Indoleamine 2,3-dioxygenase and iron are required for Mycobacterium leprae survival.

Our previous study has demonstrated that IL-10 may modulate both indoleamine 2,3-dioxygenase (IDO) and CD163 expression in lepromatous leprosy (LL) cells, favoring Mycobacterium leprae persistence through induction of regulatory pathways and iron storage. Here, we observed that in LL lesion cells there is an increase in the expression of proteins involved in iron metabolism such as hemoglobin (Hb), haptoglobin, heme oxygenase 1 and transferrin receptor 1 (TfR1) when compared to tuberculoid leprosy (BT) cells. We also found increased iron deposits and diminished expression of the iron exporter ferroportin 1 in LL lesion cells. Hemin, but not FeSO4 stimulation, was able to enhance M. leprae viability by a mechanism that involves IDO. Analysis of cell phenotype in lesions demonstrated a predominance of M2 markers in LL when compared with BT lesion cells. A positive correlation between CD163 and PPARG with the bacillary index (BI) was observed. In contrast, TNF, STAT1 and CSF2 presented a negative correlation with the BI. In summary, this study demonstrates that iron may regulate IDO expression by a mechanism that involves IL-10, which may contribute for the predominance of M2-like phenotype in LL lesions that favors the phagocytosis and maintenance of M. leprae in host cells.

Indoleamine 2. 3-dioxygenase and iron are required for Mycobacterium leprae survival

Our previous study has demonstrated that IL-10 may modulate both indoleamine 2,3-dioxygenase (IDO) and CD163 expression in lepromatous leprosy (LL) cells, favoring Mycobacterium leprae persistence through induction of regulatory pathways and iron storage. Here, we observed that in LL lesion cells there is an increase in the expression of proteins involved in iron metabolism such as hemoglobin (Hb), haptoglobin, heme oxygenase 1 and transferrin receptor 1 (TfR1) when compared to tuberculoid leprosy (BT) cells. We also found increased iron deposits and diminished expression of the iron exporter ferroportin 1 in LL lesion cells. Hemin, but not FeSO stimulation, was able to enhance M. leprae viability by a mechanism that involves IDO. Analysis of cell phenotype in lesions demonstrated a predominance of M2 markers in LL when compared with BT lesion cells. A positive correlation between CD163 and PPARG with the bacillary index (BI) was observed. In contrast, TNF, STAT1 and CSF2 presented a negative correlation with the BI. In summary, this study demonstrates that iron may regulate IDO expression by a mechanism that involves IL-10, which may contribute for the predominance of M2-like phenotype in LL lesions that favors the phagocytosis and maintenance of M. leprae in host cells.

Mujer de 51 años con astenia, pérdida de peso y anasarca / 51 years-old woman with fatigue, weight loss and anasarca

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The Complete Genome Sequence of the Emerging Pathogen Mycobacterium haemophilum Explains Its Unique Culture Requirements.

UNLABELLED: Mycobacterium haemophilum is an emerging pathogen associated with a variety of clinical syndromes, most commonly skin infections in immunocompromised individuals. M. haemophilum exhibits a unique requirement for iron supplementation to support its growth in culture, but the basis for this property and how it may shape pathogenesis is unclear. Using a combination of Illumina, PacBio, and Sanger sequencing, the complete genome sequence of M. haemophilum was determined. Guided by this sequence, experiments were performed to define the basis for the unique growth requirements of M. haemophilum. We found that M. haemophilum, unlike many other mycobacteria, is unable to synthesize iron-binding siderophores known as mycobactins or to utilize ferri-mycobactins to support growth. These differences correlate with the absence of genes associated with mycobactin synthesis, secretion, and uptake. In agreement with the ability of heme to promote growth, we identified genes encoding heme uptake machinery. Consistent with its propensity to infect the skin, we show at the whole-genome level the genetic closeness of M. haemophilum with Mycobacterium leprae, an organism which cannot be cultivated in vitro, and we identify genes uniquely shared by these organisms. Finally, we identify means to express foreign genes in M. haemophilum. These data explain the unique culture requirements for this important pathogen, provide a foundation upon which the genome sequence can be exploited to improve diagnostics and therapeutics, and suggest use of M. haemophilum as a tool to elucidate functions of genes shared with M. leprae. IMPORTANCE: Mycobacterium haemophilum is an emerging pathogen with an unknown natural reservoir that exhibits unique requirements for iron supplementation to grow in vitro. Understanding the basis for this iron requirement is important because it is fundamental to isolation of the organism from clinical samples and environmental sources. Defining the molecular basis for M. haemophilium's growth requirements will also shed new light on mycobacterial strategies to acquire iron and can be exploited to define how differences in such strategies influence pathogenesis. Here, through a combination of sequencing and experimental approaches, we explain the basis for the iron requirement. We further demonstrate the genetic closeness of M. haemophilum and Mycobacterium leprae, the causative agent of leprosy which cannot be cultured in vitro, and we demonstrate methods to genetically manipulate M. haemophilum. These findings pave the way for the use of M. haemophilum as a model to elucidate functions of genes shared with M. leprae.

