Fusion between Leishmania amazonensis and Leishmania major Parasitophorous Vacuoles: Live Imaging of Coinfected Macrophages

Protozoan parasites of the genus Leishmania alternate between flagellated, elongated extracellular promastigotes found in insect vectors, and round-shaped amastigotes enclosed in phagolysosome-like Parasitophorous Vacuoles (PVs) of infected mammalian host cells. Leishmania amazonensis amastigotes occupy large PVs which may contain many parasites; in contrast, single amastigotes of Leishmania major lodge in small, tight PVs, which undergo fission as parasites divide. To determine if PVs of these Leishmania species can fuse with each other, mouse macrophages in culture were infected with non-fluorescent L. amazonensis amastigotes and, 48 h later, superinfected with fluorescent L. major amastigotes or promastigotes. Fusion was investigated by time-lapse image acquisition of living cells and inferred from the colocalization of parasites of the two species in the same PVs. Survival, multiplication and differentiation of parasites that did or did not share the same vacuoles were also investigated. Fusion of PVs containing L. amazonensis and L. major amastigotes was not found. However, PVs containing L. major promastigotes did fuse with pre-established L. amazonensis PVs. In these chimeric vacuoles, L. major promastigotes remained motile and multiplied, but did not differentiate into amastigotes. In contrast, in doubly infected cells, within their own, unfused PVs metacyclic-enriched L. major promastigotes, but not log phase promastigotes - which were destroyed - differentiated into proliferating amastigotes. The results indicate that PVs, presumably customized by L. major amastigotes or promastigotes, differ in their ability to fuse with L. amazonensis PVs. Additionally, a species-specific PV was required for L. major destruction or differentiation – a requirement for which mechanisms remain unknown. The observations reported in this paper should be useful in further studies of the interactions between PVs to different species of Leishmania parasites, and of the mechanisms involved in the recognition and fusion of PVs

Control of Parasitophorous Vacuole Expansion by LYST/Beige Restricts the Intracellular Growth of Leishmania amazonensis

The intracellular protozoan Leishmania replicates in parasitophorous vacuoles (PV) that share many features with late endosomes/lysosomes. L. amazonensis PVs expand markedly during infections, but the impact of PV size on parasite intracellular survival is still unknown. Here we show that host cells infected with L. amazonensis upregulate transcription of LYST/Beige, which was previously shown to regulate lysosome size. Mutations in LYST/Beige caused further PV expansion and enhanced L. amazonensis replication. In contrast, LYST/Beige overexpression led to small PVs that did not sustain parasite growth. Treatment of LYST/Beige over-expressing cells with vacuolin-1 reversed this phenotype, expanding PVs and promoting parasite growth. The opposite was seen with E-64d, which reduced PV size in LYST-Beige mutant cells and inhibited L. amazonensis replication. Enlarged PVs appear to protect parasites from oxidative damage, since inhibition of nitric oxide synthase had no effect on L. amazonensis viability within large PVs, but enhanced their growth within LYST/Beige-induced small PVs. Thus, the upregulation of LYST/Beige in infected cells functions as a host innate response to limit parasite growth, by reducing PV volume and inhibiting intracellular survival

The Diverse and Dynamic Nature of Leishmania Parasitophorous Vacuoles Studied by Multidimensional Imaging

An important area in the cell biology of intracellular parasitism is the customization of parasitophorous vacuoles (PVs) by prokaryotic or eukaryotic intracellular microorganisms. We were curious to compare PV biogenesis in primary mouse bone marrow-derived macrophages exposed to carefully prepared amastigotes of either Leishmania major or L. amazonensis. While tight-fitting PVs are housing one or two L. major amastigotes, giant PVs are housing many L. amazonensis amastigotes. In this study, using multidimensional imaging of live cells, we compare and characterize the PV biogenesis/remodeling of macrophages i) hosting amastigotes of either L. major or L. amazonensis and ii) loaded with Lysotracker, a lysosomotropic fluorescent probe. Three dynamic features of Leishmania amastigote-hosting PVs are documented: they range from i) entry of Lysotracker transients within tight-fitting, fission-prone L. major amastigote-housing PVs; ii) the decrease in the number of macrophage acidic vesicles during the L. major PV fission or L. amazonensis PV enlargement; to iii) the L. amazonensis PV remodeling after homotypic fusion. The high content information of multidimensional images allowed the updating of our understanding of the Leishmania species-specific differences in PV biogenesis/remodeling and could be useful for the study of other intracellular microorganisms

