Avaliação do papel de macrófagos murinos na infecção por micobactérias ambientais

Submitted by Ana Maria Fiscina Sampaio ([email protected]) on 2012-11-30T21:17:29Z No. of bitstreams: 1 Juliana Perrone Bezerra De Menezes Avaliacao do papel... - 2005.pdf: 32348298 bytes, checksum: 109d71b2fb835421caaa135becd309de (MD5)Made available in DSpace on 2012-11-30T21:17:29Z (GMT). No. of bitstreams: 1 Juliana Perrone Bezerra De Menezes Avaliacao do papel... - 2005.pdf: 32348298 bytes, checksum: 109d71b2fb835421caaa135becd309de (MD5) Previous issue date: 2005Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil.Micobactérias ambientais podem ser encontradas em água, solo, poeira, alimentos e animais. A importância do estudo dessas micobactérias tem aumentado nos últimos anos, principalmente, devido a predisposição de pacientes com imunodeficiência à infecção por essas espécies de micobactéria. Além disso, a exposição a micobactérias ambientais pode constituir um dos fatores associados à baixa eficácia da imunização com a vacina BCG. As manifestações da doença, assim como a manutenção da infecção micobateriana, dependem da interação entre a micobactéria e o sistema imune do hospedeiro. O presente trabalho teve como objetivo avaliar a resposta de macrófagos peritoneais de camundongos susceptíveis BALB/c infectados com M intracellulare ou M fortuitum. Macrófagos peritoneais de camundongos BALB/c foram infectados por M intracellulare ou M fortuitum e as diferenças entre essas duas espécies quanto à capacidade de infectar e sobreviver no interior de macrófagos primários, tratados ou não com IFN-y, e produzir óxido nítrico foram avaliadas. Foi observado que os macrófagos infectados com M fortuitum apresentam um maior percentual de células infectadas que aqueles infectados com M. intracellulare, após 4, 24 e 48 horas de infecção. Entretanto, tanto M. fortuitum quanto M intracellulare são capazes de sobreviver no interior de macrófagos peritoneais, pois não há alteração da carga bacilar dessa duas espécies de micobactéria ao longo da infecção. Observamos ainda que M. intracellulare induziu uma maior produção de óxido nítrico por macrófagos primários infectados e tratados por IFN-y que M fortuitum. No entanto, o pré-tratamento com IFN-y não alterou o percentual de células infectadas nem a viabilidade de M intracellulare ou M. fortuitum. Os dados obtidos neste trabalho mostram que, in vitro, M. fortuitum e M. intracellulare interagem de formas distintas, levando á diferentes respostas do macrófago e a destinos intracelulares distintos. Além disso, mostramos que M intracellulare e M. fortuitum são resistentes ao óxido nítrico produzido por macrófagos após ativação por IFN-y.Environmental mycobacteria are found in water, soil, dust, food and animals. Environmental Mycobacterium importance has increased in the last few years, mostly because of immunodeficient patient predisposition to infection. Moreover, exposure to environmental mycobacteria could be associated to low levels of protection induced by immunization with BCG. Disease manifestations as well as infection outcome depend on interaction between mycobacteria and host immune system. The goal of this work was to evaluate peritoneal macrophage response, from the susceptible BALB/c mice, to M. intracellulare or M. fortuitum infection. Peritoneal inflammatory macrophages, pre-activated or not with IFN-y, were infected by M. intracellulare or M fortuitum and diferences between these two species related to the capacity to infect macrophages, to survive intracellularly and to induce NO production were evaluated. It was observed that the percentage of M. fortuitum-xnÍQoXQá cells was higher related to M. intracellulare-míecieá ones, after 4, 24 and 48 hours of infection. In addition, both M. fortuitum and M. intracellulare presented the ability to survive in peritoneal macrophages. It was also observed that in response to IFN-y activation, M. intracellulare induced higher NO production thanM fortuitum. However, pre-activation with IFN-y did not modify, neither the percentage of M. intracellulare and M. fortuitum infected cells, nor intracellular bacillum survival. These data demonstrate that, in vitro., M. fortuitum and M. intracellulare differently interact with macrophages, inducing diferent macrophage reponses and that both M. intracellulare and M fortuitum are resistant to NO production upon IFN-y activation

Using Proteomics to Understand How Leishmania Parasites Survive inside the Host and Establish Infection