Growth and adaptation of microorganisms on the cheese surface.

Microbial communities living on cheese surfaces are composed of various bacteria, yeasts and molds that interact together, thus generating the typical sensory properties of a cheese. Physiological and genomic investigations have revealed important functions involved in the ability of microorganisms to establish themselves at the cheese surface. These functions include the ability to use the cheese's main energy sources, to acquire iron, to tolerate low pH at the beginning of ripening and to adapt to high salt concentrations and moisture levels. Horizontal gene transfer events involved in the adaptation to the cheese habitat have been described, both for bacteria and fungi. In the future, in situ microbial gene expression profiling and identification of genes that contribute to strain fitness by massive sequencing of transposon libraries will help us to better understand how cheese surface communities function.

Análise de polimorfismos no gene PKLR e associação com a hanseníase / Analisys of polymorphins in the PKLR gene and association with leprosy

A hanseníase é uma doença infecciosa crônica provocada pelo patógeno intracelular obrigatório Mycobacterium leprae. Dado à baixa variabilidade desse bacilo, aliado à variedade de formas clínicas desenvolvidas na hanseníase, sugere-se que o componente genético do hospedeiro é o grande responsável pelo desenvolvimento da doença. Até o momento, polimorfismos de base única (SNPs) em diversos genes foram associados com a predisposição à hanseníase em estudos independentes em diferentes populações. Recentemente, SNPs no gene PKLR foram associados ao risco de desenvolvimento da hanseníase pelo nosso grupo. Na tentativa de melhor investigar o efeito de suscetibilidade desse gene a patógenos intracelulares, o presente estudo avaliou a associação de SNPs adicionais do PKLR com a hanseníase na população Brasileira e com a tuberculose na população de Moçambique. Os parâmetros funcionais relacionados aos marcadores do PKLR também foram avaliados. Inicialmente, foi feita uma seleção de SNPs a partir da busca nos dados do HapMap. Estes SNPs foram genotipados em um estudo de associação seguindo um desenho do tipo caso-controle na população do Rio de Janeiro. Os resultados mostraram uma associação significativa de suscetibilidade a hanseníase para os SNPs rs11264355, rs11264359, rs4620533 e rs4971072 na população do Rio de Janeiro, assim como para o haplótipo rs11264355G/rs11264359G/rs4620533G/rs49710729.

Leprosy is a chronic infectious disease caused by the obligate intracellular pathogenMycobacterium leprae. Given the low variability of the bacile with the variety of clinicalphenotype exhibited in leprosy, it is suggested that the genetic componente of the host isresponsable to leprosy development. Until now, single nucleotide polymorphisms (SNPs) inmany genes were associated with leprosy predisposition in independente studies andpopulation. Recently, SNPs in the PKLR gene were associated with leprosy susceptibility byour group. Aiming to investigate the susceptibility to intracellular pathogens, this studyevaluated the association of additional SNPs of the PKLR in a Brazilian population, followedby an case-control study with tuberculosis in a Mozambique population. Functionalparameters correlated to the polymorphic variants were also evaluated. Initially, using theHapMap population data, we performed an analysis to search for SNPs which were tested inan case-control association study. Results showed a significant susceptibility association withleprosy within SNPs rs11264355, rs11264359, rs4620533 and rs4971072 in Rio de Janeiropopulation. In addition, we demonstrated that the haplotypers11264355G/rs11264359G/rs4620533G/rs4971072G was significantly associated withleprosy susceptibility in this population...

