RESUMO
Vector-borne diseases are a leading cause of death worldwide and pose a substantial unmet medical need. Pathogens binding to host extracellular proteins (the "exoproteome") represents a crucial interface in the etiology of vector-borne disease. Here, we used bacterial selection to elucidate host-microbe interactions in high throughput (BASEHIT)-a technique enabling interrogation of microbial interactions with 3,324 human exoproteins-to profile the interactomes of 82 human-pathogen samples, including 30 strains of arthropod-borne pathogens and 8 strains of related non-vector-borne pathogens. The resulting atlas revealed 1,303 putative interactions, including hundreds of pairings with potential roles in pathogenesis, including cell invasion, tissue colonization, immune evasion, and host sensing. Subsequent functional investigations uncovered that Lyme disease spirochetes recognize epidermal growth factor as an environmental cue of transcriptional regulation and that conserved interactions between intracellular pathogens and thioredoxins facilitate cell invasion. In summary, this interactome atlas provides molecular-level insights into microbial pathogenesis and reveals potential host-directed targets for next-generation therapeutics.
Assuntos
Interações Hospedeiro-Patógeno , Humanos , Animais , Doença de Lyme/microbiologia , Doenças Transmitidas por Vetores , Interações entre Hospedeiro e Microrganismos , Borrelia burgdorferi/patogenicidade , Borrelia burgdorferi/metabolismoRESUMO
Malaria caused by Plasmodium falciparum remains the leading single-agent cause of mortality in children1, yet the promise of an effective vaccine has not been fulfilled. Here, using our previously described differential screening method to analyse the proteome of blood-stage P. falciparum parasites2, we identify P. falciparum glutamic-acid-rich protein (PfGARP) as a parasite antigen that is recognized by antibodies in the plasma of children who are relatively resistant-but not those who are susceptible-to malaria caused by P. falciparum. PfGARP is a parasite antigen of 80 kDa that is expressed on the exofacial surface of erythrocytes infected by early-to-late-trophozoite-stage parasites. We demonstrate that antibodies against PfGARP kill trophozoite-infected erythrocytes in culture by inducing programmed cell death in the parasites, and that vaccinating non-human primates with PfGARP partially protects against a challenge with P. falciparum. Furthermore, our longitudinal cohort studies showed that, compared to individuals who had naturally occurring anti-PfGARP antibodies, Tanzanian children without anti-PfGARP antibodies had a 2.5-fold-higher risk of severe malaria and Kenyan adolescents and adults without these antibodies had a twofold-higher parasite density. By killing trophozoite-infected erythrocytes, PfGARP could synergize with other vaccines that target parasite invasion of hepatocytes or the invasion of and egress from erythrocytes.
Assuntos
Apoptose/imunologia , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Malária Falciparum/imunologia , Malária Falciparum/prevenção & controle , Parasitos/imunologia , Plasmodium falciparum/citologia , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Adolescente , Adulto , Animais , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/química , Antígenos de Protozoários/imunologia , Aotidae/imunologia , Aotidae/parasitologia , Caspases/metabolismo , Criança , Estudos de Coortes , DNA de Protozoário/química , DNA de Protozoário/metabolismo , Ativação Enzimática , Eritrócitos/parasitologia , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/química , Quênia , Vacinas Antimaláricas/imunologia , Malária Falciparum/parasitologia , Masculino , Camundongos , Parasitos/citologia , Parasitos/crescimento & desenvolvimento , Plasmodium falciparum/crescimento & desenvolvimento , Proteínas de Protozoários/química , Tanzânia , Trofozoítos/citologia , Trofozoítos/crescimento & desenvolvimento , Trofozoítos/imunologia , Vacúolos/imunologiaRESUMO
The primary antigenic and virulence determinant of the human malaria parasite Plasmodium falciparum is a variant surface protein called PfEMP1. Different forms of PfEMP1 are encoded by a multicopy gene family called var, and switching between active genes enables the parasites to evade the antibody response of their human hosts. var gene switching is key for the maintenance of chronic infections; however, what controls switching is unknown, although it has been suggested to occur at a constant frequency with little or no environmental influence. var gene transcription is controlled epigenetically through the activity of histone methyltransferases (HMTs). Studies in model systems have shown that metabolism and epigenetic control of gene expression are linked through the availability of intracellular S-adenosylmethionine (SAM), the principal methyl donor in biological methylation modifications, which can fluctuate based on nutrient availability. To determine whether environmental conditions and changes in metabolism can influence var gene expression, P. falciparum was cultured in media with altered concentrations of nutrients involved in SAM metabolism. We found that conditions that influence lipid metabolism induce var gene switching, indicating that parasites can respond to changes in their environment by altering var gene expression patterns. Genetic modifications that directly modified expression of the enzymes that control SAM levels similarly led to profound changes in var gene expression, confirming that changes in SAM availability modulate var gene switching. These observations directly challenge the paradigm that antigenic variation in P. falciparum follows an intrinsic, programed switching rate, which operates independently of any external stimuli.