Submitted by Ana Maria Fiscina Sampaio ([email protected]) on 2017-03-06T16:31:32Z No. of bitstreams: 1 Veras PST Using proteomics....pdf: 2023439 bytes, checksum: 69c96bf28d97a98ea5b1656099c81ac8 (MD5)Approved for entry into archive by Ana Maria Fiscina Sampaio ([email protected]) on 2017-03-06T16:41:42Z (GMT) No. of bitstreams: 1 Veras PST Using proteomics....pdf: 2023439 bytes, checksum: 69c96bf28d97a98ea5b1656099c81ac8 (MD5)Made available in DSpace on 2017-03-06T16:41:42Z (GMT). No. of bitstreams: 1 Veras PST Using proteomics....pdf: 2023439 bytes, checksum: 69c96bf28d97a98ea5b1656099c81ac8 (MD5) Previous issue date: 2016Instituto Gonçalo Moniz/Fundação Oswaldo Cruz (IGM/FIOCRUZ); Coordenação de Aperfeiçoamento do Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo à PesquisaFundação Oswaldo Cruz. Instituto de Pesquisas Gonçalo Moniz. Laboratório de Patologia e Biointervenção. Salvador, BA, BrasilInstituto Nacional de Ciência e Tecnologia para Doenças Tropicais. INCT-DT. Salvador, BA, BrasilLeishmania is a protozoan parasite that causes a wide range of different clinical manifestations in mammalian hosts. It is a major public health risk on different continents and represents one of the most important neglected diseases. Due to the high toxicity of the drugs currently used, and in the light of increasing drug resistance, there is a critical need to develop new drugs and vaccines to control Leishmania infection. Over the past few years, proteomics has become an important tool to understand the underlying biology of Leishmania parasites and host interaction. The large-scale study of proteins, both in parasites and within the host in response to infection, can accelerate the discovery of new therapeutic targets. By studying the proteomes of host cells and tissues infected with Leishmania, as well as changes in protein profiles among promastigotes and amastigotes, scientists hope to better understand the biology involved in the parasite survival and the host-parasite interaction. This review demonstrates the feasibility of proteomics as an approach to identify new proteins involved in Leishmania differentiation and intracellular survival

Elucidating the Complex Interrelationship on Early Interactions between <em>Leishmania</em> and Macrophages

The host’s ability to eradicate or control infection caused by intracellular pathogens depends on early interactions between these microorganisms and host cells. These events are related to the organism’s nature and stage of development and host immune status. Pathogens are recognized by host cells, which respond to infection by either mounting an efficient response or becoming a replication niche. Early interactions between the protozoan Leishmania parasite and host cell receptors activate different signaling pathways that can result in microbe elimination or, alternatively, infection establishment and the migration of Leishmania infected cells to other host tissues. This chapter focuses on Leishmania-macrophage interaction via phagocytosis, which involves a range of parasite ligands characteristic of Leishmania species and parasite stage of development and diverse host cell receptors. We also discuss alternative Leishmania entry by cell invasion and review how Leishmania spp. survive and replicate within the phagocytic compartment they induce

Deciphering the Role Played by Autophagy in Leishmania Infection

Submitted by Ana Maria Fiscina Sampaio ([email protected]) on 2019-12-10T16:35:25Z No. of bitstreams: 1 Veras, P.S.T. Deciphering....pdf: 2656851 bytes, checksum: 47e32a3b983116aac9bbbfd38a5df64f (MD5)Approved for entry into archive by Ana Maria Fiscina Sampaio ([email protected]) on 2019-12-10T16:54:43Z (GMT) No. of bitstreams: 1 Veras, P.S.T. Deciphering....pdf: 2656851 bytes, checksum: 47e32a3b983116aac9bbbfd38a5df64f (MD5)Made available in DSpace on 2019-12-10T16:54:43Z (GMT). No. of bitstreams: 1 Veras, P.S.T. Deciphering....pdf: 2656851 bytes, checksum: 47e32a3b983116aac9bbbfd38a5df64f (MD5) Previous issue date: 2019Fundação de Amparo à Pesquisa do Estado da Bahia (PV, http://www.fapesb.ba.gov.br), Conselho Nacional de Pesquisa e Desenvolvimento Científico (PV, http://www.cnpq.br). This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal deNível Superior— Brasil (CAPES)—Finance Code 001. PV holds a grant fromCNPq for productivity in research (307832/2015-5).Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Laboratório Parasito-Hospedeiro e Epidemiologia. Salvador, BA, Brasil / National Institute of Science and Technology of Tropical Diseases. Salvador, BA, Brazil.Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Parasito-Hospedeiro e Epidemiologia. Salvador, BA, Brasil.Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Parasito-Hospedeiro e Epidemiologia. Salvador, BA, Brasil.In recent decades, studies have shown that, depending on parasite species and host background, autophagy can either favor infection or promote parasite clearance. To date, relatively few studies have attempted to assess the role played by autophagy in Leishmania infection. While it has been consistently shown that Leishmania spp. induce autophagy in a variety of cell types, published results regarding the effects of autophagic modulation on Leishmania survival are contradictory. The present review, after a short overview of the general aspects of autophagy, aims to summarize the current body of knowledge surrounding how Leishmania spp. adaptively interact with macrophages, the host cells mainly involved in controlling leishmaniasis. We then explore the scarce studies that have investigated interactions between these parasite species and the autophagic pathway, and finally present a critical perspective on how autophagy influences infection outcome