M. paratuberculosis and Parkinson's disease--is this a trigger.

Genetic linkage studies and genome wide analysis have provided insights into complex medical diseases. Mycobacterium avium ss. paratuberculosis (MAP) causes Johne's disease, an important enteric inflammatory disease mostly studied in ruminant animals. MAP is also the putative cause of Crohn's disease. Moreover, MAP has been linked to other inflammatory diseases: sarcoidosis, Blau syndrome, autoimmune diabetes, autoimmune thyroiditis and multiple sclerosis. Genetic studies reveal an association between Parkinson's disease (PD), leprosy and Crohn's disease and since discovered, these findings have been considered "surprising". Autophagy and ubiquitin-proteosome systems are cellular systems that both fight intracellular pathogens (xenophagy) and maintain cellular protein quality control. PD is a common neurodegenerative disease that manifests clinically as a profound movement disorder. The recognized genetic defects of PD create disruption of cellular homeostasis that result in protein folding abnormalities of PD called Lewy bodies. Those same genetic defects are associated with susceptibility to intracellular pathogens, including mycobacteria. It is now understood that PD Lewy body pathology starts in the enteric nervous system and "spreads" to the brain in a retrograde fashion via the vagus nerve. This is the same process by which prions affect the brain. Lewy body pathology of the enteric nervous system predates the Lewy body pathology of the central nervous system (CNS) by years or even decades. This article proposes that genetic defects associated with PD also result in a permissive environment for MAP infection--ineffective xenophagy. It postulates that beginning as an enteric infection, MAP--via the vagus nerve--initiates a pathologic process that results in a targeted neuroinvasion of the CNS. The article proposes that MAP infection and resultant PD pathology are due, in the genetically at-risk and age dependant, to the consumptive exhaustion of the protein quality control systems.

Increased hepcidin expression in multibacillary leprosy

Iron is essential for all organisms and its availability can control the growth of microorganisms; therefore, we examined the role of iron metabolism in multibacillary (MB) leprosy, focusing on the involvement of hepcidin. Erythrograms, iron metabolism parameters, pro-inflammatory cytokines and urinary hepcidin levels were evaluated in patients with MB and matched control subjects. Hepcidin expression in MB lesions was evaluated by quantitative polymerase chain reaction. The expression of ferroportin and hepcidin was evaluated by immunofluorescence in paucibacillary and MB lesions. Analysis of hepcidin protein levels in urine and of hepcidin mRNA and protein levels in leprosy lesions and skin biopsies from healthy control subjects showed elevated hepcidin levels in MB patients. Decreases in haematologic parameters and total iron binding capacity were observed in patients with MB leprosy. Moreover, interleukin-1 beta, ferritin, soluble transferrin receptor and soluble transferrin receptor/log ferritin index values were increased in leprosy patients. Hepcidin was elevated in lepromatous lesions, whereas ferroportin was more abundant in tuberculoid lesions. In addition, hepcidin and ferroportin were not colocalised in the biopsies from leprosy lesions. Anaemia was not commonly observed in patients with MB; however, the observed changes in haematologic parameters indicating altered iron metabolism appeared to result from a mixture of anaemia of inflammation and iron deficiency. Thus, iron sequestration inside host cells might play a role in leprosy by providing an optimal environment for the bacillus.

An enlarged, adaptable active site in CYP164 family P450 enzymes, the sole P450 in Mycobacterium leprae.