Assuntos
Malária Falciparum , Parasitos , Animais , Humanos , Plasmodium falciparum/metabolismo , Parasitos/metabolismo , Regulação da Expressão Gênica , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Malária Falciparum/parasitologia , Variação Antigênica/genéticaRESUMO
Pathogens such as Plasmodium, Babesia, and Theileria invade and multiply within host red blood cells, leading to the pathological consequences of malaria, babesiosis, and theileriosis. Establishing continuous in vitro culture systems and suitable animal models is crucial for studying these pathogens. This review spotlights the Babesia duncani in culture-in mouse (ICIM) model as a promising resource for advancing research on the biology, pathogenicity, and virulence of intraerythrocytic parasites. The model offers practical benefits, encompassing well-defined culture conditions, ease of manipulation, and a well-annotated genome. Moreover, B. duncani serves as a surrogate system for drug discovery, facilitating the evaluation of new antiparasitic drugs in vitro and in animals, elucidating their modes of action, and uncovering potential resistance mechanisms. The B. duncani ICIM model thus emerges as a multifaceted tool with profound implications, promising advancements in our understanding of parasitic biology and shaping the development of future therapies.
Assuntos
Babesia , Babesiose , Modelos Animais de Doenças , Eritrócitos , Animais , Babesia/efeitos dos fármacos , Babesia/genética , Eritrócitos/parasitologia , Camundongos , Babesiose/tratamento farmacológico , Babesiose/parasitologia , Antiparasitários/uso terapêutico , Antiparasitários/farmacologia , Humanos , VirulênciaRESUMO
Decarboxylation of phosphatidylserine (PS) to form phosphatidylethanolamine by PS decarboxylases (PSDs) is an essential process in most eukaryotes. Processing of a malarial PSD proenzyme into its active alpha and beta subunits is by an autoendoproteolytic mechanism regulated by anionic phospholipids, with PS serving as an activator and phosphatidylglycerol (PG), phosphatidylinositol, and phosphatidic acid acting as inhibitors. The biophysical mechanism underlying this regulation remains unknown. We used solid phase lipid binding, liposome-binding assays, and surface plasmon resonance to examine the binding specificity of a processing-deficient Plasmodium PSD (PkPSDS308A) mutant enzyme and demonstrated that the PSD proenzyme binds strongly to PS and PG but not to phosphatidylethanolamine and phosphatidylcholine. The equilibrium dissociation constants (Kd) of PkPSD with PS and PG were 80.4 nM and 66.4 nM, respectively. The interaction of PSD with PS is inhibited by calcium, suggesting that the binding mechanism involves ionic interactions. In vitro processing of WT PkPSD proenzyme was also inhibited by calcium, consistent with the conclusion that PS binding to PkPSD through ionic interactions is required for the proenzyme processing. Peptide mapping identified polybasic amino acid motifs in the proenzyme responsible for binding to PS. Altogether, the data demonstrate that malarial PSD maturation is regulated through a strong physical association between PkPSD proenzyme and anionic lipids. Inhibition of the specific interaction between the proenzyme and the lipids can provide a novel mechanism to disrupt PSD enzyme activity, which has been suggested as a target for antimicrobials, and anticancer therapies.