In Search of Biomarkers for Pathogenesis and Control of Leishmaniasis by Global Analyses of Leishmania-Infected Macrophages

Submitted by Ana Maria Fiscina Sampaio ([email protected]) on 2018-10-19T17:46:58Z No. of bitstreams: 1 Veras PS In search of Biomarkers....2018.pdf: 570607 bytes, checksum: 210e729195b45b2a10cdf51ab688a017 (MD5)Approved for entry into archive by Ana Maria Fiscina Sampaio ([email protected]) on 2018-10-19T18:22:19Z (GMT) No. of bitstreams: 1 Veras PS In search of Biomarkers....2018.pdf: 570607 bytes, checksum: 210e729195b45b2a10cdf51ab688a017 (MD5)Made available in DSpace on 2018-10-19T18:22:19Z (GMT). No. of bitstreams: 1 Veras PS In search of Biomarkers....2018.pdf: 570607 bytes, checksum: 210e729195b45b2a10cdf51ab688a017 (MD5) Previous issue date: 2018Fundação de Amparo à Pesquisa do Estado da Bahia (PV http://www.fapesb.ba. gov.br), Conselho Nacional de Pesquisa e Desenvolvimento Científico (PV http://www.cnpq.br). PV holds a grant from CNPq for productivity in research (307832/2015-5).Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Laboratório de Interação Parasito-Hospedeiro e Epidemiologia. Salvador, BA, Brasil / National Institute of Tropical Disease. Brasilia, DF, Brazil.Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Centro de Integração de Dados e Conhecimento para a Saúde. Salvador, BA, Brasil.Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Laboratório de Interação Parasito-Hospedeiro e Epidemiologia. Salvador, BA, Brasil.Leishmaniasis is a vector-borne, neglected tropical disease with a worldwide distribution that can present in a variety of clinical forms, depending on the parasite species and host genetic background. The pathogenesis of this disease remains far from being elucidated because the involvement of a complex immune response orchestrated by host cells significantly affects the clinical outcome. Among these cells, macrophages are the main host cells, produce cytokines and chemokines, thereby triggering events that contribute to the mediation of the host immune response and, subsequently, to the establishment of infection or, alternatively, disease control. There has been relatively limited commercial interest in developing new pharmaceutical compounds to treat leishmaniasis. Moreover, advances in the understanding of the underlying biology of Leishmania spp. have not translated into the development of effective new chemotherapeutic compounds. As a result, biomarkers as surrogate disease endpoints present several potential advantages to be used in the identification of targets capable of facilitating therapeutic interventions considered to ameliorate disease outcome. More recently, large-scale genomic and proteomic analyses have allowed the identification and characterization of the pathways involved in the infection process in both parasites and the host, and these analyses have been shown to be more effective than studying individual molecules to elucidate disease pathogenesis. RNA-seq and proteomics are large-scale approaches that characterize genes or proteins in a given cell line, tissue, or organism to provide a global and more integrated view of the myriad biological processes that occur within a cell than focusing on an individual gene or protein. Bioinformatics provides us with the means to computationally analyze and integrate the large volumes of data generated by high-throughput sequencing approaches. The integration of genomic expression and proteomic data offers a rich multi-dimensional analysis, despite the inherent technical and statistical challenges. We propose that these types of global analyses facilitate the identification, among a large number of genes and proteins, those that hold potential as biomarkers. The present review focuses on large-scale studies that have identified and evaluated relevant biomarkers in macrophages in response to Leishmania infection

