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1.
Transfusion ; 56(8): 2085-99, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27184823

RESUMO

BACKGROUND: Babesia microti is a protozoan parasite responsible for the majority of reported cases of human babesiosis and a major risk to the blood supply. Laboratory screening of blood donors may help prevent transfusion-transmitted babesiosis but there is no Food and Drug Administration-approved screening method yet available. Development of a sensitive, specific, and highly automated B. microti antibody assay for diagnosis of acute babesiosis and blood screening could have an important impact on decreasing the health burden of B. microti infection. STUDY DESIGN AND METHODS: Herein, we take advantage of recent advances in B. microti genomic analyses, field surveys of the reservoir host, and human studies in endemic areas to apply a targeted immunomic approach to the discovery of B. microti antigens that serve as signatures of active or past babesiosis infections. Of 19 glycosylphosphatidylinositol (GPI)-anchored protein candidates (BmGPI1-19) identified in the B. microti proteome, 17 were successfully expressed, printed on a microarray chip, and used to screen sera from uninfected and B. microti-infected mice and humans to determine immune responses that are associated with active and past infection. RESULTS: Antibody responses to various B. microti BmGPI antigens were detected and BmGPI12 was identified as the best biomarker of infection that provided high sensitivity and specificity when used in a microarray antibody assay. CONCLUSION: BmGPI12 alone or in combination with other BmGPI proteins is a promising candidate biomarker for detection of B. microti antibodies that might be useful in blood screening to prevent transfusion-transmitted babesiosis.


Assuntos
Antígenos de Protozoários/imunologia , Babesia microti/imunologia , Babesiose/imunologia , Biomarcadores/análise , Animais , Genoma de Protozoário/genética , Humanos , Cinética , Camundongos , Análise Serial de Proteínas
2.
Mol Microbiol ; 99(6): 999-1014, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26585333

RESUMO

Phospholipid biosynthesis is critical for the development, differentiation and pathogenesis of several eukaryotic pathogens. Genetic studies have validated the pathway for phosphatidylethanolamine synthesis from phosphatidylserine catalyzed by phosphatidylserine decarboxylase enzymes (PSD) as a suitable target for development of antimicrobials; however no inhibitors of this class of enzymes have been discovered. We show that the Plasmodium falciparum PSD can restore the essential function of the yeast gene in strains requiring PSD for growth. Genetic, biochemical and metabolic analyses demonstrate that amino acids between positions 40 and 70 of the parasite enzyme are critical for proenzyme processing and decarboxylase activity. We used the essential role of Plasmodium PSD in yeast as a tool for screening a library of anti-malarials. One of these compounds is 7-chloro-N-(4-ethoxyphenyl)-4-quinolinamine, an inhibitor with potent activity against P. falciparum, and low toxicity toward mammalian cells. We synthesized an analog of this compound and showed that it inhibits PfPSD activity and eliminates Plasmodium yoelii infection in mice. These results highlight the importance of 4-quinolinamines as a novel class of drugs targeting membrane biogenesis via inhibition of PSD activity.


Assuntos
Antimaláricos/farmacologia , Carboxiliases/antagonistas & inibidores , Carboxiliases/metabolismo , Inibidores Enzimáticos/farmacologia , Malária Falciparum/tratamento farmacológico , Plasmodium falciparum/enzimologia , Animais , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Carboxiliases/genética , Clonagem Molecular , Feminino , Malária Falciparum/microbiologia , Camundongos , Testes de Sensibilidade Parasitária , Fosfatidilserinas/metabolismo , Plasmodium falciparum/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética
3.
PLoS Pathog ; 10(12): e1004520, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25522250

