Q4QE27 Putative serine/threonine protein phosphatase type 5 52.9 6.84 High Not found 27. A0A3Q8IGW3 Maf-like protein, putative 24.6 5.29 Not found High 28. A0A3S5H7N7 MAPK, putative 43.9 7.34 Not found High 29. E9BMA7 Serine/threonine-protein kinase, putative 84.6 4.97 High Not found 30. E9AFM1 Protein kinase A catalytic subunit isoform 2 39.1 9.07 High Not found 31. E9BEL9 Ser/thr protein phosphatase 2A regulatory subunit, putative 63.8 6.06 Not found High 32. A0A3Q8IKQ7 Zinc-binding phosphatase, putative 107.7 6.84 Not found High 33. Q4QCP6 Putative zinc-binding phosphatase 107.5 7.08 Not found High 34. Q4Q0C8 Serine/threonine-protein kinase TOR 291.5 6.86 Not found High 35. E9BFW2 Adaptor complex AP-1 medium subunit, putative 36 8.5 High Not found 36. A0A3Q8IB94 Adaptor complex AP-1 medium subunit, putative 49.3 7.09 High Not found 37. E9BLH8 Rac serine-threonine kinase, putative 57.6 6.6 Not found High 38. Q4Q7M5 Putative serine-threonine kinase 57.7 6.7 Not found High 39. S6CXR4 RAC/AKT-like 57.6 6.7 Not found High 40. A0A3Q8IB30 Protein kinase A catalytic subunit 38.2 8.48 Not found High 41. A0A3Q8IEW2 Translation factor sui1, putative 12.4 9.38 High Not found 42. Q4Q701 Putative MAPK 44 7.34 Not found High 43. E9B893 Ras-like small GTPases, putative 21.7 7.4 High Not found 44. Q4QJK0 Putative ras-like small GTPases 21.7 7.4 High Not found 45. R9VZZ9 Activated protein kinase C receptor (fragment) 25.3 6.79 High Not found 46. R9VW64 Activated protein kinase C receptor (fragment) 23.4 6.98 High Not found 47. A0A3S5H5L8 Phosphatidylinositol 4-phosphate 5-kinase, putative 80.3 4.98 Not found Not found 48. Q4Q078 AP complex subunit b 82.2 5.15 Not found High 49. Q4Q6T0 Putative serine/threonine-protein kinase 84.8 4.94 High Not found (Table continues) 2742 CD40 SIGNALING AND LEISHMANIA VIRULENCE
Table I. (Continued)
Molecular Serial No. Accession No. Gene mass, kDa Calc. pI L. major LP L. major HP RNA binding proteins 1. Q4Q8I6 Putative RNA binding protein rbp16 15.1 10.11 High Not found 2. E9BLL0 RNA-binding protein, putative 67.4 6.19 Not found High 3. Q4Q7J2 Putative RNA-binding protein 67.5 6.27 Not found High 4. A0A3Q8IMV3 RNA binding protein, putative 26.5 5.57 High Not found 5. Q4QAV6 Putative ATP-dependent RNA helicase 66.7 9.64 High Not found Nucleases 1. Q4QBF5 Putative endoribonuclease L-PSP (Pb5) 16.9 5.85 High Not found 2. E9BTP6 Exoribonuclease 2, putative 100.5 8.24 Not found High 3. Q4Q1P5 Putative exoribonuclease 2 102.3 8.28 Not found High Transcription 1. Q4Q3Q4 Transcription elongation factor-like protein 50.5 5.55 Not found High 2. Q4Q3B6 Putative DNA-directed RNA polymerase subunit 17.5 4.5 High Not found 3. A0A3Q8IKT1 DNA-directed RNA polymerase III subunit, putative 17.5 4.53 High Not found 4. E9BMD9 DNA-directed RNA polymerase subunit b 133.2 7.8 Not found High 5. Q4Q6P8 DNA-directed RNA polymerase subunit b 133.8 7.87 Not found High 6. Q4QH24 Putative transcription modulator/accessory protein 117.7 9.2 Not found High
Proteasome and related proteins Downloaded from 1. E9BNE5 Proteasome regulatory non-ATP-ase subunit 8, putative 40.1 5.5 High Not found 2. Q4Q5P6 Putative 26S proteasome regulatory subunit 40.1 5.73 High Not found 3. E9BBD6 Ubiquitin-like protein 89.1 5.41 Not found High 4. A0A3Q8IEW8 Proteasome regulatory non-ATPase subunit 2, putative 107.7 5.49 Not found High 5. A0A3Q8IKW3 Ubiquitin-conjugating enzyme protein, putative 29.9 4.63 High Not found 6. Q4Q5I6 Putative ubiquitin-conjugating enzyme protein 29.9 4.7 High Not found Nuclear proteins 1. Q4Q1H0 Nuclear pore protein 96.9 6.77 High High http://www.jimmunol.org/ 2. E9BNC3 U2 small nuclear ribonucleoprotein 40K, putative 34.5 6.6 Not found High 3. Q4Q5R8 Putative U2 small nuclear ribonucleoprotein 40K 34.6 6.04 Not found High 4. E9BPS1 Small nuclear ribonucleoprotein Sm D2 11.8 9.54 High Not found 5. E9BE39 Nucleosome assembly protein, putative 39.8 4.68 Not found High 6. Q4QDH7 Putative nucleosome assembly protein 39.7 4.73 Not found High 7. Q4Q377 Putative NLI-interacting factor 41.1 7.64 High Not found Other genes 1. Q4QF74 Tryparedoxin peroxidase 21.2 6.9 High Not found 2. Q9TZS4 Peroxidoxin 22.2 7.72 High Not found
3. Q4Q063 Putative T-complex protein 1, u subunit 58.1 5.4 High Not found by guest on September 29, 2021 4. A0A3Q8I931 Tb-292 membrane associated protein-like protein 1563.8 5.25 High Not found 5. E9AF32 Putative kinesin 77 6.74 High Not found 6. Q4QFW1 HIT domain–containing protein 15.3 6.62 Peak found High 7. E9B8J4 Stomatin-like protein 39.6 7.81 High Not found 8. A0A3S5H6F3 MORN repeat, putative 23.2 6.2 High Not found 9. Q4Q5R2 Dynein L chain 10.6 6.51 High Not found 10. A0A3Q8IB76 RF-1 domain–containing protein, putative 62.3 8.91 High Not found 11. Q4QA70 Dynein L chain 10.3 7.05 High Not found 12. A0A3Q8IJN8 Snf7, putative 25.6 4.93 High Not found 13. Q4QJ98 Stomatin-like protein 39.7 8.21 High Not found 14. E9BFZ1 Centrin, putative 16.4 4.54 High Not found 15. A0A3Q8IGV8 b Prime cop protein, putative 97.7 5.19 High Not found 16. E9BPS3 Coatomer subunit b’ 97.7 5.19 High Not found 17. Q4Q3L5 Coatomer subunit b’ 97.9 5.22 High Not found 18. A0A3Q8IHU1 TIP49 C terminus/Holliday junction DNA helicase 39.8 8.68 High Not found ruvB N terminus/AAA domain (Dynein-related subfamily)/AAA domain/ATPase family associated with various cellular activities (AAA), putative 19. A0A3S5H503 Voltage-dependent anion-selective channel, putative 31.9 8.48 High Not found 20. E9AE06 ANK_REP_REGION domain–containing protein 75 5.66 Not found High 21. A0A3S5H6F4 Nucleolar protein 56, putative 52.6 8.1 Not found High 22. Q95VS9 Centrin 16.4 4.59 High Not found 23. Q4Q576 DUF3456 domain–containing protein 40.5 6.01 Not found High 24. Q4Q5L5 Putative OSM3-like kinesin 126.9 5.94 Not found High 25. Q4Q5N9 Putative PG f synthase 31.8 7.33 Not found High 26. Q4Q7S6 Rhodanese domain–containing protein 82.1 6.49 High Not found 27. Q4Q3V2 Rhodanese domain–containing protein 89.9 6.15 High Not found 28. A0A3Q8IKN9 Small myristoylated protein-1 15 5.36 High Peak found 29. Q4QC65 RF_PROK_I domain-containing protein 62.6 9.06 High Not found 30. A0A3Q8IE03 Vesicle-associated membrane protein, putative 28.1 8.95 High Not found 31. A0A3Q8IIK7 Dynein H chain and region D6 of dynein motor/Ankyrin 38.9 5.95 High Not found repeats (3 copies)/Ankyrin repeat, putative 32. A0A3Q8IEG3 C2 domain containing protein, putative 100 7.31 Not found High 33. E9AFF5 LRRcap domain-containing protein 32.8 7.15 High Not found 34. E9AG07 ANK_REP_REGION domain-containing protein 38.8 5.8 High Not found 35. Q1 3 7J8 Excreted/secreted protein 31 138.6 5.25 Not found Not found (Table continues) The Journal of Immunology 2743
