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Licensed Unlicensed Requires Authentication Published by De Gruyter October 15, 2017

Apparent isocitrate lyase activity in Leishmania amazonensis

Concepción Hernández-Chinea, Laura Maimone, Yelitza Campos, Walter Mosca and Pedro J. Romero
From the journal Acta Parasitologica

Abstract

Early reports have demonstrated the occurrence of glyoxylate cycle enzymes in several Leishmania species. However, these results have been underestimated because genes for the two key enzymes of the cycle, isocitrate lyase (ICL) and malate synthase (MS), are not annotated in Leishmania genomes. We have re-examined this issue in promastigotes of Leishmania amazonensis. Enzyme activities were assayed spectrophotometrically in cellular extracts and characterized partially. A 40 kDa band displaying ICL activity was visualized on zymograms of the extracts. By immunoblotting with mouse antibodies against ICL from Bacillus stearothermophilus, a band of approximately 40 kDa was identified, coincident with the relative molecular mass of the activity band revealed on zymograms. Indirect immunofluorescence of intact promastigotes showed that the recognized antigen is distributed as a punctuated pattern, mainly distributed beneath the subpellicular microtubules, over a diffused cytoplasmic stain. These results clearly demonstrate the existence of an apparent ICL activity in L. amazonensis promastigotes, which is associated to a 40 kDa polypeptide and distributed both diffused and as punctuate aggregates in the cytoplasm. The relevance of this activity is discussed.

Acknowledgements

This work was financially supported by Fondo Nacional de Ciencia y Tecnología (FONACIT), [grant number S1-2001000668] and Consejo de Desarrollo Científico y Humanístico de la Universidad Central de Venezuela (CDCH-UCV), [grant number PI 03-33-5462-2004] to C.H-Ch.

References

Britton K.L., Langridge S.J., Baker P.J., Weeradechapon K., Sedelnikova S.E., De Lucas J.R., et al. 2000. The crystal structure and active site location of isocitrate lyase from the fungus Aspergillus nidulans. Structure, 8, 349–362. 10.1016/S0969-2126(00)00117-9Search in Google Scholar PubMed

Chell R.M., Sundaram T.K., Wilkinson A.E.1978. Isolation and characterization of isocitrate lyase from a thermophilic Bacillus sp. Biochemical Journal, 173, 165–177. 10.1042/bj1730165Search in Google Scholar

Cioni M., Pinzauti G., Vanni P. 1981. Comparative biochemistry of the glyoxylate cycle. Comparative Biochemistry and Physiology, 70B, 1–26. 10.1016/0305-0491(81)90118-8Search in Google Scholar

Cohen-Freue G., Holzer T.R., Forney J.D., McMaster W.R. 2007. Global gene expression in Leishmania. International Journal for Parasitology, 37, 1077–1086. 10.1016/j.ijpara.2007.04.011Search in Google Scholar PubMed

Courret N., Fréhel C., Gouhier N., Pouchelet M., Prina E., Roux P., Antoine J-C. 2002. Biogenesis of Leishmania-harbouring parasitophorous vacuoles following phagocytosis of the metacyclic promastigote or amastigote stages of the parasites. Journal of Cell Science, 115, 2303–231610.1242/jcs.115.11.2303Search in Google Scholar PubMed

Dixon G.H., Kornberg H.L. 1959. Assay methods for key enzymes of the glyoxylate cycle. Biochemical Journal, 72, 3P. 10.1042/bj0720001PSearch in Google Scholar

Dunn M.F., Ramírez-Trujillo J.A., Hernández-Lucas I. 2009. Major roles of isocitrate lyase and malate synthase in bacterial and fungal pathogenesis. Microbiology, 155, 3166–3175. 10.1099/mic.0.030858-0Search in Google Scholar PubMed

Ginger M.L. 2006. Niche metabolism in parasitic protozoa. Philosophycal Transactions of the Royal Society B: Biological Sciences, 361, 101–118. 10.1098/rstb.2005.1756Search in Google Scholar PubMed PubMed Central

