RESUMEN
Leishmania species are intracellular protozoan pathogens that have evolved to successfully infect and deactivate host macrophages. How this deactivation is brought about is not completely understood. Recently, microRNAs (miRNAs) have emerged as ubiquitous regulators of macrophage gene expression that contribute to shaping the immune responses to intracellular pathogens. Conversely, several pathogens have evolved the ability to exploit host miRNA expression to manipulate host-cell phenotype. However, very little is known about the mechanisms used by intracellular pathogens to drive changes in host-cell miRNA abundance. Using miRNA expression profiling of Leishmania donovani-infected human macrophages, we show here that Leishmania infection induced a genome-wide down-regulation of host miRNAs. This repression occurred at the level of miRNA gene transcription, because the synthesis rates of primary miRNAs were significantly decreased in infected cells. miRNA repression depended on the host macrophage transcription factor c-Myc. Indeed, the expression of host c-Myc was markedly up-regulated by Leishmania infection, and c-Myc silencing reversed the miRNA suppression. Furthermore, c-Myc silencing significantly reduced intracellular survival of Leishmania, demonstrating that c-Myc is essential for Leishmania pathogenesis. Taken together, these findings identify c-Myc not only as being responsible for miRNA repression in Leishmania-infected macrophages but also as a novel and essential virulence factor by proxy that promotes Leishmania survival.
Asunto(s)
Leishmania donovani , Leishmaniasis Visceral/metabolismo , Macrófagos/metabolismo , MicroARNs/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Factores de Virulencia/metabolismo , Humanos , Leishmania donovani/metabolismo , Leishmania donovani/patogenicidad , Leishmaniasis Visceral/patología , Macrófagos/parasitología , Macrófagos/patologíaRESUMEN
Autophagy is essential for cell survival under stress and has also been implicated in host defense. Here, we investigated the interactions between Leishmania donovani, the main etiological agent of visceral leishmaniasis, and the autophagic machinery of human macrophages. Our results revealed that during early infection-and via activation of the Akt pathway-Leishmania actively inhibits the induction of autophagy. However, by 24 h, Leishmania switched from being an inhibitor to an overall inducer of autophagy. These findings of a dynamic, biphasic response were based on the accumulation of lipidated light chain 3 (LC3), an autophagosome marker, by Western blotting and confocal fluorescence microscopy. We also present evidence that Leishmania induces delayed host cell autophagy via a mechanism independent of reduced activity of the mechanistic target of rapamycin (mTOR). Notably, Leishmania actively inhibited mTOR-regulated autophagy even at later stages of infection, whereas there was a clear induction of autophagy via some other mechanism. In this context, we examined host inositol monophosphatase (IMPase), reduced levels of which have been implicated in mTOR-independent autophagy, and we found that IMPase activity is significantly decreased in infected cells. These findings indicate that Leishmania uses an alternative pathway to mTOR to induce autophagy in host macrophages. Finally, RNAi-mediated down-regulation of host autophagy protein 5 (ATG5) or autophagy protein 9A (ATG9A) decreased parasite loads, demonstrating that autophagy is essential for Leishmania survival. We conclude that Leishmania uses an alternative pathway to induce host autophagy while simultaneously inhibiting mTOR-regulated autophagy to fine-tune the timing and magnitude of this process and to optimize parasite survival.
Asunto(s)
Autofagia , Interacciones Huésped-Parásitos , Leishmania donovani/crecimiento & desarrollo , Leishmaniasis Visceral/fisiopatología , Proteína 5 Relacionada con la Autofagia/genética , Proteína 5 Relacionada con la Autofagia/metabolismo , Humanos , Leishmania donovani/genética , Leishmania donovani/fisiología , Leishmaniasis Visceral/genética , Leishmaniasis Visceral/metabolismo , Leishmaniasis Visceral/parasitología , Macrófagos/citología , Macrófagos/metabolismo , Macrófagos/parasitología , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/metabolismo , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Protozoa of the genus Leishmania infect macrophages in their mammalian hosts causing a spectrum of diseases known as the leishmaniases. The search for leishmania effectors that support macrophage infection is a focus of significant interest. One such candidate is leishmania chaperonin 10 (CPN10) which is secreted in exosomes and may have immunosuppressive properties. Here, we report for the first time that leishmania CPN10 localizes to the cytosol of infected macrophages. Next, we generated two genetically modified strains of Leishmania donovani (Ld): one strain overexpressing CPN10 (CPN10+++) and the second, a CPN10 single allele knockdown (CPN10+/-), as the null mutant was lethal. When compared with the wild-type (WT) parental strain, CPN10+/- Ld showed higher infection rates and parasite loads in human macrophages after 24 h of infection. Conversely, CPN10+++ Ld was associated with lower initial infection rates. This unexpected apparent gain-of-function for the knockdown could have been explained either by enhanced parasite internalization or by enhanced intracellular survival. Paradoxically, we found that CPN10+/- leishmania were more readily internalized than WT Ld, but also displayed significantly impaired intracellular survival. This suggests that leishmania CPN10 negatively regulates the rate of parasite uptake by macrophages while being required for intracellular survival. Finally, quantitative proteomics identified an array of leishmania proteins whose expression was positively regulated by CPN10. In contrast, many macrophage proteins involved in innate immunity were negatively regulated by CPN10. Taken together, these findings identify leishmania CPN10 as a novel effector with broad based effects on macrophage cell regulation and parasite survival.