Table I. (Continued)
Molecular Serial No. Accession No. Gene mass, kDa Calc. pI L. major LP L. major HP 36. Q4QAH3 UBA domain–containing protein 42.7 10.58 Not found High 37. Q4Q803 Putative leucine-rich repeat protein 96.2 7.97 High Not found 38. A0A3Q8IK24 Mkiaa0324 protein-like protein 56.4 12.31 Not found High 39. Q4Q5L8 AKAP7_NLS domain–containing protein 31.4 8.92 High Not found 40. Q4QES6 Dynein L chain 13.2 5.82 Not found High 41. A0A3Q8IGL2 OSM3-like kinesin, putative 127.1 5.8 Not found High 42. Q4Q2Y0 Thioredoxin domain–containing protein 47.4 7.33 Not found High 43. E9ADE3 BRO1 domain–containing protein 102.3 6.65 High Not found 44. E9BE06 Clathrin L chain 30.2 6.92 Not found Not found 45. A0A3S5H749 Clathrin L chain, putative 41.7 7.47 Not found Not found 46. E9ADU9 Letm1 RBD domain–containing protein 56.7 9.6 Not found High 47. E9BKS0 Tryparedoxin 17.2 7.12 High Not found 48. A0A3S5H6D0 Plus-3 domain/Zinc finger, C3HC4 type (RING finger) 51.3 6.47 Not found High containing protein, putative 49. E9AC00 Glutaredoxin-like protein 21.4 5.43 High Not found 50. E9BRP3 Coatomer z subunit, putative 20.2 4.83 Not found High 51. A0A3S5H6E1 Ef-hand protein 5, putative 20.8 4.89 High Not found
52. E9AFW9 C3H1-type domain-containing protein 107.3 8.84 Not found High Downloaded from 53. A0A3Q8IMD7 Tumor suppressor, Mitostatin, putative 59.1 8.97 High Not found 54. E9AEZ8 RPN13_C domain–containing protein 27.3 8.54 High Not found 55. Q25323 gp96-92 (fragment) 26.6 5.49 High Not found 56. Q4Q843 Putative gp96-92 87.1 8.73 High Not found 57. Q4QGL2 Promastigote surface Ag protein PSA 55.6 6.77 High Not found 58. Q4QGJ7 Surface Ag protein 2 39 5.02 High Not found 59. Q4QGJ2 Putative surface Ag protein 2 68.4 7.78 High Not found 60. Q4QGJ9 Putative surface Ag protein 2 61.8 5.33 High Not found http://www.jimmunol.org/ 61. Q4QGK6 Promastigote surface Ag protein 2 PSA2 39.9 4.86 High Not found 62. Q4JI42 Promastigote surface Ag 40.7 6.52 High Not found 63. Q4QGL4 Putative surface Ag protein 2 41.9 5.06 High Not found 64. A0A3S5H6M4 Surface Ag protein 2, putative 69.9 5.31 High Not found 65. Q4QGJ4 Promastigote surface Ag protein 2 PSA2 40 4.98 High Not found 66. Q4QGL5 Putative surface Ag protein 2 58.9 8.09 High Not found 67. Q4QGK0 Putative surface Ag protein 2 60.6 4.93 High Not found 68. Q4JI41 Promastigote surface Ag (fragment) 31 7.62 High Not found 69. Q4QGK2 Putative surface Ag protein 60.6 7.97 High Not found
70. Q4QGK1 Putative surface Ag protein 2 75 8.18 High Not found by guest on September 29, 2021 71. Q4QGK8 Promastigote surface Ag protein PSA 55.6 6.54 High Not found 72. Q4QGJ6 Putative surface Ag protein 2 60.8 8.22 High Not found 73. Q25339 Surface Ag P2 40.1 5.08 High Not found 74. Q25331 Promastigote surface Ag protein 2 PSA2 40 4.92 High Not found 75. E9AF66 Putative phosphoinositide-binding protein 46.5 6.55 High Not found 76. Q4QCH7 DUF667 domain–containing protein 24.7 8.85 High Not found 77. E9BR14 Centrin, putative 20.8 4.59 Not found High 78. Q4Q0H9 Mkiaa0324 protein-like protein 56.6 12.26 Not found High 79. Q4Q0L9 Putative branch point binding protein 32.9 5.14 High Not found 80. Q4Q1R1 Putative poly-zinc finger protein 2 14.6 8.13 High Not found 81. E9BTN0 Poly-zinc finger protein 2, putative 14.5 8.13 High Not found 82. Q4QHY1 RING-type domain–containing protein 51.2 6.29 Not found High 83. E9B822 Spermidine synthase, putative 32.9 5.36 High Peak found 84. Q9GSR3 Spermidine synthase 32.9 5.26 High Peak found 85. Q4Q8G9 Putative P27 protein 27.6 9.42 High Not found 86. E9AD08 TIP120 domain-containing protein 133.9 5.49 Not found High 87. Q4Q3S3 Putative dynein intermediate chain 110.4 5.76 High Peak found 88. A0A0G2YFI9 Pyridoxal kinase (fragment) 33.1 6.65 Not found High 89. E9AEI8 Putative coatomer z subunit 20.3 4.73 Not found High 90. E9AFS4 CS domain-containing protein 21.5 4.26 High Not found 91. Q4QFW3 PX domain-containing protein 51 7.46 Not found High 92. Q4Q4I8 Putative ATP-binding cassette protein subfamily F,member 3 74.5 6.73 Not found High 93. Q4Q1D0 Putative ATP-binding cassette protein subfamily 72.6 6.71 High Not found G,member 6 94. A0A3Q8IHB5 ATP-binding cassette subfamily F member 1, putative 74.7 6.47 Not found High 95. Q4QHT4 Putative ef-hand protein 5 20.7 4.82 High Not found 96. A0A3S5H7E1 Dynein L chain 1, cytoplasmic, putative 10.3 7.05 High Not found 97. Q4QGZ2 Putative kinesin 148.1 9.66 Not found High 98. Q4QB38 Putative coronin 56.6 6.64 High Not found 99. E9BGF4 Coronin 56.2 7.27 High Not found 100. Q3T1U8 Coronin-like protein 56.7 6.42 High Not found 101. Q4Q9A0 J domain–containing protein 92.7 9.42 High Not found 102. E9BGK2 Pyridoxal phosphate homeostasis protein 27.2 6.37 Not found High 103. Q4Q4U9 Putative dynein light intermediate chain 47 8.69 High Not found 104. Q4QBY1 BAG domain-containing protein 59.5 8.56 Not found High 105. Q4QGW1 Putative S-phase kinase-associated protein 21.1 4.5 High Not found (Table continues) 2744 CD40 SIGNALING AND LEISHMANIA VIRULENCE
Table I. (Continued)
Molecular Serial No. Accession No. Gene mass, kDa Calc. pI L. major LP L. major HP 106. E9AEU9 AAA domain-containing protein 39.8 8.69 High Not found 107. A0A3S5H5G2 Leucine Rich repeat/Leucine rich repeat/Leucine Rich 47 8.43 High Not found Repeat, putative 108. Q4QCJ7 CHAT domain-containing protein 159.1 6.24 Not found High 109. Q4Q1G1 Cyclin-e binding protein 1-like protein 74.5 6.55 Not found High 110. Q4Q1T6 ARL2_Bind_BART domain-containing protein 47.8 Not found High 111. Q9XZY9 ADH_N domain-containing protein 50.2 5.86 Not found High 112. Q4QAV3 Dynein intermediate-chain-like protein 75.2 6.64 High Not found 113. E9BGN9 Dynein intermediate-chain-like protein 75.1 6.68 High Not found 114. E9ADE2 WD_REPEATS_REGION domain-containing protein 93.1 6.57 Not found High 115. A0A3Q8IDD0 Epsin, putative 61.3 6.96 High Not found 116. E9BHH2 Epsin, putative 61.3 7.21 High Not found 117. E9ABZ6 BSD domain-containing protein 64.2 5.62 High Not found 118. E9BM78 UV excision repair RAD23-like protein 44.4 4.37 High Not found 119. Q4Q0W5 MTS domain-containing protein 57.5 7.08 High Not found 120. Q4QCU5 Putative glutaredoxin 12.4 9.16 Not found High 121. E9AEJ6 FCP1 homology domain-containing protein 27.2 6.14 Not found High
