IL-1R1-Dependent Signals Improve Control of Cytosolic Virulent Mycobacteria In Vivo.

Mycobacterium tuberculosis infections claim more than a million lives each year, and better treatments or vaccines are required. A crucial pathogenicity factor is translocation from phagolysosomes to the cytosol upon phagocytosis by macrophages. Translocation from the phagolysosome to the cytosol is an ESX-1-dependent process, as previously shown in vitro Here, we show that in vivo, mycobacteria also translocate to the cytosol but mainly when host immunity is compromised. We observed only low numbers of cytosolic bacilli in mice, armadillos, zebrafish, and patient material infected with M. tuberculosis, M. marinum, or M. leprae In contrast, when innate or adaptive immunity was compromised, as in severe combined immunodeficiency (SCID) or interleukin-1 receptor 1 (IL-1R1)-deficient mice, significant numbers of cytosolic M. tuberculosis bacilli were detected in the lungs of infected mice. Taken together, in vivo, translocation to the cytosol of M. tuberculosis is controlled by adaptive immune responses as well as IL-1R1-mediated signals.IMPORTANCE For decades, Mycobacterium tuberculosis has been one of the deadliest pathogens known. Despite infecting approximately one-third of the human population, no effective treatment or vaccine is available. A crucial pathogenicity factor is subcellular localization, as M. tuberculosis can translocate from phagolysosome to the cytosol in macrophages. The situation in vivo is more complicated. In this study, we establish that high-level cytosolic escape of mycobacteria can indeed occur in vivo but mainly when host resistance is compromised. The IL-1 pathway is crucial for the control of the number of cytosolic mycobacteria. The establishment that immune signals result in the clearance of cells containing cytosolic mycobacteria connects two important fields, cell biology and immunology, which is vital for the understanding of the pathology of M. tuberculosis.

Cytosolic aromatic aldehyde dehydrogenase provides benzoic acid for xanthone biosynthesis in Hypericum.

Benzoic acid is a building block of a multitude of well-known plant natural products, such as paclitaxel and cocaine. Its simple chemical structure contrasts with its complex biosynthesis. Hypericum species are rich in polyprenylated benzoic acid-derived xanthones, which have received attention due to their biological impact on human health. The upstream biosynthetic sequence leading to xanthones is still incomplete. To supply benzoic acid for xanthone biosynthesis, Hypericum calycinum cell cultures use the CoA-dependent non-ß-oxidative pathway, which starts with peroxisomal cinnamate CoA-ligase (HcCNL). Here, we use the xanthone-producing cell cultures to identify the transcript for benzaldehyde dehydrogenase (HcBD), a pivotal player in the non-ß-oxidative pathways. In addition to benzaldehyde, the enzyme efficiently catalyzes the oxidation of trans-cinnamaldehyde in vitro. The enzymatic activity is strictly dependent on the presence of NAD+ as co-factor. HcBD is localized to the cytosol upon ectopic expression of reporter fusion constructs. HcBD oxidizes benzaldehyde, which moves across the peroxisome membrane, to form benzoic acid. Increases in the HcCNL and HcBD transcript levels precede the elicitor-induced xanthone accumulation. The current work addresses a crucial step in the yet incompletely understood CoA-dependent non-ß-oxidative route of benzoic acid biosynthesis. Addressing this step may offer a new biotechnological tool to enhance product formation in biofactories.

Quantitative measurement of cytosolic penetration using the chloroalkane penetration assay.

A major barrier for drug development is ensuring molecules can access intracellular targets. This is especially true for biomolecules, which are notoriously difficult to deliver to the cytosol. Many current methods for measuring the internalization of therapeutic biomolecules are largely indirect and qualitative, and they do not offer information about subcellular localization. We recently reported a new assay, called the ChloroAlkane Penetration Assay (CAPA), that addresses some of the drawbacks of existing methods. CAPA is high-throughput, quantitative, and compartment-specific, and can be used to monitor cytosolic penetration over time and under a variety of culture conditions. We have used CAPA to investigate the cytosolic localization of peptides, proteins, and oligonucleotides. In this chapter, we discuss the materials, protocols, and troubleshooting necessary to perform CAPA and appropriately analyze the data. We end with a discussion about the applications and limitations of CAPA, and we speculate on the potential of the assay and its variations.

Functional identification and subcellular localization of NAD kinase in the protozoan parasite Giardia intestinalis / Identificación funcional y localización subcelular de la NAD quinasa en el parásito protozoario Giardia intestinalis / Identificação funcional e localização subcelular da NAD quinase no parasita protozoário Giardia intestinalis

Abstract Nicotinamide adenine dinucleotide phosphate (NADP) is an essential biomolecule that participates in the redox homeostasis and synthesis of signaling compounds. NAD kinase (NADK) (EC 2.7.1.23 / 2.7.1.86) is the only enzyme capable of synthesizing NADP. This study offers an approach to the NADP metabolism in the parasite Giardia intestinalis, the etiological agent of giardiasis, a disease of high prevalence in America, Asia and Africa. Through bioinformatics tools a NADK enzyme candidate was identified, whose tertiary structure modeling demonstrated distinctive and universal motifs of characterized NADKs. The corresponding recombinant protein (His-GINADK) was expressed in Escherichia coli BL21 (DE3) and its partial purification was achieved by nickel affinity chromatography. Functional identification, which showed NADP synthesis, was completed through enzymatic assays evaluated by RP-HPLC. A cytosolic localization of the endogenous GINADK enzyme was observed in trophozoites throughout indirect immunofluorescence analysis, using polyclonal antibodies produced in mice by its immunization with the His-GINADK protein, purified from inclusion bodies. Taken together, our results contribute to the understanding of the NADP metabolism and the physiological role of NADK in the Giardia model.

