ActinoBase: tools and protocols for researchers working on Streptomyces and other filamentous actinobacteria.

Actinobacteria is an ancient phylum of Gram-positive bacteria with a characteristic high GC content to their DNA. The ActinoBase Wiki is focused on the filamentous actinobacteria, such as Streptomyces species, and the techniques and growth conditions used to study them. These organisms are studied because of their complex developmental life cycles and diverse specialised metabolism which produces many of the antibiotics currently used in the clinic. ActinoBase is a community effort that provides valuable and freely accessible resources, including protocols and practical information about filamentous actinobacteria. It is aimed at enabling knowledge exchange between members of the international research community working with these fascinating bacteria. ActinoBase is an anchor platform that underpins worldwide efforts to understand the ecology, biology and metabolic potential of these organisms. There are two key differences that set ActinoBase apart from other Wiki-based platforms: [1] ActinoBase is specifically aimed at researchers working on filamentous actinobacteria and is tailored to help users overcome challenges working with these bacteria and [2] it provides a freely accessible resource with global networking opportunities for researchers with a broad range of experience in this field.

Integrating perspectives in actinomycete research: an ActinoBase review of 2020-21.

Last year ActinoBase, a Wiki-style initiative supported by the UK Microbiology Society, published a review highlighting the research of particular interest to the actinomycete community. Here, we present the second ActinoBase review showcasing selected reports published in 2020 and early 2021, integrating perspectives in the actinomycete field. Actinomycetes are well-known for their unsurpassed ability to produce specialised metabolites, of which many are used as therapeutic agents with antibacterial, antifungal, or immunosuppressive activities. Much research is carried out to understand the purpose of these metabolites in the environment, either within communities or in host interactions. Moreover, many efforts have been placed in developing computational tools to handle big data, simplify experimental design, and find new biosynthetic gene cluster prioritisation strategies. Alongside, synthetic biology has provided advances in tools to elucidate the biosynthesis of these metabolites. Additionally, there are still mysteries to be uncovered in understanding the fundamentals of filamentous actinomycetes' developmental cycle and regulation of their metabolism. This review focuses on research using integrative methodologies and approaches to understand the bigger picture of actinomycete biology, covering four research areas: i) technology and methodology; ii) specialised metabolites; iii) development and regulation; and iv) ecology and host interactions.

Insights Into the Ecological Role of Pseudomonas spp. in an Ant-plant Symbiosis.

In the myrmecophytic mutualistic relationship between Azteca ants and Cecropia plants both species receive protection and exchange nutrients. The presence of microorganisms in this symbiotic system has been reported, and the symbiotic role of some fungi involved in the myrmecophytic interactions has been described. In this work we focus on bacteria within this mutualism, conducting isolations and screening for antimicrobial activities, genome sequencing, and biochemical characterization. We show that Pantoea, Rhizobium, Methylobacterium, Streptomyces and Pseudomonas are the most common cultivable genera of bacteria. Interestingly, Pseudomonas spp. isolates showed potent activity against 83% of the pathogens tested in our antimicrobial activity assays, including a phytopathogenic fungus isolated from Cecropia samples. Given the predicted nitrogen limitations associated with the fungal patches within this myrmecophyte, we performed nitrogen fixation analyses on the bacterial isolates within the Proteobacteria and show the potential for nitrogen fixation in Pseudomonas strains. The genome of one Pseudomonas strain was sequenced and analyzed. The gene cluster involved in the biosynthesis of cyclic lipodepsipeptides (CLPs) was identified, and we found mutations that may be related to the loss of function in the dual epimerization/condensation domains. The compound was isolated, and its structure was determined, corresponding to the antifungal viscosinamide. Our findings of diazotrophy and production of viscosinamide in multiple Pseudomonas isolates suggests that this bacterial genus may play an important role in the Cecropia-Azteca symbiosis.

A chemiresistor sensor based on a cobalt(salen) metallopolymer for dissolved molecular oxygen.

A resistance detection device for dissolved molecular oxygen in aqueous solutions is prepared using a chemiresistor material as sensor platform. The chemiresistive circuit element is fashioned from a thin film of a cobalt-salen metallopolymer electrodeposited on a platinum electrode. Electrochemical impedance spectroscopy shows that the resistive and capacitive properties of the sensor platform depend on the presence of dissolved oxygen. The electrical circuit models are R(Q/R)(Q/R) and R(Q/R)(Q/RW) in the absence and presence of oxygen, respectively. The chemiresistor sensor exhibits good sensitivity (0.483 kΩ L mg-1), excellent reversibility and excellent linearity over a range of dissolved oxygen concentrations typically found under environmental conditions (2.72-40.9 mg L-1). The sensor fabricated in this work can potentially serve as an alternative sensor for the detection of dissolved oxygen in environmental samples.

Bioenergetic profiling of Trypanosoma cruzi life stages using Seahorse extracellular flux technology.

Energy metabolism is an attractive target for the development of new therapeutics against protozoan pathogens, including Trypanosoma cruzi, the causative agent of human Chagas disease. Despite emerging evidence that mitochondrial electron transport is essential for the growth of intracellular T. cruzi amastigotes in mammalian cells, fundamental knowledge of mitochondrial energy metabolism in this parasite life stage remains incomplete. The Clark-type electrode, which measures the rate of oxygen consumption, has served as the traditional tool to study mitochondrial energetics and has contributed to our understanding of it in T. cruzi. Here, we evaluate the Seahorse XF(e)24 extracellular flux platform as an alternative method to assess mitochondrial bioenergetics in isolated T. cruzi parasites. We report optimized assay conditions used to perform mitochondrial stress tests with replicative life cycle stages of T. cruzi using the XF(e)24 instrument, and discuss the advantages and potential limitations of this methodology, as applied to T. cruzi and other trypanosomatids.

Leishmania amazonensis promastigotes in 3D Collagen I culture: an in vitro physiological environment for the study of extracellular matrix and host cell interactions.

Leishmania amazonensis is the causative agent of American cutaneous leishmaniasis, an important neglected tropical disease. Once Leishmania amazonensis is inoculated into the human host, promastigotes are exposed to the extracellular matrix (ECM) of the dermis. However, little is known about the interaction between the ECM and Leishmania promastigotes. In this study we established L. amazonensis promastigote culture in a three-dimensional (3D) environment mainly composed of Collagen I (COL I). This 3D culture recreates in vitro some aspects of the human host infection site, enabling the study of the interaction mechanisms of L. amazonensis with the host ECM. Promastigotes exhibited "freeze and run" migration in the 3D COL I matrix, which is completely different from the conventional in vitro swimming mode of migration. Moreover, L. amazonensis promastigotes were able to invade, migrate inside, and remodel the 3D COL I matrix. Promastigote trans-matrix invasion and the freeze and run migration mode were also observed when macrophages were present in the matrix. At least two classes of proteases, metallo- and cysteine proteases, are involved in the 3D COL I matrix degradation caused by Leishmania. Treatment with a mixture of protease inhibitors significantly reduced promastigote invasion and migration through this matrix. Together our results demonstrate that L. amazonensis promastigotes release proteases and actively remodel their 3D environment, facilitating their migration. This raises the possibility that promastigotes actively interact with their 3D environment during the search for their cellular "home"-macrophages. Supporting this hypothesis, promastigotes migrated faster than macrophages in a novel 3D co-culture model.