122. A0A3Q8IF24 Protein mkt1, putative 90.5 5.25 High Not found Downloaded from 123. Q4QAF2 RRM domain-containing protein 23.9 4.64 High Not found 124. Q4QFQ1 Excreted/secreted protein 9.1 62.6 6.44 High Not found 125. E9BJ95 Dynein H chain, putative 498.3 6.81 Not found High 126. Q4Q6 3 2 RRM domain-containing protein 16.5 5.88 Not found High 127. E9BE21 Kinesin-like protein 93.7 6.52 High Not found 128. A0A3S5H751 C-terminal motor kinesin, putative 93.6 6.52 High Not found 129. O97005 TPR_REGION domain–containing protein 141.4 7.49 High Not found 130. Q4QG02 AAA domain–containing protein 104.3 9.19 High Not found http://www.jimmunol.org/ 131. A0A3S5H746 Kinesin, putative 133.1 6.95 Not found Not found 132. E9BDY3 Kinesin, putative 133 6.9 Not found Not found 133. Q4QBN6 Putative dynein H chain, cytosolic 619.7 6.64 High Not found 134. Q4QEH6 TPR_REGION domain–containing protein 36.2 6.19 Not found High 135. Q4QAF7 GCV_T domain–containing protein 40.6 7.5 Not found High 136. E9AE90 PlsC domain–containing protein 35.1 8.7 Not found High 137. Q4QE78 SAM_MT_RSMB_NOP domain–containing protein 128.3 8.75 Not found High 138. Q4Q2C8 EMC1_C domain–containing protein 87 6.54 High Not found 139. E9AFI0 CRAL-TRIO domain–containing protein 43.7 7.85 High Not found
140. Q4QAB4 GP-PDE domain–containing protein 38 8.59 High Peak found by guest on September 29, 2021 141. Q4Q6V9 UV excision repair RAD23-like protein 44 4.32 High Not found 142. Q4Q737 Reticulon-like protein 22.1 9.41 Not found High 143. A0A3Q8IFS1 Reticulon domain protein, 22 kDa potentially aggravating 22.1 8.97 Not found High protein (Paple22) 144. A0A3Q8IJI8 Putative C2 domain protein 241.3 7.4 High Not found 145. Q68RJ8 Trypanothione-dependent glyoxalase I 16.3 4.94 Peak found High 146. Q4Q9T1 WD_REPEATS_REGION domain-containing protein 126.3 5.41 High Not found 147. E9AF64 WD_REPEATS_REGION domain-containing protein 193 6.67 High Peak found 148. Q4Q8A2 DUF3342 domain-containing protein 67 7.5 Not found High 149. A0A3Q8IK57 Adaptin complex 1 subunit, putative 82.4 5.36 Not found High 150. Q4QDZ5 WD_REPEATS_REGION domain-containing protein 67.1 6.93 Not found High 151. A0A3S5H5C5 Nascent polypeptide associated complex subunit-like 18.1 4.56 High Not found protein, copy 2 152. A0A3Q8I9E6 N-terminal region of Chorein, a TM vesicle-mediated sorter, 605.8 8.24 High Not found putative 153. A0A3Q8IRU0 ABC1 family, putative 60.9 7.18 High Not found 154. A0A3Q8IDV2 C-terminal motor kinesin, putative 103.5 8.32 Not found High 155. E9BJR8 Kinesin-like protein 103.5 8.34 Not found High 156. E9BQT7 NLI-interacting factor, putative 41.1 7.03 High Not found 157. A0A3Q8IIU2 Zinc finger protein family member, putative 107.1 9.06 Not found High Calc. pI, calculated isoelectric point.
Briefly, ELISA plates were coated overnight at 4˚C with purified Abs to BCA protein assay kit (Pierce). Ag-specific Ab isotypes were measured IL-10 (2 mg/ml), IL-12 (2 mg/ml), IL-4 (1 mg/ml), or IFN-g (2 mg/ml). by ELISA on CSA-coated plates (15). Plates were washed thrice (0.05% Tween 20 in 13 PBS) and blocked for 2 h with 1% BSA. Plates were incubated overnight with cytokine standards Sample preparation for SELDI, protein chip array preparation, or culture supernatants. Plates were washed and incubated with respective and data analysis biotin-conjugated detection Abs for 1 h at 25˚C. Plates were washed and incubated with peroxidase-conjugated streptavidin (Roche Applied Science) Stationary-phase parasites were harvested by centrifugation at 2500 rpm for for 30 min, followed by washing and development with tetramethylbenzidine 10 min, washed thrice with 13 PBS, resuspended in 300 ml of lysis buffer substrate (BD Pharmingen, San Diego, CA). Reaction was stopped by the (20 mM Tris [pH 7.4], 150 mM NaCl, 0.1% Triton X-100, 1 mM EDTA, and addition of 1 N H2SO4, and absorbance was read at 450 nm. protease inhibitor mixture from Roche), and incubated at 4˚C for 1 h. Following To detect Leishmania-specific Ig, 96-well microtiter plates were coated lysis, samples were centrifuged at 12,000 rpm at 4˚C for 1 h. Supernatants overnight with leishmanial-CSA-15 mg/ml in 13 PBS at 4˚C. CSA was were collected and estimated protein content using BCA kit (Pierce). prepared by rapid freeze thaw cycles (seven cycles), followed by sonication ProteinChip Q10 anion array (Bio-Rad) was used for proteome analysis and clarification by centrifugation. The protein content was assayed by of HP and low-passage (LP) of L. major promastigotes. Ten micrograms The Journal of Immunology 2745
Table II. Protein profiles in virulent and avirulent L. major strains data analysis were performed using ProteinChip data manager software 3.0.7 (Bio-Rad). Peaks were automatically detected and normalized Serial to total ion current and mass spectra were collected over the range of No. Biological Process Count in LP Count in HP 2,000–20,000 Da mass. The background was subtracted using the de- fault software settings. For data analysis, all peaks with signal to noise Cellular functions ratio higher than 5 and valley depth 3 within a cluster mass window of 1 Cell communication 0 0 0.3% of the mass were considered. 2 Cell death 2 2 3 Cell differentiation 1 1 Proteomics analysis of virulent (LP) and avirulent (HP) strains 4 Cell division 1 1 of L. major 5 Cell growth 1 0 6 Cell organization and 151 136 Twenty-five microliter samples were taken and reduced with 5 mM biogenesis TCEP and further alkylated with 50 mM iodoacetamide, followed by 7 Cell proliferation 2 2 Trypsin (1:50, Trypsin/lysate ratio) digestion for 16 h at 37˚C. Digests were 8 Cellular component 63 52 cleaned using a C18 silica cartridge to remove the salt and dried using a movement speed vac. The dried pellet was resuspended in buffer A (5% acetonitrile, 9 Cellular homeostasis 50 48 0.1% formic acid). 10 Coagulation 0 0 All experiments were performed using EASY-nLC 1000 system (Thermo 11 Conjugation 0 0 Fisher Scientific) coupled to Thermo Fisher QExactiveequipped with 12 Defense response 3 3 nanoelectrospray ion source. One microgram peptide mixture was re- 13 Development 0 0 solved using a 25-cm PicoFrit column (360-mm outer diameter, 75-mm 14 Metabolic process 1248 1160 inner diameter, 10-mm tip) filled with 1.8 mm of C18-resin (Dr. Maisch,