Resumen El dinucleótido de adenina y nicotinamida fosfato (NADP) es una biomolécula esencial que participa en la homeostasis redox y en la síntesis de compuestos de señalización. La única enzima capaz de sintetizar NADP es la NAD Quinasa (NADK, EC 2.7.1.23 / 2.7.1.86). En este estudio se presenta un acercamiento al metabolismo del NADP en el parásito Giardia intestinalis, agente etiológico de la giardiasis, una enfermedad de alta prevalencia en América, África y Asia. Mediante herramientas bioinformáticas se identificó un candidato a NADK, cuya predicción a nivel de estructura terciaria mostró motivos característicos y universales de NADKs previamente caracterizadas. La proteína recombinante correspondiente (His-GINADK) se expresó en Escherichia coli BL21 (DE3) y se purificó parcialmente mediante cromatografía de afinidad a níquel. La síntesis de NADP por parte de la proteína His-GINADK se comprobó mediante ensayos enzimáticos evaluados por RP-HPLC. Adicionalmente, se determinó una localización subcelular citosólica en trofozoítos del parásito, empleando inmunofluorescencia indirecta y anticuerpos policlonales producidos en modelos murinos inmunizados con la proteína His-GINADK purificada a partir de cuerpos de inclusión. Los resultados obtenidos representan un avance en el entendimiento del metabolismo del NADP y de la importancia fisiológica de la NADK en el modelo de Giardia.

Resumo A nicotinamida adenina dinucleótido fosfato (NADP) é uma biomolécula essencial que participa na homeostase redox e na síntese de importantes compostos de sinalização. A NAD quinase (NADK) (EC 2.7.1.23 / 2.7.1.86) é a única enzima capaz de sintetizar o NADP. Este estudo apresenta uma abordagem do metabolismo do NADP no parasita Giardia intestinalis que causa giardíase, uma doença de alta prevalência na América, Ásia e África. Através de ferramentas de bioinformática, um candidato a enzima NADK foi identificado no parasita, cuja modelagem de estrutura terciária, demonstra motivos distintos e universais de NADKs caracterizadas. A correspondente proteína recombinante (His-GINADK) foi expressa em Escherichia coli BL21 (DE3) e a sua purificação parcial foi conseguida por cromatografia de afinidade com níquel. A identificação funcional, que mostrou a síntese de NADP, foi completada através de ensaios enzimáticos avaliados por RP-HPLC. Uma localização citosólica da enzima GINADK endógena foi observada em trofozoítos ao longo da análise de imunofluorescência indireta, utilizando anticorpos policlonais produzidos em camundongos, imunizados com a proteína His-GINADK purificada de corpos de inclusão. Em conjunto, nossos resultados contribuem para a compreensão do metabolismo do NADP e da importância fisiológica do NADK no modelo de Giardia.

Photocytotoxic copper(II) complexes of N-salicylyl-l-tryptophan and phenanthroline bases.

Four ternary copper(II) complexes of N-salicylyl-l-Tryptophan (Sal-TrpH) and phenanthroline bases of general formula [Cu(Sal-Trp)(L)], where L is 1,10-phenanthroline (phen, 1), dipyrido[3,2-d:2',3'-f]quinoxaline (dpq, 2), dipyrido[3,2-a:2',3'-c]phenazine (dppz, 3) and 2-(anthracen-1-yl)-1H-imidazo[4,5-f][1,10]phenanthroline (aip, 4), were synthesized and fully characterized. The complexes were evaluated for their affinity for biomolecules and photocytotoxic activities. Single crystal X-ray diffraction studies of complex 1 revealed that it has a square pyramidal CuN3O2 core with the phenolate oxygen of salicylaldehyde occupying the axial coordination site in the solid state. Complexes 1-4 displayed the Cu(II)-Cu(I) redox couples at ~-0.3 V vs. Ag/AgCl reference electrode in DMF-0.1 M [Bun4N](ClO4). A Cu(II)-based weak d-d band ~650 nm and a moderately strong ligand to metal charge transfer band at ~430 nm were observed in DMF-Tris-HCl buffer (pH 7.2) (1:4 v/v). The complexes are efficient binders to calf thymus DNA and model proteins such as bovine serum albumin and lysozyme. They cleave supercoiled plasmid DNA efficiently when exposed to 446 and 660 nm laser radiation. They are cytotoxic to HeLa (human cervical cancer) and MCF-7 (human breast cancer) cells showing significant enhancement of cytotoxicity upon photo-excitation with low energy visible light. The complexes are found to kill cancer cells through generation of reactive oxygen species (ROS) as confirmed by DCFDA (2',7'-dichlorofluorescin diacetate) assay. The apoptotic cell death induced by complex 4 was confirmed by Annexin V-Fluorescein isothiocyanate-Propidium iodide assay. Confocal microscopic images using 4 showed its primary cytosolic localization in the HeLa and MCF-7 cells.