RESUMO

All pathogenesis and death associated with Plasmodium falciparum malaria is due to parasite-infected erythrocytes. Invasion of erythrocytes by P. falciparum merozoites requires specific interactions between host receptors and parasite ligands that are localized in apical organelles called micronemes. Here, we identify cAMP as a key regulator that triggers the timely secretion of microneme proteins enabling receptor-engagement and invasion. We demonstrate that exposure of merozoites to a low K+ environment, typical of blood plasma, activates a bicarbonate-sensitive cytoplasmic adenylyl cyclase to raise cytosolic cAMP levels and activate protein kinase A, which regulates microneme secretion. We also show that cAMP regulates merozoite cytosolic Ca2+ levels via induction of an Epac pathway and demonstrate that increases in both cAMP and Ca2+ are essential to trigger microneme secretion. Our identification of the different elements in cAMP-dependent signaling pathways that regulate microneme secretion during invasion provides novel targets to inhibit blood stage parasite growth and prevent malaria.


Assuntos
AMP Cíclico/fisiologia , Eritrócitos/parasitologia , Malária Falciparum/fisiopatologia , Merozoítos/crescimento & desenvolvimento , Plasmodium falciparum/patogenicidade , Cálcio/fisiologia , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Eritrócitos/efeitos dos fármacos , Eritrócitos/patologia , Humanos , Concentração de Íons de Hidrogênio , Merozoítos/fisiologia , Potássio/farmacologia , Transdução de Sinais/fisiologia
4.
Proc Natl Acad Sci U S A ; 110(45): 18262-7, 2013 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-24145416

RESUMO

Efficient transmission of Plasmodium species between humans and Anopheles mosquitoes is a major contributor to the global burden of malaria. Gametocytogenesis, the process by which parasites switch from asexual replication within human erythrocytes to produce male and female gametocytes, is a critical step in malaria transmission and Plasmodium genetic diversity. Nothing is known about the pathways that regulate gametocytogenesis and only few of the current drugs that inhibit asexual replication are also capable of inhibiting gametocyte development and blocking malaria transmission. Here we provide genetic and pharmacological evidence indicating that the pathway for synthesis of phosphatidylcholine in Plasmodium falciparum membranes from host serine is essential for parasite gametocytogenesis and malaria transmission. Parasites lacking the phosphoethanolamine N-methyltransferase enzyme, which catalyzes the limiting step in this pathway, are severely altered in gametocyte development, are incapable of producing mature-stage gametocytes, and are not transmitted to mosquitoes. Chemical screening identified 11 inhibitors of phosphoethanolamine N-methyltransferase that block parasite intraerythrocytic asexual replication and gametocyte differentiation in the low micromolar range. Kinetic studies in vitro as well as functional complementation assays and lipid metabolic analyses in vivo on the most promising inhibitor NSC-158011 further demonstrated the specificity of inhibition. These studies set the stage for further optimization of NSC-158011 for development of a class of dual activity antimalarials to block both intraerythrocytic asexual replication and gametocytogenesis.


Assuntos
Inibidores Enzimáticos/farmacologia , Malária Falciparum/transmissão , Metiltransferases/metabolismo , Plasmodium falciparum/enzimologia , Reprodução Assexuada/efeitos dos fármacos , Antimaláricos/farmacologia , Feminino , Imunofluorescência , Humanos , Malária Falciparum/enzimologia , Masculino , Metiltransferases/antagonistas & inibidores , Plasmodium falciparum/crescimento & desenvolvimento , Radiometria , Serina/metabolismo
5.
PLoS One ; 8(9): e72657, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24023759

RESUMO

Babesia microti is the primary causative agent of human babesiosis, an emerging pathogen that causes a malaria-like illness with possible fatal outcome in immunocompromised patients. The genome sequence of the B. microti R1 strain was reported in 2012 and revealed a distinct evolutionary path for this pathogen relative to that of other apicomplexa. Lacking from the first genome assembly and initial molecular analyses was information about the terminal ends of each chromosome, and both the exact number of chromosomes in the nuclear genome and the organization of the mitochondrial genome remained ambiguous. We have now performed various molecular analyses to characterize the nuclear and mitochondrial genomes of the B. microti R1 and Gray strains and generated high-resolution Whole Genome maps. These analyses show that the genome of B. microti consists of four nuclear chromosomes and a linear mitochondrial genome present in four different structural types. Furthermore, Whole Genome mapping allowed resolution of the chromosomal ends, identification of areas of misassembly in the R1 genome, and genomic differences between the R1 and Gray strains, which occur primarily in the telomeric regions. These studies set the stage for a better understanding of the evolution and diversity of this important human pathogen.