15 Regulation of biological 208 185 Ammerbuch-Entringen, Germany). The peptides were loaded with buffer Downloaded from process A and eluted with a 0–40% gradient of buffer B (95% acetonitrile, 0.1% 16 Reproduction 0 0 formic acid) at a flow rate of 300 nl/min for 100 min. Mass spetrometry 17 Response to stimulus 134 124 data were acquired using a data-dependent top 10 method dynamically 18 Transport 209 179 choosing the most abundant precursor ions from the survey scan. Molecular function All samples were processed and RAW files generated were analyzed 1 Antioxidant activity 40 35 with Proteome Discoverer (v2.2) against the Uniprot Leishmania major 2 Catalytic activity 1116 1016 reference proteome database. For Sequest search, the precursor and 3 DNA binding 59 60 fragment mass tolerances were set at 10 ppm and 0.5 Da, respectively. http://www.jimmunol.org/ 4 Enzyme regulator activity 37 37 The protease used to generate peptides (i.e., enzyme specificity) was set 5 Metal ion binding 210 191 for trypsin/P (cleavage at the C terminus of “K/R: unless followed by 6 Motor activity 53 49 “P”) along with maximum missed cleavages value of two. Carbami- 7 Nucleotide binding 459 425 domethyl on cysteine as fixed modification and oxidation of methionine 8 Protein binding 394 353 and N-terminal acetylation were considered as variable modifications 9 Receptor activity 1 1 for database search. Both peptide spectrum match and protein false dis- 10 RNA binding 189 187 covery rate were set to 0.01 false discovery rate. 11 Signal transducer activity 17 14 12 Structural molecule activity 165 163 FACS–regulatory T cell analyses
13 Transcription regulator 00Popliteal lymph node T cells from primed, unprimed, or naive female BALB/c by guest on September 29, 2021 activity mice were studied for regulatory T (Treg) cells by multicolor FACS 14 Translation regulator 01analyses. After blocking with 5% FCS for 30 min at 4˚C, the cells were activity washed twice with FACS buffer (13 PBS, 10 mM HEPES buffer, and 3% FCS) 15 Transporter activity 83 75 and stained with fluorescently labeled Abs anti–CD3-PE-Cy7, anti–CD4- Cellular component PerCP-Cy5.5, anti–CD25-allophycocyanin-Cy7, anti–CD127-allophycocyanin, 1 Cell surface 0 0 and anti–GITR-PE for 45 min at 4˚C in dark and washed twice with FACS 2 Chromosome 30 31 buffer. For intracellular staining, the cells were permeabilized using 3 Cytoplasm 285 270 Cytofix/Cytoperm-Plus Kit with GolgiPlug (BD Biosciences) and washed 4 Cytoskeleton 22 21 with Perm/Wash buffer (BD Biosciences). The cells were then stained with 5 Cytosol 69 64 anti–Foxp3-PB Abs for 60 min at 4˚C in dark. Cells were acquired in the 6 Endoplasmic reticulum 24 21 CD3+CD4+CD25+CD127dim gate for Treg cells by FACSCanto II flow 7 Endosome 2 2 cytometer (BD Biosciences) and analyzed for expression of GITR and 8 Extracellular 3 2 Foxp3 using BD FACSDiva software (version 5.2; BD Biosciences). 9 Golgi 17 13 Cells stained with specific Isotype Abs were used as controls. 10 Membrane 247 194 11 Mitochondrion 73 67 Statistical analyses 12 Nucleus 121 118 13 Organelle lumen 12 12 The significance of difference between the means of two samples was 14 Proteasome 58 56 calculated from Student t test. For multiple comparisons, one-way ANOVA 15 Ribosome 150 149 with Tukey test was performed. The minimum number of mice used for in vivo 16 Spliceosomal complex 2 2 experiments was five per group. The results were plotted as mean 6 SEM 17 Vacuole 12 12 from triplicate sets of experiments. Differences were considered statisti- cally significant when *p # 0.05, **p # 0.01, ***p # 0.001. protein from each sample was added to a final volume of 50 ml in binding buffer (50 mM Tris-HCl, pH 9) and was applied to Q10 anion ProteinChip Results array. Chips were incubated for 1 h at room temperature with vigorous The proteome profiles of avirulent strains differ from those of shaking (200 rpm) and washed thrice with 150 ml of binding buffer and the virulent strains twice with deionized water at room temperature for 5 min at 200 rpm. The spots were air dried, and finally, 1 ml sinapinic acid mixture (Bio-Rad As described in Materials and Methods, using the protocol, for Laboratories: 100 ml of saturated acetonitrile and 100 ml of 1% trifluoro- Q10 anion ProteinChip–generated mass spectra that range from acetic acid was added to 5 mg of sinapinic acid) was applied as energy 2,000 to 20,000 Da, differential expressions of proteins were an- absorbing molecule. alyzed using ProteinChip data manager software 3.0.7 (Bio-Rad). The analyses were performed with duplicate samples each from three individual cultures of promastigotes of L. major LP and HP strains. The The spectra obtained from the promastigotes of two virulent and peaks with p value , 0.01 were considered as significant. SELDI was two avirulent strains (Fig. 1A, 1B) revealed four important facts: performed using Bio-Rad PCS4000 ProteinChip instrument, and all the 1) differential expression of several peaks in virulent (LP) and 2746 CD40 SIGNALING AND LEISHMANIA VIRULENCE
Table III. Identification of the proteins in the heat map (Fig. 2C)
2Log t Test t Test t Test t Test Abundance Abundance Abundance p Value q-Value Difference Statistic Ratio Ratio Log2 Accession Description LP_HP LP_HP LP_HP LP_HP (HP/LP) HP/LP LP HP R9VVD9 Thiol-specific antioxidant 2.4066 0.04184 1.7252601 15.9237 1.164 0.22 High High A0A3Q8IDG0 cytochrome C oxidase VII 2.67968 0.09066 1.7284294 21.83532 0.58 20.79 High High Q4Q4D3 Putative 60S ribosomal protein L6 2.48877 0.0544 21.7264301 217.5116 2.011 1.01 High High Q4QF84 Putative 60S ribosomal protein L6 2.48877 0.05180 21.7264301 217.5116 2.011 1.01 High High E9BG54 Tryptophanyl-tRNA synthase 2.79044 0.136 1.7292446 24.81397 0.234 22.1 High High A0A3Q8IJ66 Casein kinase 2.65730 0.08369 1.7282379 21.27811 0.509 20.97 High High Q4QE21 Aha1_N domain-containing protein 2.52233 0.068 21.7268482 18.20490 2.379 1.25 High High Q4QI66 Cathepsin L-like protease 2.4991 0.064 1.7265625 17.72254 0.206 22.28 High Low Q4QI61 Cathepsin L-like protease 2.4991 0.06044 1.7265625 17.72254 0.206 22.28 High Low P90628 Cathepsin L-like protease 2.4991 0.05726 1.7265625 17.72254 0.206 22.28 High Low Q9NHE1 Casein kinase 1 isoform 2 2.65730 0.07771 1.7282379 21.27811 0.509 20.97 High High A0A3Q8IDM3 Ankyrin repeats 2.83759 0.2176 1.7295333 26.20126 0.238 22.07 High High E9BKR5 Uncharacterized protein 2.8375 0.18133 1.7295333 26.20126 0.238 22.07 High High E9ADW8 ANK_REP_Region domain-containing 2.8375 0.15542 1.7295333 26.20126 0.238 22.07 High High protein K7P582 Cathepsin L-like protease 2.41931 0.04352 1.7254554 16.15902 0.059 24.09 High Low