Asunto(s)
Chaperonina 10/metabolismo , Endocitosis , Interacciones Huésped-Patógeno , Leishmania donovani/fisiología , Macrófagos/parasitología , Factores de Virulencia/metabolismo , Supervivencia Celular , Células Cultivadas , Chaperonina 10/genética , Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Leishmania donovani/genética , Leishmania donovani/patogenicidad , Proteómica , Proteínas Protozoarias/análisis , Factores de Virulencia/genéticaRESUMEN
Protozoan parasites of the genus Leishmania adapt to their arthropod and vertebrate hosts through the development of defined life cycle stages. Stage differentiation is triggered by environmental stress factors and has been linked to parasite chaperone activities. Using a null mutant approach we previously revealed important, nonredundant functions of the cochaperone cyclophilin 40 in L. donovani-infected macrophages. Here, we characterized in more detail the virulence defect of cyp40-/- null mutants. In vitro viability assays, infection tests using macrophages, and mixed infection experiments ruled out a defect of cyp40-/- parasites in resistance to oxidative and hydrolytic stresses encountered inside the host cell phagolysosome. Investigation of the CyP40-dependent proteome by quantitative 2D-DiGE analysis revealed up regulation of various stress proteins in the null mutant, presumably a response to compensate for the lack of CyP40. Applying transmission electron microscopy we showed accumulation of vesicular structures in the flagellar pocket of cyp40-/- parasites that we related to a significant increase in exosome production, a phenomenon previously linked to the parasite stress response. Together these data suggest that cyp40-/- parasites experience important intrinsic homeostatic stress that likely abrogates parasite viability during intracellular infection.
Asunto(s)
Ciclofilinas/deficiencia , Leishmania donovani/enzimología , Leishmaniasis Visceral/parasitología , Proteínas Protozoarias/genética , Animales , Peptidil-Prolil Isomerasa F , Ciclofilinas/genética , Electroforesis en Gel Bidimensional , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Leishmania donovani/genética , Leishmania donovani/crecimiento & desarrollo , Leishmania donovani/metabolismo , Macrófagos/parasitología , Ratones , Ratones Endogámicos C57BL , Mutación , Fenotipo , Proteínas Protozoarias/metabolismoRESUMEN
BACKGROUND: Leishmania use exosomes to communicate with their mammalian hosts and these secreted vesicles appear to contribute to pathogenesis by delivering protein virulence factors to macrophages. In other eukaryotes, exosomes were found to carry RNA cargo, such as mRNAs and small non-coding RNAs, capable of altering recipient cell phenotype. Whether leishmania exosomes also contain RNAs which they are able to deliver to bystander cells is not known. Here, we show that leishmania exosomes indeed contain RNAs and compare and contrast the RNA content of exosomes released by Leishmania donovani and Leishmania braziliensis. RESULTS: We purified RNA from exosomes collected from axenic amastigote culture supernatant and found that when compared with total leishmania RNA, exosomes mainly contained short RNA sequences. Exosomes with intact membranes were capable of protecting their RNA cargo from degradation by RNase. Moreover, exosome RNA cargo was delivered to host cell cytoplasm in vitro. Sequencing of exosomal RNA indicated that the majority of cargo sequences were derived from non-coding RNA species such as rRNA and tRNA. In depth analysis revealed the presence of tRNA-derived small RNAs, a novel RNA type with suspected regulatory functions. Northern blotting confirmed the specific and selective enrichment of tRNA-derived small RNAs in exosomes. We also identified a number of novel transcripts, which appeared to be specifically enriched in exosomes compared to total cell RNA. In addition, we observed the presence of sequences mapping to siRNA-coding regions in L. braziliensis , but not in L. donovani exosomes. CONCLUSIONS: These results show that leishmania exosomes are selectively and specifically enriched in small RNAs derived almost exclusively from non-coding RNAs. These exosomes are competent to deliver their cargo of novel, potential small regulatory RNAs to macrophages where they may influence parasite-host cell interactions. The remarkably high degree of congruence in exosomal RNA content between L. donovani and L. braziliensis, argues for the presence of a conserved mechanism for exosomal RNA packaging in leishmania. These findings open up a new avenue of research on non-canonical, small RNA pathways in this trypanosomatid, which may elucidate pathogenesis and identify novel therapeutic approaches.