Assuntos
Carboxiliases , Malária , Fosfolipídeos , Plasmodium , Motivos de Aminoácidos , Cálcio/metabolismo , Cálcio/farmacologia , Carboxiliases/antagonistas & inibidores , Carboxiliases/química , Carboxiliases/metabolismo , Precursores Enzimáticos/metabolismo , Lipossomos , Ácidos Fosfatídicos/metabolismo , Ácidos Fosfatídicos/farmacologia , Fosfatidilcolinas/metabolismo , Fosfatidilcolinas/farmacologia , Fosfatidiletanolaminas/metabolismo , Fosfatidiletanolaminas/farmacologia , Fosfatidilgliceróis/metabolismo , Fosfatidilgliceróis/farmacologia , Fosfatidilinositóis/metabolismo , Fosfatidilinositóis/farmacologia , Fosfatidilserinas/metabolismo , Fosfatidilserinas/farmacologia , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Fosfolipídeos/farmacologia , Ligação Proteica , Malária/parasitologia , Proteólise/efeitos dos fármacos , Ressonância de Plasmônio de Superfície , Plasmodium/enzimologiaRESUMO
Effective and safe therapies for the treatment of diseases caused by intraerythrocytic parasites are impeded by the rapid emergence of drug resistance and the lack of novel drug targets. One such disease is human babesiosis, which is a rapidly emerging tick-borne illness caused by Babesia parasites. In this study, we identified fosinopril, a phosphonate-containing, FDA-approved angiotensin converting enzyme (ACE) inhibitor commonly used as a prodrug for hypertension and heart failure, as a potent inhibitor of Babesia duncani parasite development within human erythrocytes. Cell biological and mass spectrometry analyses revealed that the conversion of fosinopril to its active diacid molecule, fosinoprilat, is essential for its antiparasitic activity. We show that this conversion is mediated by a parasite-encoded esterase, BdFE1, which is highly conserved among apicomplexan parasites. Parasites carrying the L238H mutation in the active site of BdFE1 failed to convert the prodrug to its active moiety and became resistant to the drug. Our data set the stage for the development of this class of drugs for the therapy of vector-borne parasitic diseases.
Assuntos
Babesia , Parasitos , Pró-Fármacos , Animais , Humanos , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Fosinopril/farmacologia , Pró-Fármacos/farmacologia , Esterases/metabolismoRESUMO
Babesia and Plasmodium pathogens, the causative agents of babesiosis and malaria, are vector-borne intraerythrocytic protozoan parasites, posing significant threats to both human and animal health. The widespread resistance exhibited by these pathogens to various classes of antiparasitic drugs underscores the need for the development of novel and more effective therapeutic strategies. Antifolates have long been recognized as attractive antiparasitic drugs as they target the folate pathway, which is essential for the biosynthesis of purines and pyrimidines, and thus is vital for the survival and proliferation of protozoan parasites. More efficacious and safer analogs within this class are needed to overcome challenges due to resistance to commonly used antifolates, such as pyrimethamine, and to address liabilities associated with the dihydrotriazines, WR99210 and JPC-2067. Here, we utilized an in vitro culture condition suitable for the continuous propagation of Babesia duncani, Babesia divergens, Babesia MO1, and Plasmodium falciparum in human erythrocytes to screen a library of 50 dihydrotriazines and 29 biguanides for their efficacy in vitro and compared their potency and therapeutic indices across different species and isolates. We identified nine analogs that inhibit the growth of all species, including the P. falciparum pyrimethamine-resistant strain HB3, with IC50 values below 10 nM, and display excellent in vitro therapeutic indices. These compounds hold substantial promise as lead antifolates for further development as broad-spectrum antiparasitic drugs.
RESUMO
Pantothenate kinase-associated neurodegeneration (PKAN) is an incurable rare genetic disorder of children and young adults caused by mutations in the PANK2 gene, which encodes an enzyme critical for the biosynthesis of coenzyme A. Although PKAN affects only a small number of patients, it shares several hallmarks of more common neurodegenerative diseases of older adults such as Alzheimer's disease and Parkinson's disease. Advances in etiological understanding and treatment of PKAN could therefore have implications for our understanding of more common diseases and may shed new lights on the physiological importance of coenzyme A, a cofactor critical for the operation of various cellular metabolic processes. The large body of knowledge that accumulated over the years around PKAN pathology, including but not limited to studies of various PKAN models and therapies, has contributed not only to progress in our understanding of the disease but also, importantly, to the crystallization of key questions that guide future investigations of the disease. In this review, we will summarize this knowledge and demonstrate how it forms the backdrop to new avenues of research.