A0A2I6J0X1 Cysteine protease b 2.46778 0.04730 1.7261519 17.09161 0.059 24.09 High Low Downloaded from Q4QI67 Cathepsin L-like protease 2.47012 0.04945 1.7261836 17.13791 0.059 24.09 High Low E9BAY0 Uncharacterized 3.1715 0.36266 1.7308841 38.51017 0.211 22.25 High High Q4QFW1 HIT domain-containing protein 2.56102 0.07253 21.7272916 219.0378 3.721 1.9 Low High A0A3Q8IHN0 Uncharacterized 3.1845 1 21.7309183 239.0883 5.009 2.32 High High E9BBK7 Uncharacterized 2.46390 0.04533 21.7260989 217.0149 1.881 0.91 High High E9ADD2 Uncharacterized 3.1845 0.544 21.7309183 239.0883 5.009 2.32 High High Q4Q3S3 Putative dynein intermediate chain 3.0027 0.272 1.7303296 31.69846 0.097 23.36 High Low Q4QIG4 Uncharacterized 2.68821 0.1088 1.7284999 22.05154 0.133 22.92 Low High http://www.jimmunol.org/ E9B9M5 Uncharacterized 2.68821 0.09890 1.7284999 22.05154 0.133 22.92 Low High Q4Q485 Uncharacterized 2.69023 0.12088 1.7285163 22.10309 0.277 21.85 High Low avirulent (HP) strains; 2) both avirulent strains showed comparable counterpart, implying that reversal of virulence in the attenuated peak profiles; 3) major upregulated proteins in HP were in the range L. major strain is improbable. of 8,000–15,000 Da (Fig. 1A, 1B); and 4) major downregulated Avirulent L. major strain shows compromised survival in proteins in HP were also in the range of 8,000–20,000 Da. These macrophages and elicits host-protective response even in a by guest on September 29, 2021 results indicated that both avirulent strains had comparable changes susceptible host in their proteome profiles but the proteins expression profiles in both avirulent strains were different from the profiles observed in virulent The promastigotes of the avirulent and the virulent L. major strains L. major strains. did not differ in their growth kinetics in culture (Fig. 3A). So, we tested whether the virulent and the avirulent strains of L. major Liquid chromatography–mass spectrometry analyses of the would differ in their ability to survive in BALB/c-derived peri- proteome profiles of the avirulent and virulent L. major strain toneal macrophages. We observed that amastigote number was As the proteome analyses by SELDI were severely limited, we significantly higher in the virulent, but not avirulent, L. major– further characterized the proteome profiles of both virulent infected macrophages (Fig. 3B, 3C), suggesting attenuation of the (5ASKH-LP) and avirulent (5ASKH-HP) strains. The total num- avirulent L. major strains. A hallmark of the disease is significant ber of proteins identified in the L. major database, considering all immune deviation whereby TH cells are polarized toward IL-4 experiments and replicates, was 3049. A total of 2263 (74.2%) of secreting TH2 cells. By contrast, TH cell polarization toward the 3049 proteins were detected in the proteomes of both virulent IFN-g secreting TH1 cells offer host protection. To assess the and avirulent strains of L. major (5ASKH), whereas 283 (9.3%) T cell response elicited by the avirulent parasites, we infected and 503 (16.5%) proteins were detected only in 5ASKH-LP and BALB/c mice with the stationary phase promastigotes of the 5ASKH-HP promastigotes, respectively (Fig. 2A). Samples were avirulent or the virulent L. major strains (2 3 106 parasites, s.c). also grouped based on principal component analysis (Fig. 2B), We observed that the BALB/c mice infected with the avirulent indicating that the two strains have remarkable differences in parasite had significantly less footpad thickness and parasite load terms of protein abundance. Some of these proteins were classified than that observed in the mice infected with virulent L. major according to their functions (Tables I–III). Another 26 proteins of parasites (Fig. 3D). The reduced infection was accompanied by difference, as represented in the heat map (Fig. 2C), between the lowered IL-10 or IL-4 and heightened IFN-g responses (Fig. 3E). virulent and avirulent strains of L. major, were also involved in These observations suggested attenuation of the L. major strains different pathways. Out of those 26 significant proteins, eight (Q4QI66, and elicitation of host-protective response even in a susceptible Q4QI61, P90628, K7P582, A0A2I6J0X1, Q4QI67, Q4Q3S3, and host. Q4Q485) were significantly highly expressed in LP, whereas three (Q4QFW1, Q4QIG4, and E9B9M5) were significantly less expressed Avirulent parasites cannot establish infection even in severely in LP (Fig. 2C, Table III). The pattern of expression of the dif- immune-compromised mice ferentially expressed proteins precisely clustered 5ASKH LP and Because the avirulent L. major strains failed to inhibit host-protective 5ASKH HP samples in two separate branches, as shown in the immune response in susceptible BALB/c mice, we investigated volcano plot (Fig. 2D). These results indicate that the attenu- if those parasites would generate immune response or establish ated L. major strain was significantly different from its virulent an infection in an immune-compromised mouse strain such as The Journal of Immunology 2747 Downloaded from http://www.jimmunol.org/
FIGURE 3. Avirulent and virulent parasites differ in terms of survival in macrophages, and avirulent L. major strains fail to establish infection in a susceptible host. (A) L. major virulent and avirulent stationary promastigotes (5 3 106) were cultured in a T25 culture flask for 11 d. The parasites were enumerated each day under the light microscope for both L. major strains. (B and C) BALB/c-derived thioglycolate-elicited macrophages were plated in eight-well chamber slides for 6 h. The macrophages were infected with 1:10 avirulent and virulent L. major promastigotes for 6 h, and
extracellular parasites were removed and cultured up to 72 h. In some cases, the kinetics of infection was performed with metacyclic promastigotes by guest on September 29, 2021 of both strains (C). The macrophages were washed, fixed, Giemsa stained, and enumerated as described in Materials and Methods. The experiments were performed thrice and the representative data from one experiment are shown. Data represented as mean 6 SEM. (D)FemaleBALB/c(n =8) mice were infected s.c. in hind footpad with 2 3 106 avirulent or virulent L. major stationary phase promastigotes of 5ASKH and LV39. Disease progression was scored weekly by evaluating the net footpad swelling (the thickness of the uninfected left footpad subtracted from the infected right footpad) by digital micrometer for 5 wk. Mice were sacrificed 5 wk after the infection, and parasite burden (insert) in lymph node cells was assessed. (E) Lymph node cells from mice, as indicated, in each group (n = 8) were pooled and stimulated with CSA (25 mg/ml) and cultured for 48 h. Culture supernatants were assessed for the production of the cytokines IL-10, IL-4, and IFN-g, as described in Materials and Methods. The experiments were performed thrice, and representative data from one experiment are shown. Error bars represent mean 6 SEM. **p # 0.01, ***p # 0.001.