Assuntos
Babesia microti/genética , Genoma Mitocondrial/genética , Genoma de Protozoário/genética , Animais
6.
J Biol Chem ; 288(28): 20558-67, 2013 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-23729665

RESUMO

The human malaria parasite Plasmodium falciparum is absolutely dependent on the acquisition of host pantothenate for its development within human erythrocytes. Although the biochemical properties of this transport have been characterized, the molecular identity of the parasite-encoded pantothenate transporter remains unknown. Here we report the identification and functional characterization of the first protozoan pantothenate transporter, PfPAT, from P. falciparum. We show using cell biological, biochemical, and genetic analyses that this transporter is localized to the parasite plasma membrane and plays an essential role in parasite intraerythrocytic development. We have targeted PfPAT to the yeast plasma membrane and showed that the transporter complements the growth defect of the yeast fen2Δ pantothenate transporter-deficient mutant and mediates the entry of the fungicide drug, fenpropimorph. Our studies in P. falciparum revealed that fenpropimorph inhibits the intraerythrocytic development of both chloroquine- and pyrimethamine-resistant P. falciparum strains with potency equal or better than that of currently available pantothenate analogs. The essential function of PfPAT and its ability to deliver both pantothenate and fenpropimorph makes it an attractive target for the development and delivery of new classes of antimalarial drugs.


Assuntos
Membrana Celular/metabolismo , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Simportadores/metabolismo , Sequência de Aminoácidos , Animais , Antimaláricos/farmacologia , Cloroquina/farmacologia , Resistência a Medicamentos/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Eritrócitos/parasitologia , Eritrócitos/ultraestrutura , Teste de Complementação Genética , Células HEK293 , Interações Hospedeiro-Parasita/efeitos dos fármacos , Humanos , Malária Falciparum/parasitologia , Microscopia de Fluorescência , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Morfolinas/metabolismo , Morfolinas/farmacologia , Mutação , Ácido Pantotênico/metabolismo , Ácido Pantotênico/farmacologia , Filogenia , Plasmodium falciparum/genética , Plasmodium falciparum/fisiologia , Proteínas de Protozoários/genética , Pirimetamina/farmacologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Simportadores/classificação , Simportadores/genética
7.
Infect Immun ; 79(10): 4019-28, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21807906

RESUMO

As arguably the most successful parasite, Chlamydia is an obligate intracellular bacterium replicating inside a vacuole of eukaryotic host cells. The chlamydial vacuole does not fuse with the defense cell organelle lysosome. We previously showed that chlamydial infection increases markers of autophagy, an innate antimicrobial activity requiring lysosomal function. However, the work presented here demonstrates that p62, an autophagy protein that is degraded in lysosomes, either remained unchanged or increased in chlamydia-infected human epithelial, mouse fibroblast, and mouse macrophage cell lines. In addition, the activities of three lysosomal enzymes analyzed were diminished in chlamydia-infected macrophages. Bafilomycin A1 (BafA), a specific inhibitor of vacuolar ATPase (vATPase) required for lysosomal function, increased the growth of the human pathogen Chlamydia trachomatis (L2) in wild-type murine fibroblasts and macrophages but inhibited growth in the autophagy-deficient ATG5(-/-) fibroblasts. BafA exhibited only slight inhibition or no effect on L2 growth in multiple human genital epithelial cell lines. In contrast to L2, the mouse pathogen Chlamydia muridarum (MoPn) was consistently inhibited by BafA in all cell lines examined, regardless of species origin and autophagy status. Finally, L2 but not MoPn grew more efficiently in the ATG5(-/-) cells than in wild-type cells. These results suggest that there are two types of vATPase-bearing organelles that regulate chlamydial infection: one supports chlamydial infection, while the other plays a defensive role through autophagy when cells are artificially infected with certain chlamydiae that have not been adapted to the host species.