Asunto(s)
Exosomas/genética , Leishmaniasis Visceral/genética , ARN Pequeño no Traducido/genética , ARN de Transferencia/genética , Animales , Secuencia de Bases , Leishmania braziliensis/genética , Leishmania braziliensis/patogenicidad , Leishmania donovani/genética , Leishmania donovani/patogenicidad , Leishmaniasis Visceral/parasitología , MicroARNs/genética , ARN Mensajero/genéticaRESUMEN
As part of an ongoing study to elucidate the SAR of bisindole alkaloid inhibitors against the evolutionary conserved MRSA pyruvate kinase (PK), we present here the synthesis and biological activity of six dihalogenated analogues of the naturally occurring sponge metabolite deoxytopsentin, including the naturally occurring dibromodeoxytopsentin. The most active compounds displayed potent low nanomolar inhibitory activity against MRSA PK with concomitant significant selectivity for MRSA PK over human PK orthologues. Computational studies suggest that these potent MRSA PK inhibitors occupy a region of the small interface of the enzyme tetramer where amino acid sequence divergence from common human PK orthologues may contribute to the observed selectivity.
Asunto(s)
Alcaloides Indólicos/síntesis química , Alcaloides Indólicos/farmacología , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Piruvato Quinasa/antagonistas & inhibidores , Secuencia de Aminoácidos , Humanos , Alcaloides Indólicos/química , Biología Marina , Staphylococcus aureus Resistente a Meticilina/enzimología , Estructura Molecular , Relación Estructura-ActividadRESUMEN
Using a genetic screen in yeast we found that Mycobacterium tuberculosis PE-PGRS62 was capable of disrupting yeast vacuolar protein sorting, suggesting effects on endosomal trafficking. To study the impact of PE-PGRS62 on macrophage function, we infected murine macrophages with Mycobacterium smegmatis expressing PE-PGRS62. Infected cells displayed phagosome maturation arrest. Phagosomes acquired Rab5, but displayed a significant defect in Rab7 and LAMP-1 acquisition. Macrophages infected with M. smegmatis expressing PE-PGRS62 also expressed two- to threefold less iNOS protein when compared with cells infected with wild-type bacteria. Consistent with this, cells infected with a Mycobacterium marinum transposon mutant for the PE-PGRS62 orthologue showed greater iNOS protein expression when compared to cells infected with wild-type organisms. Complementation restored the ability of the mutant to inhibit iNOS expression. No differences in iNOS transcript levels were observed, suggesting that PE-PGRS62 effects on iNOS expression occurred post-transcriptionally. Marked differences in colony morphology were also seen in M. smegmatis expressing PE-PGRS62 and in the M. marinum transposon mutant, suggesting that PE-PGRS62 may affect cell wall composition. These findings suggest that PE-PGRS62 supports virulence via inhibition of phagosome maturation and iNOS expression, and these phenotypes may be linked to effects on bacterial cell wall composition.
Asunto(s)
Proteínas Bacterianas/aislamiento & purificación , Macrófagos/metabolismo , Mycobacterium tuberculosis/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Animales , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Humanos , Macrófagos/microbiología , Macrófagos/patología , Ratones , Mycobacterium smegmatis/metabolismo , Mycobacterium smegmatis/patogenicidad , Mycobacterium tuberculosis/patogenicidad , Óxido Nítrico Sintasa de Tipo II/antagonistas & inhibidores , Fagosomas/metabolismo , Fagosomas/microbiologíaRESUMEN
Methicillin-resistant Staphylococcus aureus pyruvate kinase (MRSA PK) has recently been identified as a target for development of novel antibacterial agents. Testing a series of 1,2-bis(3-indolyl)ethanes against MRSA PK has led to the discovery of a potent inhibitor that is selective over human isoforms.