NOD-SCID. We infected the immune-compromised SCID mice proinflammatory and anti-inflammatory cytokine production on a Swiss background and control Swiss mice with the stationary- in macrophages infected with virulent (LP) or avirulent (HP) phase avirulent or virulent L. major (5ASKH; 2 3 106 parasites, s.c) L. major parasites. IL-10, an anti-inflammatory cytokine, was strains. We observed that only the virulent strain, but not the significantly increased in LP-infected macrophages, whereas avirulent strain, established infection in SCID mice, as evident IL-12, a proinflammatory cytokine, was higher in HP-infected from footpad thickness (Fig. 4A), parasite load (Fig. 4B), and macrophages (Fig. 5A). As L. major infection is character- lymph node weight (Fig. 4C). Because avirulent parasites did not ized by suppressed host-protective T cell response (14), we establish the infection in SCID mice, we tested whether these first assessed the role of avirulent parasites in T cell re- parasites would establish infection in single gene-deficient mice sponse. We observed that the avirulent L. major parasite-infected IL-122/2,iNOS2/2 and CD402/2, which are known to be sus- BALB/c-derived macrophages elicited significantly increased ceptible to L. major infection. It was observed that the avirulent IFN-g,aTH1 type cytokine (Fig. 5B, right panel), but reduced L. major parasites did not induce footpad swelling (Fig. 4D), IL-4, a TH2 type cytokine (Fig. 5B, left panel), production and parasite load (Fig. 4E), and lymph node hypertrophy (Fig. 4F) in reduced the number of amastigotes (Fig. 5C) in the cocultured any of these mouse strains, whereas the virulent parasites did L. major–infected macrophages. These observations indicate establish the infection. These data provided further evidence for that the avirulent 5ASKH strain elicits host-protective immune attenuation of the L. major parasites. response. Avirulent parasites elicit host-protective cytokine response Avirulent parasite fails to suppress the host-protective Because host protection is associated with reduced amastigote CD40 signaling number in infected macrophages and reciprocal regulation of Because cell signaling in Leishmania-infected macrophages is a IL-10 and IL-12 expression (12), we assessed representative functional correlate of the eventual outcome of infection (12, 13, 15), 2748 CD40 SIGNALING AND LEISHMANIA VIRULENCE Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021
FIGURE 4. Avirulent parasites cannot establish infection even in severely immune-compromised mice. (A) Female NOD SCID and SWISS mice were infected s.c. in hind footpad with 2 3 106 avirulent or virulent L. major stationary promastigotes of 5ASKH. In each group (n =5) mice were taken. Disease progression was scored weekly by evaluating the net footpad swelling (the thickness of the uninfected left footpad subtracted from the infected right footpad) by digital micrometer for 5 wk. (B) Mice were sacrificed 5 wk after the infection, and parasite burden in lymph node cells was assessed. (C) Significantly reduced lymph node weight in avirulent infected parasite. The experiments were performed twice, and representative data from one experiment are shown. Error bars represent mean 6 SEM. ***p # 0.001. (D) Female BALB/c, C57BL/6, CD402/2,IL-122/2,IL-102/2,andiNOS2/2 mice were infected s.c. in hind footpad with 2 3 106 avirulent or virulent L. major stationary promastigotes of 5ASKH. In each group (n = 5) mice were taken. Disease progression was scored weekly by evaluating the net footpad swelling (the thickness of the uninfected left footpad subtracted from the infected right footpad) by digital micrometer for 5 wk. (E) Mice were sacrificed 5 wk after the infection, and parasite burden in lymph node cells was assessed. (F) Significantly reduced lymph node weight in avirulent infected parasite. The experiments were performed twice, and representative data from one experiment are shown. Error bars represent mean 6 SEM. ***p # 0.001. we examined the CD40-induced differential phosphorylation Fig. 1B). These data indicate that involvement of PKC isoforms of membrane-proximal kinases such as Lyn and Syk, PKC in CD40 signaling in BALB/c-derived peritoneal macrophages isoforms, Ras and Ras effectors, MAPK module kinases, and are differentially modulated by virulent and avirulent L. major the phosphatases in BALB/c-derived elicited macrophages strains. Because Ras GTPases were implied in CD40 signaling 72 h after the in vitro infection with virulent or avirulent in B cells, endothelial cells, and T cells (18–20), we examined L. major strains. As we had previously performed detailed differential modulation of Ras GTPases in CD40 signaling in kinetics of these signaling intermediates and established their 5ASKH-LP or 5ASKH-HP–infected elicited peritoneal macro- peak phosphorylation or activation (e.g., Ras GTPase) time phages from BALB/c mice (14). We observed that Ras GTPases (9–17), we assessed the phosphorylation of these intermedi- were activated in virulent parasite infection with anti-CD40 Ab ates in macrophages infected with these parasites at the pre- stimulation but not by the avirulent L. major strain (Fig. 6C, viously reported peak activation time point. We observed that Supplemental Fig. 1C). Because only the active Ras GTPases the virulent L. major strain (5ASKH-LP) promoted CD40-induced show high affinity toward Ras Binding Domain (RBD) of the phosphorylation of Syk but not of Lyn (Fig. 6A, Supplemental downstream Ras effectors (11, 19) PI-3K and Raf-1 kinase Fig. 1A). Contrary to that observed with the virulent strain, (21), we examined the CD40-induced PI-3K and Raf-1 phos- avirulent L. major strain (5ASKH-HP) promoted CD40-induced phorylation in avirulent and virulent L. major infection in PKCb1andPKCd isoform phosphorylation but reduced PKCz macrophages. We observed that the avirulent L. major strain and PKCz/l isoform phosphorylation (Fig. 6B, Supplemental increased CD40-induced PI3K activation; by contrast, virulent The Journal of Immunology 2749 Downloaded from
FIGURE 5. Avirulent parasites elicit host-protective cytokine response. (A) BALB/c-derived peritoneal macrophages were infected with avirulent and virulent L. major stationary promastigotes at a ratio of one macrophage/10 parasites for 6 h, extracellular parasites were washed out, and macrophages were incubated for 48 h. Culture supernatants were collected and assessed for IL-10 and IL-12 production by ELISA. The error bars represent mean 6 SEM. Data are from one experiment representative of experiments done at least three times. ***p # 0.001. IM, infected macrophages; UIM, uninfected macrophages. (B) BALB/c-derived peritoneal macrophages were infected with avirulent and virulent L. major stationary promastigotes at a ratio of one macrophage/10 parasites for 6 h, extracellular parasites were washed out, and macrophages were in- http://www.jimmunol.org/ cubated for 36 h. After 36 h infection, macrophages were cocultured with the popliteal lymph node cells isolated from the virulent L. major– infected (fifth week–infected) BALB/c mice at 1:3 ratio (macrophage/LN cells); unstimulated (UNS) and stimulated (CSA-25 mg/ml) and cocultured cells were incubated for 48 h. Culture supernatants were collected and assessed for IL-4 and IFN-g production by ELISA. The ex- periments were performed thrice, and representative data from one experiment are shown. Error bars represent mean 6 SEM. (C)BALB/c-derived peritoneal macrophages were infected with avirulent and virulent L. major stationary promastigotes at a ratio of one macrophage/10 parasites for 6 h, extracellular parasites were washed out, and macrophages were incubatedfor36h.After36hinfectionmacrophages were cocultured with the popliteal lymph node cells isolated from the virulent L. major–infected (fifth week–infected) or naive female BALB/c mice at 1:3 ratio (macrophage/T cells); cells were incubated for 72 h. After 72 h infection, macrophages were washed, fixed, stained with Giemsa stain, and evaluated for the amastigotes per 100 macrophages. The experiments were performed thrice, and representative data from one experiment are shown. Error bars represent mean 6 SEM. *p # 0.05, ***p # 0.001. mF, macrophage. by guest on September 29, 2021
