Tao3 mediates a phenotypic switch between amoeba-adapted and mammalian-adapted forms of Cryptococcus neoformans

Comparative Medicine - OneHealth and Comparative Medicine Poster SessionMany microbes are capable of changing phenotypes more frequently than due to basal mutation rates alone, and this ability is coupled to pathogenesis. The human pathogenic yeast Cryptococcus neoformans is found in the environment in soil, pigeon guano and tree species, locations in which the organism is exposed to microbial predators. Previous research showed that co-incubation of C. neoformans with amoeba causes a switch from a yeast to a pseudohyphal form, enabling fungal survival in amoeba yet conversely reducing virulence in mammalian models of cryptococcosis. We identify the basis for pseudohyphal development in phenotypic-switched and amoeba-derived strains, to show that genes encoding proteins of the RAM (Regulation of Ace2p activity and cellular Morphogenesis) pathway bear mutations. Reversion to wild type yeast morphology can occur through multiple different mechanisms to suggest that underlying rates of spontaneous mutation control this process and thereby influence the pathogenic potential of an organism

Adaptive Response to DNA-Damaging Agents in Natural Saccharomyces cerevisiae Populations from “Evolution Canyon”, Mt. Carmel, Israel

BACKGROUND:Natural populations of most organisms, especially unicellular microorganisms, are constantly exposed to harsh environmental factors which affect their growth. UV radiation is one of the most important physical parameters which influences yeast growth in nature. Here we used 46 natural strains of Saccharomyces cerevisiae isolated from several natural populations at the "Evolution Canyon" microsite (Nahal Oren, Mt. Carmel, Israel). The opposing slopes of this canyon share the same geology, soil, and macroclimate, but they differ in microclimatic conditions. The interslope differences in solar radiation (200%-800% more on the "African" slope) caused the development of two distinct biomes. The south-facing slope is sunnier and has xeric, savannoid "African" environment while the north-facing slope is represented by temperate, "European" forested environment. Here we studied the phenotypic response of the S. cerevisiae strains to UVA and UVC radiations and to methyl methanesulfonate (MMS) in order to evaluate the interslope effect on the strains' ability to withstand DNA-damaging agents. METHODOLOGY/PRINCIPAL FINDINGS:We exposed our strains to the different DNA-damaging agents and measured survival by counting colony forming units. The strains from the "African" slope were more resilient to both UVA and MMS than the strains from the "European" slope. In contrast, we found that there was almost no difference between strains (with similar ploidy) from the opposite slopes, in their sensitivity to UVC radiation. These results suggest that the "African" strains are more adapted to higher solar radiation than the "European" strains. We also found that the tetraploids strains were more tolerant to all DNA-damaging agents than their neighboring diploid strains, which suggest that high ploidy level might be a mechanism of adaptation to high solar radiation. CONCLUSIONS/SIGNIFICANCE:Our results and the results of parallel studies with several other organisms, suggest that natural selection appears to select, at a microscale, for adaptive complexes that can tolerate the higher UV radiation on the "African" slope

Root-TRAPR: a modular plant growth device to visualize root development and monitor growth parameters, as applied to an elicitor response of Cannabis sativa

Background Plant growth devices, for example, rhizoponics, rhizoboxes, and ecosystem fabrication (EcoFAB), have been developed to facilitate studies of plant root morphology and plant-microbe interactions in controlled laboratory settings. However, several of these designs are suitable only for studying small model plants such as Arabidopsis thaliana and Brachypodium distachyon and therefore require modification to be extended to larger plant species like crop plants. In addition, specific tools and technical skills needed for fabricating these devices may not be available to researchers. Hence, this study aimed to establish an alternative protocol to generate a larger, modular and reusable plant growth device based on different available resources. Results Root-TRAPR (Root-Transparent, Reusable, Affordable three-dimensional Printed Rhizo-hydroponic) system was successfully developed. It consists of two main parts, an internal root growth chamber and an external structural frame. The internal root growth chamber comprises a polydimethylsiloxane (PDMS) gasket, microscope slide and acrylic sheet, while the external frame is printed from a three-dimensional (3D) printer and secured with nylon screws. To test the efficiency and applicability of the system, industrial hemp (Cannabis sativa) was grown with or without exposure to chitosan, a well-known plant elicitor used for stimulating plant defense. Plant root morphology was detected in the system, and plant tissues were easily collected and processed to examine plant biological responses. Upon chitosan treatment, chitinase and peroxidase activities increased in root tissues (1.7- and 2.3-fold, respectively) and exudates (7.2- and 21.6-fold, respectively). In addition, root to shoot ratio of phytohormone contents were increased in response to chitosan. Within 2 weeks of observation, hemp plants exhibited dwarf growth in the Root-TRAPR system, easing plant handling and allowing increased replication under limited growing space. Conclusion The Root-TRAPR system facilitates the exploration of root morphology and root exudate of C. sativa under controlled conditions and at a smaller scale. The device is easy to fabricate and applicable for investigating plant responses toward elicitor challenge. In addition, this fabrication protocol is adaptable to study other plants and can be applied to investigate plant physiology in different biological contexts, such as plant responses against biotic and abiotic stresses

