The long-term goal of this application is to uncover strategies used by the protozoan parasite Leishmania to survive and replicate within the harsh environment of macrophage phagolysosomes. Parasite access to iron within this compartment is suspected to play an important role, but little is known about the specific molecular mechanisms used by Leishmania to acquire iron intracellularly. Through database searches, we recently identified two identical Leishmania genes in tandem (LIT1) with strong similarity to IRT1, a well- characterized plasma membrane iron transporter from Arabidopsis thaliana. Our initial studies indicate that Leishmania amazonensis LIT1 is expressed predominantly by intracellular amastigotes, and that expression of this transporter on the parasite's surface is regulated by iron availability. LIT1 null mutants do not grow intracellularly in macrophages and are avirulent for mice, strongly suggesting that iron acquisition through this transporter is a critical limiting step for the establishment of intracellular infections by Leishmania. Investigating host cell responses, we also found that infection with L. amazonensis upregulates the expression of LYST, a macrophage cytosolic protein that regulates lysosomal size. Parasitophorous vacuole enlargement favors Leishmania intracellular growth, suggesting that LYST upregulation functions as a host cell innate mechanism to limit infection. We propose to characterize and extend these novel findings, with the following specific aims: 1) Determine if LIT1 functions as a ferrous iron transporter, and define the conditions regulating its surface expression in L. amazonensis; 2) Assess the requirement for LIT1-mediated iron transport in the intracellular survival and growth of Leishmania amastigotes; 3) Elucidate the role of parasitophorous vacuole expansion in the intracellular survival and replication of amastigotes in fibroblasts and macrophages. These studies will provide a detailed understanding of adaptive mechanisms utilized by Leishmania to overcome host cell defenses, including the process by which the parasites acquire iron in the hostile environment of macrophage phagolysosomes. The information derived from this project will fill a large gap in our understanding of the cell biology of Leishmania infections, and will facilitate the development of novel therapeutic approaches for this serious human disease. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI067979-02
Application #
7431777
Study Section
Special Emphasis Panel (ZRG1-IDM-M (02))
Program Officer
Mcgugan, Glen C
Project Start
2007-06-01
Project End
2011-05-31
Budget Start
2008-06-01
Budget End
2009-05-31
Support Year
2
Fiscal Year
2008
Total Cost
$405,786
Indirect Cost
Name
Yale University
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
06520
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Yuan, Xiaojing; Hamza, Iqbal (2018) Cys Links Heme: Stereo-orientation of Heme Transfer in Cytochrome c Biogenesis. J Mol Biol 430:1081-1083
Chen, Andy Jing; Yuan, Xiaojing; Li, Junjie et al. (2018) Label-Free Imaging of Heme Dynamics in Living Organisms by Transient Absorption Microscopy. Anal Chem 90:3395-3401
Sarkar, Amrita; Khan, Yousuf A; Laranjeira-Silva, Maria Fernanda et al. (2018) Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites. J Vis Exp :
de Menezes, Juliana Perrone Bezerra; Koushik, Amrita; Das, Satarupa et al. (2017) Leishmania infection inhibits macrophage motility by altering F-actin dynamics and the expression of adhesion complex proteins. Cell Microbiol 19:
Gouveia, ZĂ©lia; Carlos, Ana R; Yuan, Xiaojing et al. (2017) Characterization of plasma labile heme in hemolytic conditions. FEBS J 284:3278-3301
Mittra, Bidyottam; Laranjeira-Silva, Maria Fernanda; Miguel, Danilo Ciccone et al. (2017) The iron-dependent mitochondrial superoxide dismutase SODA promotes Leishmania virulence. J Biol Chem 292:12324-12338
Mittra, Bidyottam; Laranjeira-Silva, Maria Fernanda; Perrone Bezerra de Menezes, Juliana et al. (2016) A Trypanosomatid Iron Transporter that Regulates Mitochondrial Function Is Required for Leishmania amazonensis Virulence. PLoS Pathog 12:e1005340
Soares, Miguel P; Hamza, Iqbal (2016) Macrophages and Iron Metabolism. Immunity 44:492-504
Renberg, Rebecca L; Yuan, Xiaojing; Samuel, Tamika K et al. (2015) The Heme Transport Capacity of LHR1 Determines the Extent of Virulence in Leishmania amazonensis. PLoS Negl Trop Dis 9:e0003804

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