Assuntos
Doenças Neurodegenerativas , Neurodegeneração Associada a Pantotenato-Quinase , Animais , Coenzima A/genética , Coenzima A/metabolismo , Humanos , Mutação , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/terapia , Neurodegeneração Associada a Pantotenato-Quinase/genética , Neurodegeneração Associada a Pantotenato-Quinase/metabolismo , Neurodegeneração Associada a Pantotenato-Quinase/patologia , Neurodegeneração Associada a Pantotenato-Quinase/terapia , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismoRESUMO
BACKGROUND: Protozoan parasites are known to attach specific and diverse group of proteins to their plasma membrane via a GPI anchor. In malaria parasites, GPI-anchored proteins (GPI-APs) have been shown to play an important role in host-pathogen interactions and a key function in host cell invasion and immune evasion. Because of their immunogenic properties, some of these proteins have been considered as malaria vaccine candidates. However, identification of all possible GPI-APs encoded by these parasites remains challenging due to their sequence diversity and limitations of the tools used for their characterization. METHODS: The FT-GPI software was developed to detect GPI-APs based on the presence of a hydrophobic helix at both ends of the premature peptide. FT-GPI was implemented in C ++and applied to study the GPI-proteome of 46 isolates of the order Haemosporida. Using the GPI proteome of Plasmodium falciparum strain 3D7 and Plasmodium vivax strain Sal-1, a heuristic method was defined to select the most sensitive and specific FT-GPI software parameters. RESULTS: FT-GPI enabled revision of the GPI-proteome of P. falciparum and P. vivax, including the identification of novel GPI-APs. Orthology- and synteny-based analyses showed that 19 of the 37 GPI-APs found in the order Haemosporida are conserved among Plasmodium species. Our analyses suggest that gene duplication and deletion events may have contributed significantly to the evolution of the GPI proteome, and its composition correlates with speciation. CONCLUSION: FT-GPI-based prediction is a useful tool for mining GPI-APs and gaining further insights into their evolution and sequence diversity. This resource may also help identify new protein candidates for the development of vaccines for malaria and other parasitic diseases.
Assuntos
Proteínas Ligadas por GPI , Plasmodium falciparum , Plasmodium vivax , Proteoma , Proteínas de Protozoários , Proteínas Ligadas por GPI/genética , Plasmodium falciparum/genética , Plasmodium vivax/genética , Proteoma/análise , Proteínas de Protozoários/genéticaRESUMO
Human babesiosis is an emerging tick-borne malaria-like illness caused by Babesia parasites following their development in erythrocytes. Here, we show that a mutation in the Babesia microti mitochondrial cytochrome b (Cytb) that confers resistance to the antibabesial drug ELQ-502 decreases parasite fitness in the arthropod vector. Interestingly, whereas the mutant allele does not affect B. microti fitness during the mammalian blood phase of the parasite life cycle and is genetically stable as parasite burden increases, ELQ-502-resistant mutant parasites developing in the tick vector are genetically unstable with a high rate of the wild-type allele emerging during the nymphal stage. Furthermore, we show that B. microti parasites with this mutation are transmitted from the tick to the host, raising the possibility that the frequency of Cytb resistance mutations may be decreased by passage through the tick vector, but could persist in the environment if present when ticks feed.
Assuntos
Antiprotozoários/farmacologia , Babesia/genética , Babesiose/tratamento farmacológico , Babesiose/transmissão , Citocromos b/genética , Resistência a Medicamentos/genética , Ixodes , Quinolonas/farmacologia , Carrapatos , Animais , Babesia/efeitos dos fármacos , Babesia/crescimento & desenvolvimento , Babesiose/diagnóstico , Citocromos b/efeitos dos fármacos , Eritrócitos/parasitologia , Humanos , Mutação , ParasitosRESUMO
Human babesiosis is a malaria-like illness caused by tick-borne intraerythrocytic Babesia parasites of the Apicomplexa phylum. Whereas several species of Babesia can cause severe disease in humans, the ability to propagate Babesia duncani both in vitro in human erythrocytes and in mice makes it a unique pathogen to study Babesia biology and pathogenesis. Here we report an optimized B. duncani in culture-in mouse (ICIM) model that combines continuous in vitro culture of the parasite with a precise model of lethal infection in mice. We demonstrate that B. duncani-infected erythrocytes as well as free merozoites can cause lethal infection in C3H/HeJ mice. Highly reproducible parasitemia and survival outcomes could be established using specific parasite loads in different mouse genetic backgrounds. Using the ICIM model, we discovered 2 new endochin-like quinolone prodrugs (ELQ-331 and ELQ-468) that alone or in combination with atovaquone are highly efficacious against B. duncani and Babesia microti.