Non‐linear analysis of suspension bridges with flexible and rigid cables

One of the main problems related to the design of suspension bridges is stabilisation of their initial form. The tendency of suspension bridges to deform is generally determined by the kinematical displacements of the suspension cable caused by asymmetrical loads rather than by the elastic deformations. There are some suspension bridges when the so‐called rigid (stiff in bending) cables instead of usual flexible cables are suggested for stabilisation of their initial form. The analysis methods of such suspension bridges with rigid cables are underdeveloped. For the analysis of classical suspension bridges analytical models can be applied. However, in case of concentrated forces, the numerical techniques are preferred. The article presents analytical expressions for the calculation of internal forces and displacements of suspension bridges with a rigid cable. The article also discusses the discrete calculation model for classical suspension bridges. Santrauka Viena iš pagrindiniu kabamuju tiltu projektavimo problemu yra pradinus ju formos stabilizavimas. Kabamuju tiltu deformatyvuma lemia iš esmes ne tiek tampriosios deformacijos, kiek asimetriniu apkrovu sukelti kinematiniai kabamojo lyno poslinkiai. Yra žinomi kabamieji tiltai, kuriu pradinei formai stabilizuoti siūloma vietoje iprastiniu lanksčiuju lynu taikyti vadinamuosius standžius lynus. Tokiu kabamuju tiltu su standžiaislynais skaičiavimo metodai nera iki galo parengti. Klasikiniams tiltams su lanksčiu lynu skaičiuoti taikomi daugiausia kontinualūs modeliai, kurie esant tam tikrai tilto sandarai ar veikiant sutelktoms apkrovoms nera pakankamai tikslūs. Straipsnyje pateikiamos analizines išraiškos kabamuju tiltu su standžiu lynu iražoms ir poslinkiams apskaičiuoti, aptariamas diskretusis klasikiniu kabamuju tiltu skaičiavimo modelis. Published Published Online: 24 Jun 2011 Reikšminiai žodžiai: kabamasis tiltas, lankstus lynas, standus lynas, netiesine analize, kontinualus ir diskretinis modeliai, iražos ir poslinkiai

Statistical analysis of reinforced concrete bridges in Estonia

This paper introduces a possible way to use a multivariate methodology, called principal component analysis, to reduce the dimensionality of condition state database of bridge elements, collected during visual inspections. Attention is paid to the condition assessment of bridges in Estonian national roads and collected data, which plays an important role in the selection of correct statistical technique and obtaining reliable results. Additionally, detailed overview of typical road bridges and examples of collected information is provided. Statistical analysis is carried out by most natural reinforced concrete bridges in Estonia and comparison is made among different typologies. The introduced multivariate technique algorithms are presented and collated in two different formulations, with contrast on unevenness in variables and taking into account the missing data. Principal components and weighing factors, which are calculated for bridges with different typology, also have differences in results and element groups where variation is retainedTU1406 – Quality Specifications for Roadway Bridges, standardiza- tion at a European level (BridgeSpec), a COST Action sup- ported by EU Framework Programme Horizon 2020info:eu-repo/semantics/publishedVersio

The role of the de novo pyrimidine biosynthetic pathway in Cryptococcus neoformans high temperature growth and virulence