Assuntos
Autofagia , Infecções por Chlamydia/patologia , Chlamydia muridarum/patogenicidade , Chlamydia trachomatis/patogenicidade , Organelas/enzimologia , ATPases Vacuolares Próton-Translocadoras/metabolismo , Animais , Linhagem Celular , Infecções por Chlamydia/metabolismo , Infecções por Chlamydia/microbiologia , Chlamydia muridarum/efeitos dos fármacos , Chlamydia muridarum/crescimento & desenvolvimento , Chlamydia trachomatis/efeitos dos fármacos , Chlamydia trachomatis/crescimento & desenvolvimento , Inibidores Enzimáticos/farmacologia , Células Epiteliais/microbiologia , Fibroblastos/microbiologia , Células HeLa , Humanos , Lisossomos/enzimologia , Lisossomos/metabolismo , Macrolídeos/farmacologia , Macrófagos/microbiologia , Camundongos , ATPases Vacuolares Próton-Translocadoras/antagonistas & inibidores
8.
Microbiology (Reading) ; 157(Pt 9): 2569-2581, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21719536

RESUMO

Chlamydia trachomatis, an obligate intracellular bacterium, is a highly prevalent human pathogen. Hydroxamic-acid-based matrix metalloprotease inhibitors can effectively inhibit the pathogen both in vitro and in vivo, and have exhibited therapeutic potential. Here, we provide genome sequencing data indicating that peptide deformylase (PDF) is the sole target of the inhibitors in this organism. We further report molecular mechanisms that control chlamydial PDF (cPDF) expression and inhibition efficiency. In particular, we identify the σ66-dependent promoter that controls cPDF gene expression and demonstrate that point mutations in this promoter lead to resistance by increasing cPDF transcription. Furthermore, we show that substitution of two amino acids near the active site of the enzyme alters enzyme kinetics and protein stability.


Assuntos
Amidoidrolases/antagonistas & inibidores , Amidoidrolases/genética , Chlamydia trachomatis/genética , Regulação Bacteriana da Expressão Gênica , Região 5'-Flanqueadora , Amidoidrolases/química , Substituição de Aminoácidos , Sequência de Bases , Domínio Catalítico , Chlamydia trachomatis/efeitos dos fármacos , Chlamydia trachomatis/enzimologia , Dipeptídeos/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Inibidores Enzimáticos/farmacologia , Estabilidade Enzimática/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Cinética , Mutação/genética , Regiões Promotoras Genéticas , Transcrição Gênica
9.
FEMS Microbiol Lett ; 292(2): 240-9, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19187200

RESUMO

Autophagy, a eukaryotic cellular activity leading to the degradation of cellular components, serves as a defense mechanism against facultative intracellular bacteria as well as a growth niche for the obligate intracellular bacterium Coxiella burnetii. We here demonstrate that the obligate intracellular bacterial pathogen Chlamydia trachomatis lymphogranuloma venereum strongly induced autophagy in the middle of the chlamydial developmental cycle (24 h after infection), a time point with maximal level of chlamydial replication, but not during the early stages with low overall chlamydial metabolism (before 8 h). No autophagy induction was evident in cells exposed to heat- and UV-inactivated elementary bodies (EBs, the infectious form of Chlamydia) or to inocula from which EBs had been removed before inoculation. Blocking chlamydial development with chloramphenicol also prevented autophagy induction in cells infected with infectious EBs. It appears that autophagy is activated primarily in response to the metabolic stress consequent to chlamydial replication. However, autophagy-defective ATG5(-/-) cells supported chlamydial development as efficiently as autophagy-proficient ATG5(+/+) cells.