CYP164 family P450 enzymes are found in only a subset of mycobacteria and include CYP164A1, which is the sole P450 found in Mycobacterium leprae, the causative agent of leprosy. This has previously led to interest in this enzyme as a potential drug target. Here we describe the first crystal structure of a CYP164 enzyme, CYP164A2 from Mycobacterium smegmatis. CYP164A2 has a distinctive, enlarged hydrophobic active site that extends above the porphyrin ring toward the access channels. Unusually, we find that CYP164A2 can simultaneously bind two econazole molecules in different regions of the enlarged active site and is accompanied by the rearrangement and ordering of the BC loop. The primary location is through a classic interaction of the azole group with the porphyrin iron. The second econazole molecule is bound to a unique site and is linked to a tetracoordinated metal ion complexed to one of the heme carboxylates and to the side chains of His 105 and His 364. All of these features are preserved in the closely homologous M. leprae CYP164A1. The computational docking of azole compounds to a homology model of CYP164A1 suggests that these compounds will form effective inhibitors and is supported by the correlation of parallel docking with experimental binding studies of CYP164A2. The binding of econazole to CYP164A2 occurs primarily through the high-spin "open" conformation of the enzyme (K(d) [dissociation constant] of 0.1 µM), with binding to the low-spin "closed" form being significantly hindered (K(d) of 338 µM). These studies support previous suggestions that azole derivatives may provide an effective strategy to improve the treatment of leprosy.

Increased hepcidin expression in multibacillary leprosy.

Iron is essential for all organisms and its availability can control the growth of microorganisms; therefore, we examined the role of iron metabolism in multibacillary (MB) leprosy, focusing on the involvement of hepcidin. Erythrograms, iron metabolism parameters, pro-inflammatory cytokines and urinary hepcidin levels were evaluated in patients with MB and matched control subjects. Hepcidin expression in MB lesions was evaluated by quantitative polymerase chain reaction. The expression of ferroportin and hepcidin was evaluated by immunofluorescence in paucibacillary and MB lesions. Analysis of hepcidin protein levels in urine and of hepcidin mRNA and protein levels in leprosy lesions and skin biopsies from healthy control subjects showed elevated hepcidin levels in MB patients. Decreases in haematologic parameters and total iron binding capacity were observed in patients with MB leprosy. Moreover, interleukin-1 beta, ferritin, soluble transferrin receptor and soluble transferrin receptor/log ferritin index values were increased in leprosy patients. Hepcidin was elevated in lepromatous lesions, whereas ferroportin was more abundant in tuberculoid lesions. In addition, hepcidin and ferroportin were not colocalised in the biopsies from leprosy lesions. Anaemia was not commonly observed in patients with MB; however, the observed changes in haematologic parameters indicating altered iron metabolism appeared to result from a mixture of anaemia of inflammation and iron deficiency. Thus, iron sequestration inside host cells might play a role in leprosy by providing an optimal environment for the bacillus.

O envolvimento do ferro na modulação da resposta imune em pacientes multibacilares da Hanseníase / The involvement of iron in modulating the immune response in patients with multibacillary leprosy

A forma lepromatosa da Hanseníase caracteriza-se pela baixa ou ausente resposta imune específica aos antígenos do Mycobacterium leprae e intensa proliferação bacilar. Dentre as células hospedeiras, os macrófagos parecem ter um papel crucial no direcionamento do perfil de resposta ao M. leprae. Muitas das vias de imunossupressão da resposta imune celular parecem estar envolvidas com o metabolismo do ferro e seus carreadores, como a expressão aumentada de IDO em macrófagos lepromatosos e de heme-oxigenase 1 em macrófagos M2. Anteriormente, o nosso grupo demonstrou que macrófagos de pacientes lepromatosos apresentam uma maior expressão do receptor scavenger CD163, quando comparados aos tuberculóides. O CD163 reconhece o complexo Hemoglobina-Haptoglobina e nós hipotetizamos que o M. leprae adaptou-se para aumentara expressão de CD163, a fim de aumentar estoques de ferro em macrófagos, contribuindo para sua persistência. Assim, o objetivo do presente trabalho foi investigar o envolvimento de ferro na imunopatogênese da hanseníase. Nós observamos que biópsias de lesão de pele de pacientes lepromatosos apresentam uma maior expressão de proteínas relacionadas à captação e metabolismo de ferro, assim como uma maior deposição de ferro na forma de ferritina e hemossiderina nos macrófagos espumosos, onde se localizam os bacilos. A adição de ferro exógeno, na forma de tratamento com sulfato ferroso, foi capaz de reduzir a expressão e atividade de indoleamina 2,3-dioxigenase induzida pelo M. leprae em monócitos de doadores saudáveis. A adição de ferro livre também foi capaz de modular a produção de citocinas, aumentando a produção de IL-12p70 e IL-10 em culturas de monócitos, e de IL-6 em culturas de células mononucleares do sangue periférico. O aumento dos receptores CD163 e receptor de transferrina 1 em macrófagos lepromatosos foram associados com o aumento dos estoques de ferro em biópsias de pele de pacientes lepromatosos. Em adição, o tratamento com ferro livre foi capaz aumentar vias proinflamatórias em leucócitos estimulados com M. leprae. Isto indica que a homeostase do ferro no hospedeiro no momento da infecção é importante para a definição do desfecho da Hanseníase, e que o ferro pode ter um papel duplo dependendo do momento e da forma que este está apresentado, podendo criar um ambiente favorável para a micobactéria ou induzindo a produção de citocinas próinflamatórias que podem contribuir para ativação de vias antimicrobianas em macrófagos.