In Search of Biomarkers for Pathogenesis and Control of Leishmaniasis by Global Analyses of Leishmania-Infected Macrophages

Leishmaniasis is a vector-borne, neglected tropical disease with a worldwide distribution that can present in a variety of clinical forms, depending on the parasite species and host genetic background. The pathogenesis of this disease remains far from being elucidated because the involvement of a complex immune response orchestrated by host cells significantly affects the clinical outcome. Among these cells, macrophages are the main host cells, produce cytokines and chemokines, thereby triggering events that contribute to the mediation of the host immune response and, subsequently, to the establishment of infection or, alternatively, disease control. There has been relatively limited commercial interest in developing new pharmaceutical compounds to treat leishmaniasis. Moreover, advances in the understanding of the underlying biology of Leishmania spp. have not translated into the development of effective new chemotherapeutic compounds. As a result, biomarkers as surrogate disease endpoints present several potential advantages to be used in the identification of targets capable of facilitating therapeutic interventions considered to ameliorate disease outcome. More recently, large-scale genomic and proteomic analyses have allowed the identification and characterization of the pathways involved in the infection process in both parasites and the host, and these analyses have been shown to be more effective than studying individual molecules to elucidate disease pathogenesis. RNA-seq and proteomics are large-scale approaches that characterize genes or proteins in a given cell line, tissue, or organism to provide a global and more integrated view of the myriad biological processes that occur within a cell than focusing on an individual gene or protein. Bioinformatics provides us with the means to computationally analyze and integrate the large volumes of data generated by high-throughput sequencing approaches. The integration of genomic expression and proteomic data offers a rich multi-dimensional analysis, despite the inherent technical and statistical challenges. We propose that these types of global analyses facilitate the identification, among a large number of genes and proteins, those that hold potential as biomarkers. The present review focuses on large-scale studies that have identified and evaluated relevant biomarkers in macrophages in response to Leishmania infection

The iron-dependent mitochondrial superoxide dismutase SODA promotesLeishmania virulence

Menezes, Juliana Perrone Bezerra de. “Documento produzido em parceria ou por autor vinculado à Fiocruz, mas não consta à informação no documento”.Submitted by Ana Maria Fiscina Sampaio ([email protected]) on 2018-03-26T14:14:29Z No. of bitstreams: 1 Mittra B The iron-dependent mitochondrial superoxide....pdf: 3577331 bytes, checksum: 4212fdc48af97da40a383cd14d24ead8 (MD5)Approved for entry into archive by Ana Maria Fiscina Sampaio ([email protected]) on 2018-03-26T14:34:09Z (GMT) No. of bitstreams: 1 Mittra B The iron-dependent mitochondrial superoxide....pdf: 3577331 bytes, checksum: 4212fdc48af97da40a383cd14d24ead8 (MD5)Made available in DSpace on 2018-03-26T14:34:09Z (GMT). No. of bitstreams: 1 Mittra B The iron-dependent mitochondrial superoxide....pdf: 3577331 bytes, checksum: 4212fdc48af97da40a383cd14d24ead8 (MD5) Previous issue date: 2017National Institutes of Health Grant R01 AI067979 (to N. W. A.).University of Maryland. Department of Cell Biology and Molecular Genetics. Maryland, USAUniversity of Maryland. Department of Cell Biology and Molecular Genetics. Maryland, USAUniversity of Maryland. Department of Cell Biology and Molecular Genetics. Maryland, USAUniversity of Maryland. Department of Cell Biology and Molecular Genetics. Maryland, USAUniversity of Maryland. Department of Cell Biology and Molecular Genetics. Maryland, USALeishmaniasis is one of the leading globally neglected diseases, affecting millions of people worldwide.Leishmaniainfection depends on the ability of insect-transmitted metacyclic promastigotes to invade mammalian hosts, differentiate into amastigotes, and replicate inside macrophages. To counter the hostile oxidative environment inside macrophages, these protozoans contain anti-oxidant systems that include iron-dependent superoxide dismutases (SODs) in mitochondria and glycosomes. Increasing evidence suggests that in addition to this protective role,Leishmaniamitochondrial SOD may also initiate H2O2-mediated redox signaling that regulates gene expression and metabolic changes associated with differentiation into virulent forms. To investigate this hypothesis, we examined the specific role of SODA, the mitochondrial SOD isoform inLeishmania amazonensisOur inability to generateL. amazonensis SODAnull mutants and the lethal phenotype observed following RNAi-mediated silencing of theTrypanosoma brucei SODAortholog suggests that SODA is essential for trypanosomatid survival.L. amazonensismetacyclic promastigotes lacking oneSODAallele failed to replicate in macrophages and were severely attenuated in their ability to generate cutaneous lesions in mice. Reduced expression of SODA also resulted in mitochondrial oxidative damage and failure ofSODA/ΔsodApromastigotes to differentiate into axenic amastigotes. SODA expression above a critical threshold was also required for the development of metacyclic promastigotes, asSODA/ΔsodAcultures were strongly depleted in this infective form and more susceptible to reactive oxygen species (ROS)-induced stress. Collectively, our data suggest that SODA promotesLeishmaniavirulence by protecting the parasites against mitochondrion-generated oxidative stress and by initiating ROS-mediated signaling mechanisms required for the differentiation of infective forms