Asunto(s)
Antibacterianos/química , Inhibidores Enzimáticos/química , Etano/química , Piruvato Quinasa/antagonistas & inhibidores , Antibacterianos/metabolismo , Antibacterianos/farmacología , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Etano/metabolismo , Etano/farmacología , Humanos , Indoles/química , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Unión Proteica , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/metabolismo , Piruvato Quinasa/metabolismoRESUMEN
A novel series of bis-indoles derived from naturally occurring marine alkaloid 4 were synthesized and evaluated as inhibitors of methicillin-resistant Staphylococcus aureus (MRSA) pyruvate kinase (PK). PK is not only critical for bacterial survival which would make it a target for development of novel antibiotics, but it is reported to be one of the most highly connected 'hub proteins' in MRSA, and thus should be very sensitive to mutations and making it difficult for the bacteria to develop resistance. From the co-crystal structure of cis-3-4-dihydrohamacanthin B (4) bound to S. aureus PK we were able to identify the pharmacophore needed for activity. Consequently, we prepared simple direct linked bis-indoles such as 10b that have similar anti-MRSA activity as compound 4. Structure-activity relationship (SAR) studies were carried out on 10b and led us to discover more potent compounds such as 10c, 10d, 10k and 10 m with enzyme inhibiting activities in the low nanomolar range that effectively inhibited the bacteria growth in culture with minimum inhibitory concentrations (MIC) for MRSA as low as 0.5 µg/ml. Some potent PK inhibitors, such as 10b, exhibited attenuated antibacterial activity and were found to be substrates for an efflux mechanism in S. aureus. Studies comparing a wild type S. aureus with a construct (S. aureus LAC Δpyk::Erm(R)) that lacks PK activity confirmed that bactericidal activity of 10d was PK-dependant.
Asunto(s)
Staphylococcus aureus Resistente a Meticilina/química , Piruvato Quinasa/antagonistas & inhibidores , Piruvato Quinasa/uso terapéutico , Humanos , Staphylococcus aureus Resistente a Meticilina/aislamiento & purificación , Estructura Molecular , Infecciones Estafilocócicas/microbiología , Relación Estructura-ActividadRESUMEN
Leishmania disease expression has been linked to IL-10. In this study, we investigated the regulation of IL-10 production by macrophages infected with Leishmania donovani. Infection of either murine or human macrophages brought about selective phosphorylation of Akt-2 in a PI3K-dependent manner. These events were linked to phosphorylation and inactivation of glycogen synthase kinase-3ß (GSK-3ß) at serine 9, as the latter was abrogated by inhibition of either PI3K or Akt. One of the transcription factors that is negatively regulated by GSK-3ß is CREB, which itself positively regulates IL-10 expression. Infection of macrophages with leishmania induced phosphorylation of CREB at serine 133, and this was associated with enhanced CREB DNA binding activity and induction of IL-10. Similar to phosphorylation of GSK-3ß, both phosphorylation of CREB at serine 133 and CREB DNA binding activity were abrogated in cells treated with inhibitors of either PI3K or Akt prior to infection. Furthermore, disruption of this pathway either by inhibition of Akt or by overexpression of GSK-3ß markedly attenuated IL-10 production in response to leishmania. Thus, GSK-3ß negatively regulates myeloid cell IL-10 production in response to leishmania. Switching off GSK-3ß promotes disease pathogenesis.
Asunto(s)
Regulación hacia Abajo/inmunología , Glucógeno Sintasa Quinasa 3/inmunología , Interleucina-10/inmunología , Leishmania donovani/inmunología , Leishmaniasis Visceral/inmunología , Macrófagos/inmunología , Fosfatidilinositol 3-Quinasas/inmunología , Animales , Línea Celular , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/inmunología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Activación Enzimática/inmunología , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Humanos , Interleucina-10/biosíntesis , Leishmania donovani/metabolismo , Leishmaniasis Visceral/metabolismo , Macrófagos/metabolismo , Macrófagos/parasitología , Ratones , Ratones Endogámicos BALB C , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación/inmunología , Proteínas Proto-Oncogénicas c-akt/inmunología , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
Novel classes of antimicrobials are needed to address the emergence of multidrug-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA). We have recently identified pyruvate kinase (PK) as a potential novel drug target based upon it being an essential hub in the MRSA interactome (Cherkasov, A., Hsing, M., Zoraghi, R., Foster, L. J., See, R. H., Stoynov, N., Jiang, J., Kaur, S., Lian, T., Jackson, L., Gong, H., Swayze, R., Amandoron, E., Hormozdiari, F., Dao, P., Sahinalp, C., Santos-Filho, O., Axerio-Cilies, P., Byler, K., McMaster, W. R., Brunham, R. C., Finlay, B. B., and Reiner, N. E. (2011) J. Proteome Res. 10, 1139-1150; Zoraghi, R., See, R. H., Axerio-Cilies, P., Kumar, N. S., Gong, H., Moreau, A., Hsing, M., Kaur, S., Swayze, R. D., Worrall, L., Amandoron, E., Lian, T., Jackson, L., Jiang, J., Thorson, L., Labriere, C., Foster, L., Brunham, R. C., McMaster, W. R., Finlay, B. B., Strynadka, N. C., Cherkasov, A., Young, R. N., and Reiner, N. E. (2011) Antimicrob. Agents Chemother. 