Assuntos
Babesia , Parasitos , Pró-Fármacos , Quinolonas , Carrapatos , Animais , Atovaquona/farmacologia , Babesia/genética , Humanos , Camundongos , Camundongos Endogâmicos C3H , VirulênciaRESUMO
The apicomplexan pathogen Babesia microti is responsible for most cases of human babesiosis worldwide. The disease, which presents as a malaria-like illness, is potentially fatal in immunocompromised or elderly patients, making the need for its accurate and early diagnosis an urgent public health concern. B. microti is transmitted primarily by Ixodes ticks but can also be transmitted via blood transfusion. The parasite completes its asexual reproduction in the host red blood cell, where each invading merozoite develops and multiplies to produce four daughter parasites. While various techniques, such as microscopy, PCR, and indirect fluorescence, have been used over the years for babesiosis diagnosis, detection of the secreted B. microti immunodominant antigen BmGPI12 using specific polyclonal antibodies was found to be the most effective method for the diagnosis of active infection and for evaluation of clearance following drug treatment. Here, we report the development of a panel of 16 monoclonal antibodies against BmGPI12. These antibodies detected secreted BmGPI12 in the plasma of infected humans. Antigen capture assays identified a combination of two monoclonal antibodies, 4C8 and 1E11, as a basis for a monoclonal antibody-based BmGPI12 capture assay (mGPAC) to detect active B. microti infection. Using a collection of 105 previously characterized human plasma samples, the mGPAC assay showed 97.1% correlation with RNA-based PCR (transcription-mediated amplification [TMA]) for positive and negative samples. The mGPAC assay also detected BmGPI12 in the plasma of six babesiosis patients at the time of diagnosis but not in three matched posttreatment samples. The mGPAC assay could thus be used alone or in combination with other assays for accurate detection of active B. microti infection.
Assuntos
Babesia microti , Babesiose , Idoso , Anticorpos Monoclonais , Antígenos de Protozoários , Babesia microti/genética , Babesiose/diagnóstico , Humanos , RNARESUMO
The positioning of chromosomes in the nucleus of a eukaryotic cell is highly organized and has a complex and dynamic relationship with gene expression. In the human malaria parasite Plasmodium falciparum, the clustering of a family of virulence genes correlates with their coordinated silencing and has a strong influence on the overall organization of the genome. To identify conserved and species-specific principles of genome organization, we performed Hi-C experiments and generated 3D genome models for five Plasmodium species and two related apicomplexan parasites. Plasmodium species mainly showed clustering of centromeres, telomeres, and virulence genes. In P. falciparum, the heterochromatic virulence gene cluster had a strong repressive effect on the surrounding nuclear space, while this was less pronounced in Plasmodium vivax and Plasmodium berghei, and absent in Plasmodium yoelii In Plasmodium knowlesi, telomeres and virulence genes were more dispersed throughout the nucleus, but its 3D genome showed a strong correlation with gene expression. The Babesia microti genome showed a classical Rabl organization with colocalization of subtelomeric virulence genes, while the Toxoplasma gondii genome was dominated by clustering of the centromeres and lacked virulence gene clustering. Collectively, our results demonstrate that spatial genome organization in most Plasmodium species is constrained by the colocalization of virulence genes. P. falciparum and P. knowlesi, the only two Plasmodium species with gene families involved in antigenic variation, are unique in the effect of these genes on chromosome folding, indicating a potential link between genome organization and gene expression in more virulent pathogens.