L. major strain increased CD40-induced Raf-1 activation (Fig. 6D, phosphatase activation. Because SHP-1 plays important roles Supplemental Fig. 2A). These data suggest that the virulence of in the Leishmania-induced pathogenesis (23–26), in particular, the L. major parasites has association with differential modula- by inhibiting innate inflammatory responses through termina- tion of the Ras GTPase activation and Ras effector specificity. tion of some signaling pathways (27, 28), we examined SHP-1 Next, we studied whether the virulent and avirulent strains activation in virulent or avirulent L. major–infected anti-CD40 of L. major would differentially modulate the kinases in the Ab-treated macrophages. We observed CD40-induced SHP-1 MAPK module. We observed that the avirulent parasites enhanced phosphorylation in virulent, but not in avirulent, L. major– CD40-induced p38MAPK, but reduced ERK1/2, phosphory- infected macrophages (Fig. 6F, Supplemental Fig. 2C). These data lation, whereas the virulent parasites reversed this MAPK suggest that L. major enhances CD40-induced Raf–MEK–ERK phosphorylation profile (Fig. 6E, Supplemental Fig. 2B). As signaling but inhibits MKK-p38MAPK signaling as a mean to CD40 signaling through the Ras–Raf–MEK–ERK axis was express virulence. modulated in L. major–infected macrophages (11), we ex- amined the MEK1/2 and MKK3/6 activation in CD40-induced Virulent and avirulent L. major differentially affect CD40 BALB/c peritoneal macrophages infected in vitro with viru- relocation between detergent-resistant and detergent-soluble lent or avirulent L. major parasites. We observed that aviru- membrane domains lent parasite-infected macrophages had higher CD40-induced Because ligand-bound receptors laterally diffuse to generate MKK-3/6, but less MEK-1/2, phosphorylation but virulent an oligomeric complex in the detergent-resistant membrane parasite activated a reverse profile (Fig. 6E, Supplemental domain (13), we examined whether the virulent and the avir- Fig. 2B). ulent parasites differ in terms of CD40 relocation in response As MAPKs activation is regulated by phosphatases such as to an agonistic anti-CD40 Ab that acts as a ligand for CD40 SHP-1 (16) and dual-specific phosphatases MKP-1 and MKP-3 (12). We observed that the infection of macrophages with the (3, 22), we next checked MKP-1 and MKP-3 phosphorylation virulent L. major parasitepreventedCD40migrationtothe in avirulent or virulent L. major–infected BALB/c-derived detergent-resistant membrane domain, whereas the avirulent elicited macrophages. We observed that MKP-3 was more phos- parasite failed to block the migration of CD40 (Fig. 7). The phorylated in avirulent parasite-infected macrophages, whereas observation suggests that the avirulent parasites’ inability to MKP-1 was phosphorylated more in virulent parasite-infected selectively modulate CD40 signaling is perhaps due to their macrophages (Fig. 6F, Supplemental Fig. 2C), suggesting inability to prevent CD40 relocation to the detergent-membrane expression of virulence through modulation of dual-specific domain. 2750 CD40 SIGNALING AND LEISHMANIA VIRULENCE Downloaded from http://www.jimmunol.org/ FIGURE 6. Avirulent parasites fail to promote the disease-exacerbative, or suppress the host-protective, CD40 signaling. BALB/c-derived peritoneal macrophages were either left uninfected (UIM) or infected with avirulent or virulent L. major 5 ASKH (one macrophage/10 parasites) for 72 h, followed by anti-CD40 Ab (3 mg/ml, clone 3/23) stimulation for 15 min. (A) Macrophages were lysed and assessed for phosphorylation of p-Lyn and p-Syk or total Lyn and Syk. (B) Macrophages were lysed and assessed for phosphorylation of p-PKCb1, p-PKCd,p-PKCz,andp-PKCz/l isoforms; total PKCb1, PKCd,PKCz,andPKCz/l isoforms; and b-actin by immunoblotting. (C) BALB/c-derived peritoneal macrophages were either left uninfected (UIM) or infected (IM) with avirulent or virulent L. major 5 ASKH (one macrophage per 10 parasites) for 72 h, followed by isotype control (rat IgG2ak,3mg/ml) or anti-CD40 Ab (3 mg/ml, clone 3/23) stimulation for 7 min, as previously reported standardized kinetic experiments, and protein was isolated by using Active Ras pull-down assay, as described in Materials and Methods. Pan-Ras was used as loading control. The experiments were performed thrice, and the representative data from one experiment are shown. (D) BALB/c-derived peritoneal by guest on September 29, 2021 macrophages were either left uninfected (UIM) or infected with avirulent or virulent L. major 5 ASKH (one macrophage per 10 parasites) for 72 h, followedbyanti-CD40Ab(3mg/ml, clone 3/23) stimulation for 15 min, as previously reported standardized kinetic experiments. Macrophages were lysed and assessed for phosphorylation of p-Raf and p-PI3K or total PI3K and Raf. (E) Macrophages were lysed and assessed for phos- phorylation of p-p38MAPK, p-ERK1/2, p-MEK1/2, and p-MKK3/6 or total p38MAPK, ERK1/2, MEK1/2, and MKK3/6. (F) Macrophages were lysed and assessed for phosphorylation of p–MKP-1, p–MKP-3, p–SHP-1, or total MKP-1, MKP-3, SHP-1, and b-actin by immunoblotting. The experiments were performed thrice and the representative data from one experiment are shown.
Avirulent L. major priming elicits host-protection against (data not shown). Given that the virulent and the avirulent challenge with virulent parasites strains differ in many proteins, it is expected not to revert to a virulent parasite. As avirulent parasites were observed to induce TH1typeof response in vitro in macrophage–T cell cocultures and could not survive in susceptible BALB/c mice, we primed these mice Discussion with low-dose (5 3 103) avirulent L. major stationary phase In this article, we describe that the naturally grown avirulent promastigotes, followed by challenge infection with virulent L. major parasites from their respective virulent strains are (2 3 106) L. major promastigotes. It was observed that the unable to selectively impair CD40 signaling and thereby elicit a priming elicited host-protective immune response resulting in host-protective response. The observations not only establish less foot pad swelling (Fig. 8A), lower parasite burden (Fig. 8B), two naturally grown avirulent L. major strains with prophy- and reduced lymph node weight (Fig. 8C) in the primed mice. lactic host-protective functions but also show that the parasite The T cells response resulted in higher IFN-g and lower IL-4 virulence is linked to the parasite’s ability to selectively impair production in the primed mice, as compared with the control CD40 signaling functions. As signaling modulated the relative mice (Fig. 8D). The leishmanial Ag-specific Ab response was resistance or susceptibility to infection or responsiveness to dominated by IgG2a isotype (Fig. 8E). Amastigote numbers Ags, the role of parasite virulence in the modulation of host were reduced (Fig. 8F) in the L. major–infected macrophages, cell signaling was examined. It is possible that the virulent and which were cocultured with T cells from the primed mice. The the avirulent parasites differ in terms of glycosylation of the primed mice had significantly fewer Treg cells than that ob- cell membrane proteins. As a result, the lectin-type receptors served in the control mice (Fig. 8G). These results suggested like CLRs may recognize the ligands differently causing a that the avirulent L. major strains generated by long continuous differential activation of the host cells. As a consequence of a cultures of the respective virulent L. major strains did elicit little bias in the signaling pathway, the subsequent activation host-protective T cell response. The mice infected with low through CD40 may tilt the signaling further toward the pre- number of avirulent parasites had never showed any infections activated pathway. The virulent and the avirulent parasites The Journal of Immunology 2751
receptor for which the reciprocity, a unique property regulat- ing cellular responsiveness, has been worked out (3, 11–13, 30). CD40–CD40L interactions have been show to crucial roles in eliciting host-protective immune response (8–17). Although the CD40 signaling cascade is being revealed, the roles of PKC isoforms have remained undefined in avirulent or virulent infection and CD40 signaling. Albeit controversial, PKC is implicated in CD40 signaling in B cells (31, 32). In human monocytes, CD40 stimulation results in increased total PKC activity and translocation from cytosol to membrane (33). Members of the PKC family are involved actively in regulation of macrophage functions necessary for host defense or suscepti- bility to bacterial infections (34, 35). In Leishmania infection, an impairment of PKC-dependent protein phosphorylation of PKC substrate proteins (36) reduced expressions of MARCKS-related protein (37), c-fos, and TNF-a (38) and impaired oxidative FIGURE 7. Avirulent parasites fail to block CD40 relocation to de- burst (39). Macrophages expressed eight PKC isoforms (17), tergent-resistant membrane (DRMs) domain. BALB/c-derived peritoneal and L. major infection differentially regulated CD40 dose- macrophages were either left uninfected (UIM) or infected with avir- dependent phosphorylation and membrane translocation of PKC Downloaded from ulent (HP-Lm) or virulent (LP-Lm) L. major 5 ASKH (one macrophage isoforms (17). We showed that avirulent L. major infection of per 10 parasites) for 72 h followed by indicated doses of anti-CD40 macrophages for 72 h resulted in increased phosphorylation A Ab for 7 min at 37˚C. ( ) Cells were lysed and detergent-resistant of PKCbIandPKCd isoforms, whereas phosphorylation of (DRM) and detergent-soluble (Soluble) membrane fractions were PKCz and PKCz/l isoforms was decreased. It is possible that isolated. Fractions were subjected to immunoblotting with CD40- specific Ab. (B) The relative amount of CD40 relocated (recovered) in the avirulent L. major parasites are unable to intercept CD40 arbitrary units from indicated membrane fractions is plotted as a function partitioning to detergent-resistant membrane microdomain. http://www.jimmunol.org/ of the concentration of anti-CD40 used for stimulation. The experi- As a result, the CD40-induced IL-12 production or signaling ments were performed thrice, and representative data from one experiment through PKCb1 or