Advances in Development of New Treatment for Leishmaniasis.

Submitted by Ana Maria Fiscina Sampaio ([email protected]) on 2016-03-23T12:18:59Z No. of bitstreams: 1 Menezes JPB Advances in ....pdf: 614749 bytes, checksum: 4fd5da201129ed240d6cd34951442a85 (MD5)Approved for entry into archive by Ana Maria Fiscina Sampaio ([email protected]) on 2016-03-23T12:31:49Z (GMT) No. of bitstreams: 1 Menezes JPB Advances in ....pdf: 614749 bytes, checksum: 4fd5da201129ed240d6cd34951442a85 (MD5)Made available in DSpace on 2016-03-23T12:31:49Z (GMT). No. of bitstreams: 1 Menezes JPB Advances in ....pdf: 614749 bytes, checksum: 4fd5da201129ed240d6cd34951442a85 (MD5) Previous issue date: 2015Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Laboratório de Patologia e Biointervenção. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Laboratório de Patologia e Biointervenção. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Laboratório de Patologia e Biointervenção. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Laboratório de Patologia e Biointervenção. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Laboratório de Patologia e Biointervenção. Salvador, BA, BrasilLeishmaniasis is a neglected infectious disease caused by several different species of protozoan parasites of the genus Leishmania. Current strategies to control this disease are mainly based on chemotherapy.Despite being available for the last 70 years, leishmanial chemotherapy has lack of efficiency, since its route of administration is difficult and it can cause serious side effects, which results in the emergence of resistant cases.The medical-scientific community is facing difficulties to overcome these problems with new suitable and efficient drugs, as well as the identification of new drug targets. The availability of the complete genome sequence of Leishmania has given the scientific community the possibility of large-scale analysis, which may lead to better understanding of parasite biology and consequent identification of novel drug targets. In this review we focus on how high-throughput analysis is helping us and other groups to identify novel targets for chemotherapeutic interventions.We further discuss recent data produced by our group regarding the use of the high-throughput techniques and how this helped us to identify and assess the potential of new identified targets

Leishmania infection inhibits macrophage motility by altering F-actin dynamics and the expression of adhesion complex proteins