Solution structure of the Mycobacterium tuberculosis EsxG·EsxH complex: functional implications and comparisons with other M. tuberculosis Esx family complexes.

Mycobacterium tuberculosis encodes five type VII secretion systems that are responsible for exporting a number of proteins, including members of the Esx family, which have been linked to tuberculosis pathogenesis and survival within host cells. The gene cluster encoding ESX-3 is regulated by the availability of iron and zinc, and secreted protein products such as the EsxG·EsxH complex have been associated with metal ion acquisition. EsxG and EsxH have previously been shown to form a stable 1:1 heterodimeric complex, and here we report the solution structure of the complex, which features a core four-helix bundle decorated at both ends by long, highly flexible, N- and C-terminal arms that contain a number of highly conserved residues. Despite clear similarities in the overall backbone fold to the EsxA·EsxB complex, the structure reveals some striking differences in surface features, including a potential protein interaction site on the surface of the EsxG·EsxH complex. EsxG·EsxH was also found to contain a specific Zn(2+) binding site formed from a cluster of histidine residues on EsxH, which are conserved across obligate mycobacterial pathogens including M. tuberculosis and Mycobacterium leprae. This site may reflect an essential role in zinc ion acquisition or point to Zn(2+)-dependent regulation of its interaction with functional partner proteins. Overall, the surface features of both the EsxG·EsxH and the EsxA·EsxB complexes suggest functions mediated via interactions with one or more target protein partners.

Blue rubber bleb nevus syndrome: prominent oral findings.

Blue rubber bleb nevus syndrome is a rare disorder characterized by multiple cutaneous venous malformations in association with visceral lesions, most commonly affecting the gastrointestinal tract. Oral cavity lesions occur in 59 to 64% of cases. We report a unique presentation of this syndrome in a 25-year-old male patient with prominent oral findings. This is a sporadic case, started during early childhood, progressively increasing in number and size. Oral vascular lesions were part of gastrointestinal involvement. Associated cardiac abnormalities were also observed. An early diagnosis of this syndrome is required as it gets complicated with bleeding, anemia and other systemic complications.

[Transferrin microheterogeneity in patients treated by maintenance haemodialysis]. / Mikroheterogennosc transferyny u chorych leczonych powtarzana hemodializa.