p38MAPK remains intact. In fact, CD40- are shown. induced Ras activation or Ras expression was also altered (14). Therefore, the virulence of the parasite can alter the host cell signaling in a way that promotes the parasite survival. How- differ in terms of lipophosphoglycan (LPG) content. High ever, how exactly the signaling pathways are differentially LPG may bind to TLR2 causing lowered cholesterol and less modulated by the virulent or the avirulent parasites remain TRAF 3 availability (29). As a result, the subsequent CD40 to be investigated. The initial recognition may alter the host stimulation leads to ERK-1/2 pathway. This CD40 signal- cell metabolism affecting membrane cholesterol content. As by guest on September 29, 2021 ing bias significantly influences the outcome of L. major the membrane cholesterol content goes down in virulent parasite infection. infection, the CD40 signaling is switched toward ERK-1/2 pathway In the current study, we reveal several novel facts about (13). These observations support the contribution of phosphatase avirulent and virulent Leishmania major. First, the growth kinetics and kinases in avirulent or virulent L. major–infected macrophages of avirulent promastigotes did not differ from the virulent strains, and CD40 signaling. Finally, although transcriptomic analyses whereas amastigote number was significantly reduced in the of Leishmania in different stages of development have been avirulent L. major–infected macrophages indicating attenuation previously reported (40–42), our work is not in conflict with of the virulent strains. Thus, avirulent parasites are differentially those reports. It is very clear that the strains reported were targeted by the macrophages. Second, the anti-inflammatory cy- indeed attenuated and not a developmental signature, as pre- tokine (IL-10) was significantly increased in virulent parasite- viously reported. In addition, Leishmania strain attenuation by infected macrophages, whereas the proinflammatory cytokine single gene deletion was also reported (43, 44). Given that we (IL-12) was higher in avirulent parasite-infected macrophages. have characterized the avirulent strains proteomically, which, The avirulent parasite fails to established infection in susceptible to our knowledge, were not previously reported, it is unlikely host. Macrophage–T cell coculture data suggested TH2type that the strains will show reversal to virulence. The improb- of response by virulent parasite-infected macrophages whereas ability of the reversal of attenuation is due to large change in avirulent parasite-infected macrophages resulted in IFN-g-secreting proteome profiles, and the described attenuated strain may TH1 response and reduced amastigotes number but reduced serve as a potential vaccine strain. Although leishmanization IL-4-secreting TH2 response, indicating an association between used to be practiced in many countries to vaccinate the people virulence and ability to induce IL-4 and avirulent-parasite-induced with killed parasites (45), the major problem is insufficient host-protective T cell responses. Third, avirulent parasite does protection offered by the killed parasites. In contrast, devel- not show any infectivity even in immunocompromised and opment of attenuated parasites by single gene deletion (46) or different gene-deficient mice. Fourth, considering these re- immunization with chimeric peptides (47, 48) brings limited strictive issues, an attenuated L. major strain is argued to work success. The failure could stem from the presence of multiple better in a vaccine protocol. The two avirulent L. major strains copies, even truncated but functional, of the targeted gene(s) show comparable changes in proteome profiles, as compared that could possibly compensate for the eliminated copy of the with their corresponding virulent strains. Fifth, responsiveness gene. In contrast, choice of limited numbers of immunodo- of a receptor to stimuli depends on various signaling inter- minant peptides may not necessarily cover the required T cell mediates that act together in an integrated manner to bring a repertoire and may not cover the crypticity of the immunodo- cellular response. CD40, a transmembrane receptor, regulates minant epitopes in a chronic infection. The attenuated strains a wide range of immune responses and is the only known also satisfy the criteria of substantially reduced survival in host, 2752 CD40 SIGNALING AND LEISHMANIA VIRULENCE Downloaded from http://www.jimmunol.org/
FIGURE 8. Avirulent L. major priming elicits host protection against challenge with virulent parasites. (A) L. major (avirulent strain 5ASKH) stationary- 3 3 phase promastigotes (5 10 per mouse in saline 50 ml) were infected s.c. in hind footpad of female BALB/c mice for priming, After 60 d, primed mice by guest on September 29, 2021 were challenged with stationary-phase promastigotes L. major (virulent strain 5ASKH, 2 3 106 per mouse), and in each group (n = 8) mice were taken. Disease progression was scored weekly by evaluating the net footpad swelling (the thickness of the uninfected left footpad subtracted from the infected right footpad) by digital micrometer for 5 wk. (B) Mice were sacrificed 5 wk after the infection, and parasite burden in lymph node cells was assessed. (C) Significantly reduced lymph node weight in primed mice. (D) IL-4 and IFN-g productions by pooled popliteal lymph node cells (in 48-h culture supernatants) in response to CSA (25 mg/ml) and (E) serum IgM, IgG1, and IgG2a assessed by ELISA. (F) BALB/c-derived peritoneal macrophages were infected with virulent L. major stationary promastigotes at a ratio of one macrophage/10 parasites for 6 h, extracellular parasites were washed out, and macrophages were incubated for 36 h. After 36 h, infection macrophages were cocultured with the popliteal lymph node T cells isolated from the virulent L. major–infected or primed + infected (fifth week–infected) or naive BALB/c mice at 1:3 ratio (macrophages/T cells); cells were incubated for 72 h. After 72 h infection, macrophages were washed, fixed, stained with Giemsa stain, and evaluated for the amastigotes per 100 macrophages. The experiments were performed twice, and representative data from one experiment are shown. Error bars represent mean 6 SEM. (G) FACS analyses of Treg cells from primed and unprimed pooled popliteal lymph node T cells. T cells stained with Treg cell–specific Abs were acquired on CD3+CD4+CD25+CD127dim gating and analyzed for GITR versus Foxp3. Values inside graphs represent percentage of cells. The experiments were performed twice, and representative data from one experiment are shown. Error bars represent mean 6 SEM. **p # 0.01, ***p # 0.001. MF, macrophage. inability to impair the cell signaling that correlates with pro- 5. Maue¨l, J. 2002. Vaccination against Leishmania infections. Curr. Drug Targets Immune Endocr. Metabol. Disord. 2: 201–226. tection and elicitation of host-protective T cell response. 6. Zutshi, S., S. Kumar, P. Chauhan, Y. Bansode, A. Nair, S. Roy, A. Sarkar, and B. Saha. 2019. Anti-Leishmanial vaccines: assumptions, approaches, and an- nulments. Vaccines (Basel) 7: E156. Disclosures 7. Kamanaka, M., P. Yu, T. Yasui, K. Yoshida, T. Kawabe, T. Horii, T. Kishimoto, The authors have no financial conflicts of interest. and H. Kikutani. 1996. Protective role of CD40 in Leishmania major infection at two distinct phases of cell-mediated immunity. Immunity 4: 275–281. 8. Campbell, K. A., P. J. Ovendale, M. K. Kennedy, W. C. Fanslow, S. G. Reed, and C. R. Maliszewski. 1996. CD40 ligand is required for protective cell-mediated References immunity to Leishmania major. Immunity 4: 283–289. 1. Murray, H. W., J. D. Berman, C. R. Davies, and N. G. Saravia. 2005. Advances 9. Soong, L., J. C. Xu, I. S. Grewal, P. Kima, J. Sun, B. J. Longley, Jr., in leishmaniasis. Lancet 366: 1561–1577. N. H. Ruddle, D. McMahon-Pratt, and R. A. Flavell. 1996. Disruption of 2. Olivier, M., D. J. Gregory, and G. Forget. 2005. Subversion mechanisms by CD40-CD40 ligand interactions results in an enhanced susceptibility to which Leishmania parasites can escape the host immune response: a signaling Leishmania amazonensis infection. Immunity 4: 263–273. point of view. Clin. Microbiol. Rev. 18: 293–305. 10. Srivastava, S., S. P. Pandey, M. K. Jha, H. S. Chandel, and B. Saha. 2013. 3. Srivastava, N., R. Sudan, and B. Saha. 2011. CD40-modulated dual- Leishmania expressed lipophosphoglycan interacts with toll-like receptor specificity phosphatases MAPK phosphatase (MKP)-1 and MKP-3 (TLR)-2 to decrease TLR-9 expression and reduce anti-leishmanial responses. reciprocally regulate Leishmania major infection. J. Immunol. 186: Clin. Exp. Immunol. 172: 403–409. 5863–5872. 11. Sarma, U., A. Sareen, M. Maiti, V. Kamat, R. Sudan, S. Pahari, N. Srivastava, 4. Forget, G., D. J. Gregory, L. A. Whitcombe, and M. Olivier. 2006. Role of host S. Roy, S. Sinha, I. Ghosh, et al. 2012. Modeling and experimental analyses protein tyrosine phosphatase SHP-1 in Leishmania donovani-induced inhibition reveals signaling plasticity in a bi-modular assembly of CD40 receptor activated of nitric oxide production. Infect. Immun. 74: 6272–6279. kinases. PLoS One 7: e39898. The Journal of Immunology 2753
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