55, 2042-2053). Screening of an extract library of marine invertebrates against MRSA PK resulted in the identification of bis-indole alkaloids of the spongotine (A), topsentin (B, D), and hamacanthin (C) classes isolated from the Topsentia pachastrelloides as novel bacterial PK inhibitors. These compounds potently and selectively inhibited both MRSA PK enzymatic activity and S. aureus growth in vitro. The most active compounds, cis-3,4-dihyrohyrohamacanthin B (C) and bromodeoxytopsentin (D), were identified as highly potent MRSA PK inhibitors (IC(50) values of 16-60 nM) with at least 166-fold selectivity over human PK isoforms. These novel anti-PK natural compounds exhibited significant antibacterial activities against S. aureus, including MRSA (minimal inhibitory concentrations (MIC) of 12.5 and 6.25 µg/ml, respectively) with selectivity indices (CC(50)/MIC) >4. We also report the discrete structural features of the MRSA PK tetramer as determined by x-ray crystallography, which is suitable for selective targeting of the bacterial enzyme. The co-crystal structure of compound C with MRSA PK confirms that the latter is a target for bis-indole alkaloids. It elucidates the essential structural requirements for PK inhibitors in "small" interfaces that provide for tetramer rigidity and efficient catalytic activity. Our results identified a series of natural products as novel MRSA PK inhibitors, providing the basis for further development of potential novel antimicrobials.
Asunto(s)
Alcaloides/química , Antiinfecciosos/química , Proteínas Bacterianas , Inhibidores Enzimáticos/química , Indoles/química , Staphylococcus aureus Resistente a Meticilina/enzimología , Piruvato Quinasa , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/química , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Humanos , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Piruvato Quinasa/antagonistas & inhibidores , Piruvato Quinasa/química , Relación Estructura-ActividadRESUMEN
Specialized secretion systems are used by numerous bacterial pathogens to export virulence factors into host target cells. Leishmania and other eukaryotic intracellular pathogens also deliver effector proteins into host cells; however, the mechanisms involved have remained elusive. In this report, we identify exosome-based secretion as a general mechanism for protein secretion by Leishmania, and show that exosomes are involved in the delivery of proteins into host target cells. Comparative quantitative proteomics unambiguously identified 329 proteins in Leishmania exosomes, accounting for >52% of global protein secretion from these organisms. Our findings demonstrate that infection-like stressors (37 degrees C +/- pH 5.5) upregulated exosome release more than twofold and also modified exosome protein composition. Leishmania exosomes and exosomal proteins were detected in the cytosolic compartment of infected macrophages and incubation of macrophages with exosomes selectively induced secretion of IL-8, but not TNF-alpha. We thus provide evidence for an apparently broad-based mechanism of protein export by Leishmania. Moreover, we describe a mechanism for the direct delivery of Leishmania molecules into macrophages. These findings suggest that, like mammalian exosomes, Leishmania exosomes function in long-range communication and immune modulation.
Asunto(s)
Comunicación Celular , Exosomas/metabolismo , Leishmania donovani/citología , Leishmania donovani/metabolismo , Macrófagos/parasitología , Proteínas Protozoarias/metabolismo , Vías Secretoras , Animales , Biomarcadores/metabolismo , Medios de Cultivo Condicionados/metabolismo , Exosomas/ultraestructura , Espacio Extracelular/metabolismo , Respuesta al Choque Térmico , Concentración de Iones de Hidrógeno , Interleucina-8/metabolismo , Leishmania donovani/patogenicidad , Leishmania donovani/ultraestructura , Macrófagos/citología , Macrófagos/metabolismo , Macrófagos/ultraestructura , Modelos Biológicos , Transporte de Proteínas , Proteómica , Temperatura , Factores de Virulencia/metabolismoRESUMEN
The release of exosomes and other microvesicles by diverse prokaryotic and eukaryotic cells and organisms was first appreciated early in the 20th century. The functional properties of these organelles, however, have only recently been the focus of rigorous investigation. In this review, we discuss the release of microvesicles of varying complexity by diverse microbial pathogens. This includes vesicle secretion by Gram-negative bacteria, eukaryotic parasites of the kinetoplast lineage and opportunistic fungal pathogens of both the ascomycetes and basidiomycetes lineages. We also discuss vesicle release from mammalian cells brought about as a result of infection with bacteria, viruses and prions. In addition, we review the evidence showing that in their specific microenvironments, release of these organelles from diverse pathogens contributes to pathogenesis. Germane to this and based upon recent findings with Leishmania, we propose a model whereby exosome release by an intracellular pathogen serves as a general mechanism for effector molecule delivery from eukaryotic pathogen to host cell cytosol. These new findings linking exosomes and other microvesicles to infection biology have important implications for understanding the immune response to infection and for the design of research strategies aimed at the development of novel therapeutics and vaccines.