Henderson B., Martin A. 2011. Bacterial Virulence in the Moonlight: Multitasking Bacterial Moonlighting Proteins Are Virulence Determinants in Infectious Disease. Infection and Immunity, 79, 3476–3491. 10.1128/IAI.00179-11Search in Google Scholar PubMed PubMed Central

Höner Zu Bentrup K., Miczak A., Swenson D.L., Russell D.G. 1999. Characterization of Activity and Expression of Isocitrate Lyase in Mycobacterium avium and Mycobacterium tuberculosis. Journal of Bacteriology, 181, 7161–716710.1128/JB.181.23.7161-7167.1999Search in Google Scholar PubMed PubMed Central

Hou W-C., Chen H-J., Lin Y-W., Chen Y-C., Yang L-L., Lee M-H. 2001. Activity staining of isocitrate lyase after electrophoresis on either native or sodium dodecyl sulfate polyacrylamide gels. Electrophoresi,s 22, 2653–2655. 10.1002/15222683(200108)22:13<2653::AID-ELPS2653>3.0.CO;2-OSearch in Google Scholar

Hoyt J.C., Johnson K.E., Reeves H.C. 1991. Purification and Characterization of Acinetobacter calcoaceticus Isocitrate Lyase. Journal of Bacteriology, 173, 6844–684810.1128/jb.173.21.6844-6848.1991Search in Google Scholar PubMed

Ivens A.C., Peacock C.S., Worthey E.A., Murphy L., Aggarwal G., Berriman M., et al. 2005. The genome of the kinetoplastid parasite, Leishmania major. Science, 309, 436–442. 10.1126/science.1112680Search in Google Scholar

Jeffery C.J. 1999. Moonlighting proteins. Trends in Biochemical Sciences, 24, 8–11. 10.1016/S0968-0004(98)01335-8Search in Google Scholar PubMed

Keegan F.P., Blum J.J. 1993. Incorporation of label from acetate and laurate into the mannan of Leishmania donovani via the glyoxylate cycle. Journal of Eukaryotic Microbiology, 40, 730–732. 10.1111/j.1550-7408.1993.tb04467.xSearch in Google Scholar

Lorenz M.C., Fink G.R. 2001. The glyoxylate cycle is required for fungal virulence. Nature, 412, 83–86. 10.1038/35083594Search in Google Scholar PubMed

Lorenz M.C., Fink G.R. 2002. Life and death in a macrophage: role of the glyoxylate cycle in virulence. Eukaryotic Cell, 1, 657–662. 10.1128/EC.1.5.657-662.2002Search in Google Scholar

Lowry O., Rosebrough N., Farr A., Randall R. 1951. Protein measurement with the Folin phenol reagent. The Journal of Biological Chemistry, 193, 265–27510.1016/S0021-9258(19)52451-6Search in Google Scholar

McFadden B.A., Purohit S. 1977. Itaconate, an isocitrate lyase-directed inhibitor in Pseudomonas indigofera. Journal of Bacteriology, 131, 136–14410.1128/jb.131.1.136-144.1977Search in Google Scholar PubMed

McKinney J.D., Höner zu Bentrup K., Muñoz-Elias E.J., Miczak A., Chen B., Chan W.T., et al. 2000. Persistence of Mycobacterium tuberculosis in macrophages and mice requires the glyoxylate shunt enzyme isocitrate lyase. Nature, 406, 735–738. 10.1038/35021074Search in Google Scholar PubMed

Mosca W., Navarro N., Campos Y., Briceño L. 2006. Proliferation and bystander suppression induced by antigens of Trypanosoma cruzi. Evaluation with a modification of the T cell blot technique. Investigacion Clinica, 47, 265–282Search in Google Scholar