Submitted by Ana Maria Fiscina Sampaio ([email protected]) on 2017-03-27T16:56:40Z No. of bitstreams: 1 Menezes JPB Leishmania infection....pdf: 951342 bytes, checksum: 30124d1fbf1b03f069b985fab99d013c (MD5)Approved for entry into archive by Ana Maria Fiscina Sampaio ([email protected]) on 2017-03-27T17:11:00Z (GMT) No. of bitstreams: 1 Menezes JPB Leishmania infection....pdf: 951342 bytes, checksum: 30124d1fbf1b03f069b985fab99d013c (MD5)Made available in DSpace on 2017-03-27T17:11:00Z (GMT). No. of bitstreams: 1 Menezes JPB Leishmania infection....pdf: 951342 bytes, checksum: 30124d1fbf1b03f069b985fab99d013c (MD5) Previous issue date: 2017National Institutes of Health grant R01 AI67979 to NWA. WL and SD were supported by National Institutes of Health grant R01 GM085574. JPBN was supported by a Ciências sem Fronteiras fellowship (CNPq).University of Maryland. Department of Cell Biology and Molecular Genetics. Maryland, USA / Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Laboratório de Patologia e Biointervenção. Salvador, BA, BrasilUniversity of Maryland. Department of Cell Biology and Molecular Genetics. Maryland, USAUniversity of Maryland. Department of Physics. Maryland, USAUniversity of Maryland. Department of Physics. Maryland, USAUniversity of Maryland. Department of Cell Biology and Molecular Genetics. Maryland, USAUniversity of Maryland. Department of Cell Biology and Molecular Genetics. Maryland, USAUniversity of Maryland. Department of Physics. Maryland, USAUniversity of Maryland. Department of Cell Biology and Molecular Genetics. Maryland, USALeishmania is an intracellular protozoan parasite that causes a broad spectrum of clinical manifestations, ranging from self-healing skin lesions to fatal visceralizing disease. As the host cells of choice for all species of Leishmania, macrophages are critical for the establishment of infections. How macrophages contribute to parasite homing to specific tissues and how parasites modulate macrophage function are still poorly understood. In this study, we show that Leishmania amazonensis infection inhibits macrophage roaming motility. The reduction in macrophage speed is not dependent on particle load or on factors released by infected macrophages. L. amazonensis-infected macrophages also show reduced directional migration in response to the chemokine MCP-1. We found that infected macrophages have lower levels of total paxillin, phosphorylated paxillin, and phosphorylated focal adhesion kinase when compared to noninfected macrophages, indicating abnormalities in the formation of signaling adhesion complexes that regulate motility. Analysis of the dynamics of actin polymerization at peripheral sites also revealed a markedly enhanced F-actin turnover frequency in L. amazonensis-infected macrophages. Thus, Leishmania infection inhibits macrophage motility by altering actin dynamics and impairing the expression of proteins that function in plasma membrane-extracellular matrix interactions

Deletion of one <i>LMIT1</i> allele impairs temperature/pH-induced differentiation of promastigotes into axenic amastigotes.

<p>Late-log phase promastigotes were washed, resuspended in pH 4.5 at 2x10⁵ parasites/ml, and cultured at 32°C. (A) Numbers of wild type (WT), single knockout (<i>LMIT1/Δlmit1</i>) and complemented single knockout (<i>LMIT1/Δlmit1+LMIT1)</i> parasites following shift to amastigote medium. The data represent the mean +/- SD of triplicate determinations and are representative of four independent experiments. (B–D) wild type (WT), single knockout (<i>LMIT1/Δlmit1</i>) and complemented single knockout (<i>LMIT1/Δlmit1+LMIT1</i>) parasites incubated for 48 h in amastigote medium at 32°C were subjected to: (B) Quantification of viable rounded forms with a short flagellum. At least 400 FDA-stained parasites were counted microscopically in each sample. The data represent the mean ± SD of quadruplicate determinations. (C) SEM analysis of the morphology of parasites. Bars = 2 μm. (D) Determination of cell viability and membrane integrity by staining with FDA (green) and PI (red). Bars = 27 μm. (E) The iron content of whole cells and mitochondrial fractions was determined in parasites collected 24 h after induction of axenic differentiation (pH 4.5/ 32°C). Data represents the mean ± SD of three independent experiments (Student’s <i>t</i> test *p = 0.037, **<i>p</i> = 0.004). (F) Aconitase activity was determined in mitochondrial fractions from parasites collected 24 h after induction of axenic differentiation (pH 4.5/ 32°C). The data represent the mean ± SD of three independent experiments (**p≤ 0.008). (G) Determination of mitochondrial membrane potential (ΔΨ_m) with JC-1 with and without prior treatment with the mitochondrial uncoupler CCCP. The data represents the mean ± SD of three independent experiments (Student’s <i>t</i> test * <i>p</i> = 0.01; **<i>p</i> = 0.002). ((H) Viable amastigotes obtained by temperature/pH- induced axenic differentiation in wild type (WT), single knockout (<i>LMIT1/Δlmit1</i>) and complemented single knockout (<i>LMIT1/Δlmit1+LMIT1</i>) cultures were tested for their ability to infect BMMs. BMMs were infected and either fixed immediately or after further incubation for 24, 48 or 72 h and the number of intracellular parasites was determined microscopically. The data represent the mean ± SD of triplicate determinations and are representative of more than three independent experiments.</p