Asunto(s)
Ascomicetos/metabolismo , Basidiomycota/metabolismo , Exosomas/metabolismo , Bacterias Gramnegativas/metabolismo , Kinetoplastida/metabolismo , Mamíferos/metabolismo , Animales , Fenotipo , Factores de Virulencia/metabolismoRESUMEN
A novel series of hydrazones were synthesized and evaluated as inhibitors of methicillin-resistant Staphylococcus aureus (MRSA) pyruvate kinase (PK). PK has been identified as one of the most highly connected 'hub proteins' in MRSA. PK has been shown to be critical for bacterial survival which makes it a potential target for development of novel antibiotics and the high degree of connectivity implies it should be very sensitive to mutations and thus less able to develop resistance. PK is not unique to bacteria and thus a critical requirement for such a PK inhibitor would be that it does not inhibit the homologous human enzyme(s) at therapeutic concentrations. Several MRSA PK inhibitors (including 8d) were identified using in silico screening combined with enzyme assays and were found to be selective for bacterial enzyme compared to four human PK isoforms (M1, M2, R and L). However these lead compounds did not show significant inhibitory activity for MRSA growth presumably due to poor bacterial cell penetration. Structure-activity relationship (SAR) studies were carried out on 8d and led us to discover more potent compounds with enzyme inhibiting activities in the low nanomolar range and some were found to effectively inhibit bacteria growth in culture with minimum inhibitory concentrations (MIC) as low as 1 µg/mL. These inhibitors bind in two elongated flat clefts found at the minor interfaces in the homo-tetrameric enzyme complex and the observed SAR is in keeping with the size and electronic constraints of these binding sites. Access to the corresponding sites in the human enzyme is blocked.
Asunto(s)
Antiinfecciosos/química , Antiinfecciosos/farmacología , Staphylococcus aureus Resistente a Meticilina/enzimología , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Piruvato Quinasa/antagonistas & inhibidores , Humanos , Modelos Moleculares , Piruvato Quinasa/metabolismo , Relación Estructura-ActividadRESUMEN
We investigated the properties of leishmania exosomes with respect to influencing innate and adaptive immune responses. Exosomes from Leishmania donovani modulated human monocyte cytokine responses to IFN-γ in a bimodal fashion by promoting IL-10 production and inhibiting that of TNF-α. Moreover, these vesicles were inhibitory with respect to cytokine responses (IL-12p70, TNF-α, and IL-10) by human monocyte-derived dendritic cells. Exosomes from wild-type (WT) L. donovani failed to prime monocyte-derived dendritic cells to drive the differentiation of naive CD4 T cells into IFN-γ-producing Th1 cells. In contrast, vesicles from heat shock protein (HSP)100(-/-) L. donovani showed a gain-of-function and proinflammatory phenotype and promoted the differentiation of naive CD4 lymphocytes into Th1 cells. Proteomic analysis showed that exosomes from WT and HSP100(-/-) leishmania had distinct protein cargo, suggesting that packaging of proteins into exosomes is dependent in part on HSP100. Treatment of C57BL/6 mice with WT L. donovani exosomes prior to challenge with WT organisms exacerbated infection and promoted IL-10 production in the spleen. In contrast, HSP100(-/-) exosomes promoted spleen cell production of IFN-γ and did not adversely affect hepatic parasite burdens. Furthermore, the proparasitic properties of WT exosomes were not species specific because BALB/c mice exposed to Leishmania major exosomes showed increased Th2 polarization and exacerbation of disease in response to infection with L. major. These findings demonstrate that leishmania exosomes are predominantly immunosuppressive. Moreover, to our knowledge, this is the first evidence to suggest that changes in the protein cargo of exosomes may influence the impact of these vesicles on myeloid cell function.