Fungal infections are often difficult to treat due to the inherent similarities between fungal and animal cells and the resulting host toxicity from many antifungal compounds. Cryptococcus neoformans is an opportunistic fungal pathogen of humans that causes life-threatening disease, primarily in immunocompromised patients. Since antifungal therapy for this microorganism is limited, many investigators have explored novel drug targets aim at virulence factors, such as the ability to grow at mammalian physiological temperature (37 degrees C). To address this issue, we used the Agrobacterium tumefaciens gene delivery system to create a random insertion mutagenesis library that was screened for altered growth at elevated temperatures. Among several mutants unable to grow at 37 degrees C, we explored one bearing an interruption in the URA4 gene. This gene encodes dihydroorotase (DHOase) that is involved in the de nova synthesis of pyrimidine ribonucleotides. Loss of the C. neoformans Ura4 protein, by targeted gene interruption, resulted in an expected uracil/uridine auxotrophy and an unexpected high temperature growth defect. in addition, the ura4 mutant displayed phenotypic defects in other prominent virulence factors (melanin, capsule and phospholipase) and reduced stress response compared to wild type and reconstituted strains. Accordingly, this mutant had a decreased survival rate in macrophages and attenuated virulence in a murine model of cryptococcal infection. Quantitative PCR analysis suggests that this biosynthetic pathway is induced during the transition from 30 degrees C to 37 degrees C, and that transcriptional regulation of de nova and salvage pyrimidine pathway are under the control of the Ura4 protein. (C) 2014 the Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-SA license (http://creativecommons.org/licenses/by-nc-sa/3.0/).Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)NIHUniversidade Federal de São Paulo, Dept Ciencias Biol, BR-09972270 Diadema, SP, BrazilUniv Brasilia, Fac Ceilandia, Brasilia, DF, BrazilDuke Univ, Sch Med, Dept Med, Durham, NC 27706 USAUniversidade Federal de São Paulo, Dept Ciencias Biol, BR-09972270 Diadema, SP, BrazilFAPESP: 2007/50536-3FAPESP: 2011/50953-9NIH: AI050128NIH: AI074677Web of Scienc

Automated Analysis of Cryptococcal Macrophage Parasitism Using GFP-Tagged Cryptococci

The human fungal pathogens Cryptococcus neoformans and C. gattii cause life-threatening infections of the central nervous system. One of the major characteristics of cryptococcal disease is the ability of the pathogen to parasitise upon phagocytic immune effector cells, a phenomenon that correlates strongly with virulence in rodent models of infection. Despite the importance of phagocyte/Cryptococcus interactions to disease progression, current methods for assaying virulence in the acrophage system are both time consuming and low throughput. Here, we introduce the first stable and fully characterised GFP–expressing derivatives of two widely used cryptococcal strains: C. neoformans serotype A type strain H99 and C. gattii serotype B type strain R265. Both strains show unaltered responses to environmental and host stress conditions and no deficiency in virulence in the macrophage model system. In addition, we report the development of a method to effectively and rapidly investigate macrophage parasitism by flow cytometry, a technique that preserves the accuracy of current approaches but offers a four-fold improvement in speed

Two Origins for the Gene Encoding α-Isopropylmalate Synthase in Fungi

BACKGROUND: The biosynthesis of leucine is a biochemical pathway common to prokaryotes, plants and fungi, but absent from humans and animals. The pathway is a proposed target for antimicrobial therapy. METHODOLOGY/PRINCIPAL FINDINGS: Here we identified the leuA gene encoding alpha-isopropylmalate synthase in the zygomycete fungus Phycomyces blakesleeanus using a genetic mapping approach with crosses between wild type and leucine auxotrophic strains. To confirm the function of the gene, Phycomyces leuA was used to complement the auxotrophic phenotype exhibited by mutation of the leu3+ gene of the ascomycete fungus Schizosaccharomyces pombe. Phylogenetic analysis revealed that the leuA gene in Phycomyces, other zygomycetes, and the chytrids is more closely related to homologs in plants and photosynthetic bacteria than ascomycetes or basidiomycetes, and suggests that the Dikarya have acquired the gene more recently. CONCLUSIONS/SIGNIFICANCE: The identification of leuA in Phycomyces adds to the growing body of evidence that some primary metabolic pathways or parts of them have arisen multiple times during the evolution of fungi, probably through horizontal gene transfer events

Purification, crystallization and preliminary X-ray analysis of adenylosuccinate synthetase from the fungal pathogen Cryptococcus neoformans

With increasingly large immunocompromised populations around the world, opportunistic fungal pathogens such as Cryptococcus neoformans are a growing cause of morbidity and mortality. To combat the paucity of antifungal compounds, new drug targets must be investigated. Adenylosuccinate synthetase is a crucial enzyme in the ATP de novo biosynthetic pathway, catalyzing the formation of adenylosuccinate from inosine monophosphate and aspartate. Although the enzyme is ubiquitous and well characterized in other kingdoms, no crystallographic studies on the fungal protein have been performed. Presented here are the expression, purification, crystallization and initial crystallographic analyses of cryptococcal adenylosuccinate synthetase. The crystals had the symmetry of space group P2(1)2(1)2(1) and diffracted to 2.2 angstrom resolution