Assuntos
Genoma de Protozoário/genética , Heterocromatina/genética , Malária Falciparum/genética , Plasmodium falciparum/genética , Animais , Centrômero/genética , Regulação da Expressão Gênica/genética , Genômica , Humanos , Malária Falciparum/parasitologia , Plasmodium berghei/genética , Plasmodium berghei/patogenicidade , Plasmodium falciparum/patogenicidade , Plasmodium knowlesi/genética , Plasmodium knowlesi/patogenicidade , Plasmodium vivax/genética , Plasmodium vivax/patogenicidade , Telômero/genética , Toxoplasma/genética , Toxoplasma/patogenicidadeRESUMO
Human PANK1, PANK2, and PANK3 genes encode several pantothenate kinase isoforms that catalyze the phosphorylation of vitamin B5 (pantothenic acid) to phosphopantothenate, a critical step in the biosynthesis of the major cellular cofactor, Coenzyme A (CoA). Mutations in the PANK2 gene, which encodes the mitochondrial pantothenate kinase (PanK) isoform, have been linked to pantothenate-kinase associated neurodegeneration (PKAN), a debilitating and often fatal progressive neurodegeneration of children and young adults. While the biochemical properties of these enzymes have been well-characterized in vitro, their expression in a model organism such as yeast in order to probe their function under cellular conditions have never been achieved. Here we used three yeast mutants carrying missense mutations in the yeast PanK gene, CAB1, which are associated with defective growth at high temperature and iron, mitochondrial dysfunction, increased iron content, and oxidative stress, to assess the cellular function of human PANK genes and functional conservation of the CoA-controlled processes between humans and yeast. Overexpression of human PANK1 and PANK3 in these mutants restored normal cellular activity whereas complementation with PANK2 was partial and could only be achieved with an isoform, PanK2mtmΔ, lacking the mitochondrial transit peptide. These data, which demonstrate functional conservation of PanK activity between humans and yeast, set the stage for the use of yeast as a model system to investigate the impact of PKAN-associated mutations on the metabolic pathways altered in this disease.
Assuntos
Estresse Oxidativo , Neurodegeneração Associada a Pantotenato-Quinase , Saccharomyces cerevisiae , Humanos , Homeostase , Ferro/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Estresse Oxidativo/genética , Neurodegeneração Associada a Pantotenato-Quinase/metabolismo , Ácido Pantotênico , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Phosphatidylserine decarboxylases (PSDs) catalyze the conversion of phosphatidylserine (PS) to phosphatidylethanolamine (PE), a critical step in membrane biogenesis and a potential target for development of antimicrobial and anti-cancer drugs. PSD activity has typically been quantified using radioactive substrates and products. Recently, we described a fluorescence-based assay that measures the PSD reaction using distyrylbenzene-bis-aldehyde (DSB-3), whose reaction with PE produces a fluorescence signal. However, DSB-3 is not widely available and also reacts with PSD's substrate, PS, producing an adduct with lower fluorescence yield than that of PE. Here, we report a new fluorescence-based assay that is specific for PSD and in which the presence of PS causes only negligible background. This new assay uses 1,2-diacetyl benzene/ß-mercaptoethanol, which forms a fluorescent iso-indole-mercaptide conjugate with PE. PE detection with this method is very sensitive and comparable with detection by radiochemical methods. Model reactions examining adduct formation with ethanolamine produced stable products of exact masses (m/z) of 342.119 and 264.105. The assay is robust, with a signal/background ratio of 24, and can readily detect formation of 100 pmol of PE produced from Escherichia coli membranes, Candida albicans mitochondria, or HeLa cell mitochondria. PSD activity can easily be quantified by sequential reagent additions in 96- or 384-well plates, making it readily adaptable to high-throughput screening for PSD inhibitors. This new assay now enables straightforward large-scale screening for PSD inhibitors against pathogenic fungi, antibiotic-resistant bacteria, and neoplastic mammalian cells.
Assuntos
Carboxiliases/análise , Corantes Fluorescentes/síntese química , Espectrometria de Fluorescência/métodos , Acetofenonas/química , Candida albicans/metabolismo , Carboxiliases/metabolismo , Membrana Celular/metabolismo , Etanolamina , Fluorescência , Células HeLa , Ensaios de Triagem em Larga Escala , Humanos , Mercaptoetanol/química , Mitocôndrias , Fosfatidiletanolaminas/metabolismo , Fosfatidilserinas/metabolismo , Estirenos/químicaRESUMO
In fungi, ergosterol is an essential component of the plasma membrane. Its biosynthesis from acetyl-CoA is the primary target of the most commonly used antifungal drugs. Here, we show that the pantothenate kinase Cab1p, which catalyzes the first step in the metabolism of pantothenic acid for CoA biosynthesis in budding yeast (Saccharomyces cerevisiae), significantly regulates the levels of sterol intermediates and the activities of ergosterol biosynthesis-targeting antifungals. Using genetic and pharmacological analyses, we show that altered pantothenate utilization dramatically alters the susceptibility of yeast cells to ergosterol biosynthesis inhibitors. Genome-wide transcription and MS-based analyses revealed that this regulation is mediated by changes both in the expression of ergosterol biosynthesis genes and in the levels of sterol intermediates. Consistent with these findings, drug interaction experiments indicated that inhibition of pantothenic acid utilization synergizes with the activity of the ergosterol molecule-targeting antifungal amphotericin B and antagonizes that of the ergosterol pathway-targeting antifungal drug terbinafine. Our finding that CoA metabolism controls ergosterol biosynthesis and susceptibility to antifungals could set the stage for the development of new strategies to manage fungal infections and to modulate the potency of current drugs against drug-sensitive and -resistant fungal pathogens.