Asunto(s)
Inmunidad Adaptativa/inmunología , Células Dendríticas/microbiología , Exosomas/inmunología , Inmunidad Innata/inmunología , Leishmania donovani/inmunología , Monocitos/microbiología , Animales , Antígenos de Protozoos/inmunología , Diferenciación Celular/inmunología , Separación Celular , Citocinas/biosíntesis , Citocinas/inmunología , Células Dendríticas/inmunología , Electroforesis en Gel Bidimensional , Endopeptidasa Clp , Citometría de Flujo , Proteínas de Choque Térmico/inmunología , Proteínas de Choque Térmico/metabolismo , Humanos , Leishmania donovani/metabolismo , Leishmaniasis/inmunología , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Monocitos/inmunología , Proteínas Protozoarias/inmunología , Proteínas Protozoarias/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Linfocitos T/citología , Linfocitos T/inmunologíaRESUMEN
The incorporation of nanoparticles (NPs) in industrial and biomedical applications has increased significantly in recent years, yet their hazardous and toxic effects have not been studied extensively. Here, we studied the effects of 24 nm silver NPs (AgNPs) on a panel of bacteria isolated from medical devices used in a hospital intensive care unit. The cytotoxic effects were evaluated in macrophages and the expression of the inflammatory cytokines IL-6, IL-10 and TNF-α were quantified. The effects of NPs on coagulation were tested in vitro in plasma-based assays. We demonstrated that 24 nm AgNPs were effective in suppressing the growth of clinically relevant bacteria with moderate to high levels of antibiotic resistance. The NPs had a moderate inhibitory effect when coagulation was initiated through the intrinsic pathway. However, these NPs are cytotoxic to macrophages and are able to elicit an inflammatory response. Thus, beneficial and potential harmful effects of 24 nm AgNPs on biomedical devices must be weighed in further studies in vivo. From the Clinical Editor: The authors of this study demonstrate that gallic acid reduced 24 nm Ag NPs are effective in suppressing growth of clinically relevant antibiotic resistant bacteria. However, these NPs also exhibit cytotoxic properties to macrophages and may trigger an inflammatory response. Thus, the balance of beneficial and potential harmful effects must be weighed carefully in further studies.
Asunto(s)
Antibacterianos/farmacología , Coagulación Sanguínea/efectos de los fármacos , Inflamación/patología , Nanopartículas del Metal/toxicidad , Plata/farmacología , Plata/toxicidad , Bacterias/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Línea Celular , Citocinas/metabolismo , Humanos , Mediadores de Inflamación/metabolismo , Luz , Macrófagos/citología , Macrófagos/efectos de los fármacos , Nanopartículas del Metal/ultraestructura , Pruebas de Sensibilidad Microbiana , Tamaño de la Partícula , Dispersión de RadiaciónRESUMEN
Mortality attributable to infection with methicillin-resistant Staphylococcus aureus (MRSA) has now overtaken the death rate for AIDS in the United States, and advances in research are urgently needed to address this challenge. We report the results of the systematic identification of protein-protein interactions for the hospital-acquired strain MRSA-252. Using a high-throughput pull-down strategy combined with quantitative proteomics to distinguish specific from nonspecific interactors, we identified 13,219 interactions involving 608 MRSA proteins. Consecutive analyses revealed that this protein interaction network (PIN) exhibits scale-free organization with the characteristic presence of highly connected hub proteins. When clinical and experimental antimicrobial targets were queried in the network, they were generally found to occupy peripheral positions in the PIN with relatively few interacting partners. In contrast, the hub proteins identified in this MRSA PIN that are essential for network integrity and stability have largely been overlooked as drug targets. Thus, this empirical MRSA-252 PIN provides a rich source for identifying critical proteins essential for network stability, many of which can be considered as prospective antimicrobial drug targets.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Staphylococcus aureus Resistente a Meticilina/química , Staphylococcus aureus Resistente a Meticilina/metabolismo , Mapeo de Interacción de Proteínas/métodos , Animales , Proteínas Bacterianas/genética , Humanos , Espectrometría de Masas , Proteómica/métodos , Proteínas Recombinantes de Fusión/metabolismo , Infecciones Estafilocócicas/metabolismoRESUMEN
Novel classes of antimicrobials are needed to address the challenge of multidrug-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA). Using the architecture of the MRSA interactome, we identified pyruvate kinase (PK) as a potential novel drug target based upon it being a highly connected, essential hub in the MRSA interactome. Structural modeling, including X-ray crystallography, revealed discrete features of PK in MRSA, which appeared suitable for the selective targeting of the bacterial enzyme. In silico library screening combined with functional enzymatic assays identified an acyl hydrazone-based compound (IS-130) as a potent MRSA PK inhibitor (50% inhibitory concentration [IC50] of 0.1 µM) with >1,000-fold selectivity over human PK isoforms. Medicinal chemistry around the IS-130 scaffold identified analogs that more potently and selectively inhibited MRSA PK enzymatic activity and S. aureus growth in vitro (MIC of 1 to 5 µg/ml). These novel anti-PK compounds were found to possess antistaphylococcal activity, including both MRSA and multidrug-resistant S. aureus (MDRSA) strains. These compounds also exhibited exceptional antibacterial activities against other Gram-positive genera, including enterococci and streptococci. PK lead compounds were found to be noncompetitive inhibitors and were bactericidal. In addition, mutants with significant increases in MICs were not isolated after 25 bacterial passages in culture, indicating that resistance may be slow to emerge. These findings validate the principles of network science as a powerful approach to identify novel antibacterial drug targets. They also provide a proof of principle, based upon PK in MRSA, for a research platform aimed at discovering and optimizing selective inhibitors of novel bacterial targets where human orthologs exist, as leads for anti-infective drug development.