Mottram J.C., Coombs G.H. 1985. Leishmania mexicana: enzyme activities of amastigotes and promastigotes and their inhibition by antimonials and arsenicals. Experimental Parasitology, 59, 151–160. 10.1016/0014-4894(85)90067-0Search in Google Scholar PubMed

Mukada A.J. 1977. Tricarboxylic acid and glyoxylate cycles in the Leishmaniae. Acta Tropica, 34, 167–175Search in Google Scholar PubMed

Munir E., Hattori T., Shimada M. 2002. Purification and characterization of isocitrate lyase from the wood-destroying basidiomycete Fomitopsis palustris grown on glucose. Archives of Biochemistry and Biophysics, 399, 225–231. 10.1006/abbi.2002.2770Search in Google Scholar PubMed

Opperdoes F.R., Coombs G.H. 2007. Metabolism of Leishmania: proven and predicted. Trends in Parasitology, 23, 149–158. 10.1016/j.pt.2007.02.004Search in Google Scholar PubMed

Orengo C.A., Jones D.T., Thornton J.M. 1994. Protein superfamilies and domain superfolds. Nature, 372, 631–634. 10.1038/372631a0Search in Google Scholar PubMed

Reinscheid D.J., Eikmanns B.J., Sahm H. 1994. Characterization of the isocitrate lyase gene from Corynebacterium glutamicum and biochemical analysis of the enzyme. Journal of Bacteriology, 176, 3474–348310.1128/jb.176.12.3474-3483.1994Search in Google Scholar PubMed PubMed Central

Rosenzweig D., Smith D., Opperdoes F., Stern S., Olafson R.W., Zilberstein D. 2008. Retooling Leishmania metabolism: from sand fly gut to human macrophage The FASEB Journal, 22, 590–602. 10.1096/fj.07-9254comSearch in Google Scholar PubMed

Russell D.G., Xu S., Chakraborty P. 1992. Intracellular trafficking and the parasitophorous vacuole of Leishmania mexicana-infected macrophages. Journal of Cell Science, 103, 1193–121010.1242/jcs.103.4.1193Search in Google Scholar PubMed

Schaible U.E., Schlesinger P.H., Steinberg T.H., Mangel W.F., Kobayashi T., Russell D.G. 1999. Parasitophorous vacuoles of Leishmania mexicana acquire macromolecules from the host cell cytosol via two independent routes. Journal of Cell Science, 112, 681–69310.1242/jcs.112.5.681Search in Google Scholar PubMed

Schmidt G., Stahmann K-P., Sahm H. 1996. Inhibition of purified isocitrate lyase identified itaconate and oxalate as potential antimetabolites for the riboflavin overproducer Ashbya gossypii. Microbiology, 142, 411–417. 10.1099/13500872-142-2411Search in Google Scholar

Simon M.W., Martin E., Mukkada A.J. 1978. Evidence for a functional glyoxylate cycle in the Leishmaniae. Journal of Bacteriology, 135, 895–89910.1128/jb.135.3.895-899.1978Search in Google Scholar PubMed PubMed Central

Singh N., Kumar M., Singh R.K. 2012. Leishmaniasis: Current status of available drugs and new potential drug targets. Asian Pacific Journal of Tropical Medicine, 5, 485–497. 10.1016/S1995-7645(12)60084-4Search in Google Scholar PubMed

Towbin H., Staehlin T., Gordon J. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences, 76, 4350–435410.1073/pnas.76.9.4350Search in Google Scholar

Vanni P., Giachetti E., Pinzauti G., McFadden B.A. 1990. Comparative structure, function and regulation of isocitrate lyase, an important assimilatory enzyme. Comparative Biochemistry and Physiology, B95, 431–458. 10.1016/0305-0491(90)90002-BSearch in Google Scholar

Received: 2016-8-15
Revised: 2017-2-22
Accepted: 2017-6-23
Published Online: 2017-10-15
Published in Print: 2017-12-20

© 2017 W. Stefański Institute of Parasitology, PAS

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