Assuntos
Farmacorresistência Fúngica/genética , Ergosterol/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Esteróis/metabolismo , Anfotericina B/farmacologia , Antifúngicos/farmacologia , Coenzima A/biossíntese , Coenzima A/efeitos dos fármacos , Ergosterol/biossíntese , Ergosterol/genética , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Genoma Fúngico/efeitos dos fármacos , Ácido Pantotênico/biossíntese , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Esteróis/biossíntese , Terbinafina/farmacologiaRESUMO
Phosphatidylserine decarboxylases (PSDs) catalyze the decarboxylation of phosphatidylserine to generate phosphatidylethanolamine, a critical step in phospholipid metabolism in both prokaryotes and eukaryotes. Most PSDs are membrane-bound, and classical radioisotope-based assays for determining their activity in vitro are not suitable for high-throughput drug screening. The finding that the PkPSD from Plasmodium knowlesi can be purified in a soluble and active form and the recent development of a fluorescence-based distyrylbenzene-bis-aldehyde (DSB-3) assay to measure PSD activity in vitro have laid the groundwork for screening chemical libraries for PSD inhibitors. Using this assay, here we conducted a high-throughput screen of a structurally diverse 130,858-compound library against PkPSD. Further characterization of the hits identified in this screening yielded five PkPSD inhibitors with IC50 values ranging from 3.1 to 42.3 µm Lead compounds were evaluated against the pathogenic yeast Candida albicans in the absence or presence of exogenous ethanolamine, and YU253467 and YU254403 were identified as inhibiting both native C. albicans PSD mitochondrial activity and C. albicans growth, with an MIC50 of 22.5 and 15 µg/ml without ethanolamine and an MIC50 of 75 and 60 µg/ml with ethanolamine, respectively. Together, these results provide the first proof of principle for the application of DSB-3-based fluorescent readouts in high-throughput screening for PSD inhibitors. The data set the stage for future analyses to identify more selective and potent PSD inhibitors with antimicrobial or antitumor activities.
Assuntos
Carboxiliases/antagonistas & inibidores , Inibidores Enzimáticos/análise , Corantes Fluorescentes/química , Ensaios de Triagem em Larga Escala , Estirenos/química , Candida albicans/efeitos dos fármacos , Carboxiliases/genética , Carboxiliases/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Etanolamina/farmacologia , Humanos , Concentração Inibidora 50 , Fosfatidilserinas/metabolismo , Plasmodium knowlesi/enzimologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
The life cycle of malaria parasites in both their mammalian host and mosquito vector consists of multiple developmental stages that ensure proper replication and progeny survival. The transition between these stages is fueled by nutrients scavenged from the host and fed into specialized metabolic pathways of the parasite. One such pathway is used by Plasmodium falciparum, which causes the most severe form of human malaria, to synthesize its major phospholipids, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. Much is known about the enzymes involved in the synthesis of these phospholipids, and recent advances in genetic engineering, single-cell RNA-Seq analyses, and drug screening have provided new perspectives on the importance of some of these enzymes in parasite development and sexual differentiation and have identified targets for the development of new antimalarial drugs. This Minireview focuses on two phospholipid biosynthesis enzymes of P. falciparum that catalyze phosphoethanolamine transmethylation (PfPMT) and phosphatidylserine decarboxylation (PfPSD) during the blood stages of the parasite. We also discuss our current understanding of the biochemical, structural, and biological functions of these enzymes and highlight efforts to use them as antimalarial drug targets.