Asunto(s)
Antibacterianos/farmacología , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/enzimología , Piruvato Quinasa/metabolismo , Secuencia de Aminoácidos , Electroforesis en Gel de Poliacrilamida , Humanos , Staphylococcus aureus Resistente a Meticilina/genética , Datos de Secuencia Molecular , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Piruvato Quinasa/química , Piruvato Quinasa/genética , Homología de Secuencia de AminoácidoRESUMEN
Parasites of Leishmania genus have developed sophisticated strategies allowing them to deactivate their host macrophage to promote their survival. It has become clear that miRNAs play important roles in shaping innate and adaptive immune responses toward pathogens. It is not surprising that several pathogens including Leishmania have evolved the ability to regulate host macrophage miRNA expression in order to manipulate host cell phenotypes to their advantage. However, very little is known about the mechanisms used by intracellular pathogens to drive changes in host cell miRNA abundance. In this review, Leishmania exploitation of macrophage transcription factor c-Myc as a critical proxy virulence factor to regulate abundance of macrophage miRNAs influencing macrophage physiology to promote its survival will be discussed.
Asunto(s)
Regulación de la Expresión Génica , Interacciones Huésped-Parásitos/genética , Leishmania/fisiología , Macrófagos/metabolismo , Macrófagos/parasitología , MicroARNs/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Animales , Humanos , MicroARNs/metabolismoRESUMEN
Novel antimicrobial targets are urgently needed to overcome rising antibiotic resistance of important human pathogens including methicillin-resistant Staphylococcus aureus (MRSA). Here we report the essentiality and kinetic properties of MRSA pyruvate kinase (PK). Targetron-mediated gene disruption demonstrated PK is essential for S. aureus growth and survival, suggesting that this protein may be a potential drug target. The presence of the pfk (6-phosphofructokinase)-pyk operon in MRSA252, and the nonessential nature of PFK shown by targetron, further emphasized the essential role of PK in cell viability. The importance of PK in bacterial growth was confirmed by showing that its enzymatic activity peaked during the logarithmic phase of S. aureus growth. PK from Staphylococcus and several other species of bacteria have an extra C-terminal domain (CT) containing a phosphoenolpyruvate (PEP) binding motif. To elucidate the possible structure and function of this sequence, the quaternary structures and kinetic properties of the full-length MRSA PK and truncated MRSA PK lacking the CT domain were characterized. Our results showed that (1) MRSA PK is an allosteric enzyme with homotetramer architecture activated by AMP or ribose 5-phosphate (R5P), but not by fructose 1,6-bisphosphate (FBP), which suggests a different mode of allosteric regulation when compared with human isozymes, (2) the CT domain is not required for the tetramerization of the enzyme; homotetramerization occurred in a truncated PK lacking the domain, (3) truncated enzyme exhibited high affinity toward both PEP and ADP and exhibited hyperbolic kinetics toward PEP in the presence of activators (AMP and R5P) consistent with kinetic properties of full-length enzyme, indicating that the CT domain is not required for substrate binding or allosteric regulation observed in the holoenzyme, (4) the kinetic efficiency (k(cat)/S(0.5)) of truncated enzyme was decreased by 24- and 16-fold, in ligand-free state, toward PEP and ADP, respectively, but was restored by 3-fold in AMP-bound state, suggesting that the sequence containing the CT domain (Gly(473)-Leu(585)) plays a substantial role in enzyme activity and comformational stability, and (5) full-length MRSA PK activity was stimulated at low concentrations of ATP (e.g., 1 mM) and inhibited by inorganic phosphate and high concentrations of FBP (10 mM) and ATP (e.g., >2.5 mM), whereas for truncated enzyme, stimulation at low concentrations of ATP was lost. These findings suggest that the CT domain is involved in maintaining the specificity of allosteric regulation of MRSA PK by AMP, R5P, and ATP. The CT extension also encodes a protein domain with homology to enzyme I of the Escherichia coli sugar-PTS system, suggesting that MRSA PK may also exert an important regulatory role in sugar transport metabolism. These findings yield new insights into MRSA PK function and mode of allosteric regulation which may aid in the development of clinically important drugs targeting this enzyme and further define the role of the extra C-terminal domain in modulating the enzyme's activity.