Transferrin (Tf) plays a crucial role in iron transport in the human body. In physiologically status Tf occurs in four variants. The concentration of Tf and its variants is changing in several conditions, especially during acute phase response, which may take place in end-stage renal disease (ESRD). The aim of this study was the assessment of the associations between changes of glycosylation of transferrin and selected blood cell count and iron metabolism parameters in ESRD patients treated by maintenance hemodialysis (mHD) with long lasting anaemia despite treatment by rHuEPO. Studies were carried out on a group of 55 ESRD patients treated by HD (ESRD group). The reference values were obtained from a group of 20 healthy volunteers (HV group). The Tf concentration was determined by the rocket immunoelectrophoresis according to Laurell. To estimate the microheterogenity of Tf the crossed affinoimmunoelectrophoresis according to Bog-Hansen was used. The concentration of selected hematological variables, and selected liver metabolism variables was assessed by routine laboratory tests, while the concentration of cytokines was measured by ELISA method, and selected acute phase proteins, i.e. C-reactive protein, by immunonephelometry method. Tf in studied sera was divided into four variants Tf1, Tf2, Tf3, Tf4. The percentage contents of variants in HV was Tf1=4%, Tf2=9%, Tf3=82%, Tf4=4%. In the sera of ESRD group the percentage contents of the Tf1, Tf2, Tf4 variants increased to 9%, 16%, 10% respectively, while the Tf3 variant decreased to 65%. Serum concentration of Tf was lower (1441 +/- 526 mg/L) in ESRD patients (p<0.0001) vs. 3539 +/- 955 mg/L in HV group. During this study we have revealed that the quantity and the quality of Tf is changing in ESRD patients. The observed correlations between concentration of Tf3 and selected hematological variables and also between Tf3 and rHuEPO, suggests that the decrease concentration of the Tf3 may lead to an impaired iron transport to the cells and may be one of the factors affecting anaemia in patients treated by HD and rHuEPO.

Metal ion transport and regulation in Mycobacterium tuberculosis.

The regulation of metal ion concentrations is central to the physiology of the interaction between pathogenic bacteria and their hosts. Apart from the NRAMP orthologue, MntH, metal ion transporters in Mycobacterium tuberculosis have not been studied. Mn, the physiological substrate of MntH in other bacteria, may play an important role as a structural and redox-active cofactor in a wide range of metabolic processes. Fe, Cu and Zn play structural and catalytic roles in metalloenzymes involved in oxidative stress responses. Fe and Mg are required for growth in macrophages. Genomic analyses reveal 28 sequences encoding a broad repertoire of putative metal ion transporters (or transporter subunits), representing 24% of all transporters in this organism. These comprise 8 families of secondary active transporters and 3 families of primary active transporters, including 12,P, type ATPases. Potential metal ion specificities include K+, Na+, Cu2+, Cd2+, Zn2+, Mn2+, Mg2+, Ca2+, Co2+, Ni2+, Fe2+/3+, Hg2+, AsO2- and AsO4(2-). 17 of these transporters are also encoded as complete open reading frames in Mycobacterium leprae, suggesting a role in intracellular survival. Iron transcriptionally regulates a diverse set of genes via the iron-dependent DNA-binding proteins, Fur and IdeR. Changes in Fe and Mg concentrations signal entry into the intracellular compartment and potentially trigger up-regulation of virulence determinants. The plethora of putative transport systems encoded by the M. tuberculosis genome contrasts strikingly with the paucity of experimental data on these systems. The detailed analysis of the temporal pattern of M. tuberculosis transporter gene expression during infection will provide important insights into the basic biology of intracellular parasitism and may help to shape novel therapeutic strategies.

[Influence of manganese on iron accumulation and flavinogenesis in yeast Debaryomyces hansenii]. / Bplyv marhantsiu na akumuliatsiiu zaliza i flavinohenez u drizhdzhiv Debaryomyces hansenii.

In response to MnCl2 introduction the yeast Debaryomyces hansenii shows the increase of intracellular iron content and riboflavin biosynthesis level. Under iron deficiency the yeast cells sensitivity to manganese ions increases in spite of redundant synthesis of riboflavin. High concentration of iron in the culture results in the lower toxicity of Mn2+. Iron accumulation favours the cell resistance to manganese. Manganese causes the redundant synthesis of riboflavin as well as iron accumulation in streptonigrin-resistant mutants with a lowered iron content in cells. The Mn(2+)-resistant strains are phenotypically similar to the previously described Rib80(-)-mutants of D. hansenii with high iron content and riboflavin biosynthesis level. This suggests that riboflavin redundant synthesis and iron accumulation in the yeast cells are regulated by a common Mn(2+)-depending factor. A new method for isolation of the Rib80(-)-mutants is described. The method is based on the positive selection of manganese-resistant clones.