Attenuated Leishmania induce pro-inflammatory mediators and influence leishmanicidal activity by p38 MAPK dependent phagosome maturation in Leishmania donovani co-infected macrophages

Promastigote form of Leishmania, an intracellular pathogen, delays phagosome maturation and resides inside macrophages. But till date limited study has been done to manipulate the phagosomal machinery of macrophages to restrict Leishmania growth. Attenuated Leishmania strain exposed RAW 264.7 cells showed a respiratory burst and enhanced production of pro-inflammatory mediators. The augmentation of pro-inflammatory activity is mostly attributed to p38 MAPK and p44/42 MAPK. In our study, these activated macrophages are found to induce phagosome maturation when infected with pathogenic Leishmania donovani. Increased co-localization of carboxyfluorescein succinimidyl ester labeled pathogenic L. donovani with Lysosome was found. Moreover, increased co-localization was observed between pathogenic L. donovani and late phagosomal markers viz. Rab7, Lysosomal Associated Membrane Protein 1, Cathepsin D, Rab9, and V-ATPase which indicate phagosome maturation. It was also observed that inhibition of V-type ATPase caused significant hindrance in attenuated Leishmania induced phagosome maturation. Finally, it was confirmed that p38 MAPK is the key player in acidification and maturation of phagosome in attenuated Leishmania strain preexposed macrophages. To our knowledge, this study for the first time reported an approach to induce phagosome maturation in L. donovani infected macrophages which could potentiate short-term prophylactic response in futur

Immune and inflammatory responses to Leishmania amazonensis isolated from different clinical forms of human leishmaniasis in CBA mice

Leishmania amazonensis causes different diseases depending on the host and parasitic virulence factors. In this study, CBA mice were infected with L. amazonensis isolates from patients with localized (Ba125), diffuse cutaneous (Ba276) or visceral leishmaniasis (Ba109). Mice infected with Ba125 and Ba276 progressed rapidly and lesions displayed an infiltrate rich in parasitized macrophages and were necrotic and ulcerated. Ba109 induced smaller lesions and a mixed inflammatory infiltrate without necrosis or ulceration. Ba109 induced an insidious disease with lower parasite load in CBA mice, similar to human disease. Levels of IFN-γ, IL-4 and IL-10 did not differ among the groups. Because all groups were unable to control the infection, expression of IL-4 associated with low production of IFN-γ in the early phase of infection may account for susceptibility, but others factors may contribute to the differences observed in inflammatory responses and infection progression. Evaluation of some parasitic virulence factors revealed that Ba276 exhibits higher ecto-ADPase and 5'-nucleotidase activities compared to the Ba109 and Ba125 strains. Both Ba276 and Ba125 had higher arginase activity in comparison to Ba109. Finally, these data suggest that the differences in enzyme activities among parasites can account for differences in host inflammatory responses and infection progression