Assuntos
Autofagia , Chlamydia trachomatis/crescimento & desenvolvimento , Células Epiteliais/microbiologia , Células Epiteliais/fisiologia , Animais , Contagem de Colônia Microbiana , Células HeLa , Humanos , Camundongos , Camundongos Knockout
10.
J Biol Chem ; 281(24): 16691-9, 2006 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-16565079

RESUMO

Chlamydia trachomatis is an obligate intracellular bacterium responsible for a number of human diseases. The mechanism underlying the intracellular parasitology of Chlamydiae remains poorly understood. In searching for host factors required for chlamydial infection, we discovered that C. trachomatis growth was effectively inhibited with GM6001 and TAPI-0, two compounds known as specific inhibitors of matrix metalloproteases. The inhibition was independent of chlamydial entry of the cell, suggesting that the loss of extracellular metalloprotease activities of the host cell is unlikely to be the mechanism for the growth suppression. Nucleotide sequences of candidate metalloprotease genes remained unchanged in a chlamydial variant designated GR10, which had been selected for resistance to the inhibitors. Nevertheless, GR10 displayed a single base mutation in the presumable promoter region of the gene for peptide deformylase (PDF), a metal-dependent enzyme that removes the N-formyl group from newly synthesized bacterial proteins. The mutation correlated with an increased PDF expression level and resistance to actinonin, a known PDF inhibitor with antibacterial activity, as compared with the parental strain. Recombinant chlamydial PDF was covalently labeled with a hydroxamate-based molecular probe designated AspR1, which was developed for the detection of metalloproteases. The AspR1 labeling of the chlamydial PDF became significantly less efficient in the presence of excessive amounts of GM6001 and TAPI-0. Finally, the PDF enzyme activity was efficiently inhibited with GM6001 and TAPI-0. Taken together, our results suggest that the metalloprotease inhibitors suppress chlamydial growth by targeting the bacterial PDF. These findings have important biochemical and medical implications.


Assuntos
Amidoidrolases/metabolismo , Chlamydia trachomatis/metabolismo , Dipeptídeos/farmacologia , Inibidores Enzimáticos/farmacologia , Ácidos Hidroxâmicos/farmacologia , Metaloproteases/antagonistas & inibidores , Linhagem Celular Tumoral , Clonagem Molecular , Células HeLa , Humanos , Ácidos Hidroxâmicos/química , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína
11.
Mol Cell Biol ; 24(14): 6162-71, 2004 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15226420

RESUMO

The immunoglobulin M heavy-chain locus contains two poly(A) sites which are alternatively expressed during B-cell differentiation. Despite its promoter proximal location, the secretory poly(A) site is not expressed in undifferentiated cells. Crucial to the activation of the secretory poly(A) site during B-cell differentiation are changes in the binding of cleavage stimulatory factor 64K to GU-rich elements downstream of the poly(A) site. What regulates this change is not understood. The secretory poly(A) site contains two downstream GU-rich regions separated by a 29-nucleotide sequence. Both GU-rich regions are necessary for binding of the specific cleavage-polyadenylation complex. We demonstrate here that U1A binds two (AUGCN(1-3)C) motifs within the 29-nucleotide sequence and inhibits the binding of cleavage stimulatory factor 64K and cleavage at the secretory poly(A) site.


Assuntos
Cadeias Pesadas de Imunoglobulinas/metabolismo , Imunoglobulina M/metabolismo , Poliadenilação , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , Proteínas de Ligação a RNA , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Animais , Composição de Bases , Fator Estimulador de Clivagem/genética , Fator Estimulador de Clivagem/metabolismo , Sequência Conservada , Células HeLa , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Imunoglobulina M/genética , Mutação , Conformação de Ácido Nucleico , Ligação Proteica , Estrutura Terciária de Proteína , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Precursores de RNA/química , Precursores de RNA/genética , Ribonucleoproteína Nuclear Pequena U1/genética
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