Withanolides and related steroids

Since the isolation of the first withanolides in the mid-1960s, over 600 new members of this group of compounds have been described, with most from genera of the plant family Solanaceae. The basic structure of withaferin A, a C28 ergostane with a modified side chain forming a δ-lactone between carbons 22 and 26, was considered for many years the basic template for the withanolides. Nowadays, a considerable number of related structures are also considered part of the withanolide class; among them are those containing γ-lactones in the side chain that have come to be at least as common as the δ-lactones. The reduced versions (γ and δ-lactols) are also known. Further structural variations include modified skeletons (including C27 compounds), aromatic rings and additional rings, which may coexist in a single plant species. Seasonal and geographical variations have also been described in the concentration levels and types of withanolides that may occur, especially in the Jaborosa and Salpichroa genera, and biogenetic relationships among those withanolides may be inferred from the structural variations detected. Withania is the parent genus of the withanolides and a special section is devoted to the new structures isolated from species in this genus. Following this, all other new structures are grouped by structural types. Many withanolides have shown a variety of interesting biological activities ranging from antitumor, cytotoxic and potential cancer chemopreventive effects, to feeding deterrence for several insects as well as selective phytotoxicity towards monocotyledoneous and dicotyledoneous species. Trypanocidal, leishmanicidal, antibacterial, and antifungal activities have also been reported. A comprehensive description of the different activities and their significance has been included in this chapter. The final section is devoted to chemotaxonomic implications of withanolide distribution within the Solanaceae. Overall, this chapter covers the advances in the chemistry and biology of withanolides over the last 16 years.Fil: Misico, Rosana Isabel. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Orgánica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Unidad de Microanálisis y Métodos Físicos Aplicados a la Química Orgánica (i); ArgentinaFil: Nicotra, V.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Instituto Multidisciplinario de Biología Vegetal (p); Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Química Orgánica; ArgentinaFil: Oberti, Juan Carlos María. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Instituto Multidisciplinario de Biología Vegetal (p); Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Química Orgánica; ArgentinaFil: Barboza, Gloria Estela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Instituto Multidisciplinario de Biología Vegetal (p); Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Farmacia; ArgentinaFil: Gil, Roberto Ricardo. University Of Carnegie Mellon; Estados UnidosFil: Burton, Gerardo. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Orgánica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Unidad de Microanálisis y Métodos Físicos Aplicados a la Química Orgánica (i); Argentin

Approaches in biotechnological applications of natural polymers

Natural polymers, such as gums and mucilage, are biocompatible, cheap, easily available and non-toxic materials of native origin. These polymers are increasingly preferred over synthetic materials for industrial applications due to their intrinsic properties, as well as they are considered alternative sources of raw materials since they present characteristics of sustainability, biodegradability and biosafety. As definition, gums and mucilages are polysaccharides or complex carbohydrates consisting of one or more monosaccharides or their derivatives linked in bewildering variety of linkages and structures. Natural gums are considered polysaccharides naturally occurring in varieties of plant seeds and exudates, tree or shrub exudates, seaweed extracts, fungi, bacteria, and animal sources. Water-soluble gums, also known as hydrocolloids, are considered exudates and are pathological products; therefore, they do not form a part of cell wall. On the other hand, mucilages are part of cell and physiological products. It is important to highlight that gums represent the largest amounts of polymer materials derived from plants. Gums have enormously large and broad applications in both food and non-food industries, being commonly used as thickening, binding, emulsifying, suspending, stabilizing agents and matrices for drug release in pharmaceutical and cosmetic industries. In the food industry, their gelling properties and the ability to mold edible films and coatings are extensively studied. The use of gums depends on the intrinsic properties that they provide, often at costs below those of synthetic polymers. For upgrading the value of gums, they are being processed into various forms, including the most recent nanomaterials, for various biotechnological applications. Thus, the main natural polymers including galactomannans, cellulose, chitin, agar, carrageenan, alginate, cashew gum, pectin and starch, in addition to the current researches about them are reviewed in this article.. }To the Conselho Nacional de Desenvolvimento Cientfíico e Tecnológico (CNPq) for fellowships (LCBBC and MGCC) and the Coordenação de Aperfeiçoamento de Pessoal de Nvíel Superior (CAPES) (PBSA). This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit, the Project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462) and COMPETE 2020 (POCI-01-0145-FEDER-006684) (JAT)