A secreted serine protease of Paracoccidioides brasiliensis and its interactions with fungal proteins

<p>Abstract</p> <p>Background</p> <p><it>Paracoccidioides brasiliensis </it>is a thermodimorphic fungus, the causative agent of paracoccidioidomycosis (PCM). Serine proteases are widely distributed and this class of peptidase has been related to pathogenesis and nitrogen starvation in pathogenic fungi.</p> <p>Results</p> <p>A cDNA (<it>Pb</it>sp) encoding a secreted serine protease (<it>Pb</it>SP), was isolated from a cDNA library constructed with RNAs of fungal yeast cells recovered from liver of infected mice. Recombinant <it>Pb</it>SP was produced in <it>Escherichia coli</it>, and used to develop polyclonal antibodies that were able to detect a 66 kDa protein in the <it>P. brasiliensis </it>proteome. <it>In vitro </it>deglycosylation assays with endoglycosidase H demonstrated that <it>Pb</it>SP is a <it>N</it>-glycosylated molecule. The <it>Pb</it>sp transcript and the protein were induced during nitrogen starvation. The <it>Pb</it>sp transcript was also induced in yeast cells infecting murine macrophages. Interactions of <it>Pb</it>SP with <it>P. brasiliensis </it>proteins were evaluated by two-hybrid assay in the yeast <it>Saccharomyces cerevisiae</it>. <it>Pb</it>SP interacts with a peptidyl prolyl cis-trans isomerase, calnexin, HSP70 and a cell wall protein PWP2.</p> <p>Conclusions</p> <p>A secreted subtilisin induced during nitrogen starvation was characterized indicating the possible role of this protein in the nitrogen acquisition. <it>Pb</it>SP interactions with other <it>P. brasiliensis </it>proteins were reported. Proteins interacting with <it>Pb</it>SP are related to folding process, protein trafficking and cytoskeleton reorganization.</p

How good are RGB cameras retrieving colors of natural scenes and paintings?—A study based on hyperspectral imaging

RGB digital cameras (RGB) compress the spectral information into a trichromatic system capable of approximately representing the actual colors of objects. Although RGB digital cameras follow the same compression philosophy as the human eye (OBS), the spectral sensitivity is different. To what extent they provide the same chromatic experiences is still an open question, especially with complex images. We addressed this question by comparing the actual colors derived from spectral imaging with those obtained with RGB cameras. The data from hyperspectral imaging of 50 natural scenes and 89 paintings was used to estimate the chromatic differences between OBS and RGB. The corresponding color errors were estimated and analyzed in the color spaces CIELAB (using the color difference formulas ΔE*ab and CIEDE2000), Jzazbz, and iCAM06. In CIELAB the most frequent error (using ΔE*ab) found was 5 for both paintings and natural scenes, a similarity that held for the other spaces tested. In addition, the distribution of errors across the color space shows that the errors are small in the achromatic region and increase with saturation. Overall, the results indicate that the chromatic errors estimated are close to the acceptance error and therefore RGB digital cameras are able to produce quite realistic colors of complex scenarios.This work was supported by the Portuguese Foundation for Science and Technology (FCT) in the framework of the Strategic Funding UIDB/04650/2020

The second International Symposium on Fungal Stress: ISFUS

The topic of ‘fungal stress’ is central to many important disciplines, including medical mycology, chronobiology, plant and insect pathology, industrial microbiology, material sciences, and astrobiology. The International Symposium on Fungal Stress (ISFUS) brought together researchers, who study fungal stress in a variety of fields. The second ISFUS was held in May 8-11 2017 in Goiania, Goiás, Brazil and hosted by the Instituto de Patologia Tropical e Saúde Pública at the Universidade Federal de Goiás. It was supported by grants from CAPES and FAPEG. Twenty-seven speakers from 15 countries presented their research related to fungal stress biology. The Symposium was divided into seven topics: 1. Fungal biology in extreme environments; 2. Stress mechanisms and responses in fungi: molecular biology, biochemistry, biophysics, and cellular biology; 3. Fungal photobiology in the context of stress; 4. Role of stress in fungal pathogenesis; 5. Fungal stress and bioremediation; 6. Fungal stress in agriculture and forestry; and 7. Fungal stress in industrial applications. This article provides an overview of the science presented and discussed at ISFUS-2017.Sao Paulo Research Foundation (FAPESP) 2010/06374-1, 2013/50518-6, 2014/01229-4Brazilian National Council for Scientific and Technological Development (CNPq) PQ2 302312/2011-0, PQ1D 308436/2014-8Coordenação de Aperfeiçoãmento de Pessoal de Nível Superior (CAPES) PAEP 88881.123209/2016-01Fundação de Amparo à Pesquisa do Estado de Goiás Brazil 20171026700011

Assessing Helicobacter pylori motility and biofilm formation in subinhibitory concentrations of antimicrobials.

Numerous studies have shown that subinhibitory concentrations of antimicrobials can alter bacterial virulence factors. This study evaluates motility and biofilm formation by H. pylori 43504 grown in subinhibitory concentrations of amoxicillin (AMX), clarithromycin (CLA), or tetracycline (TET). For the swimming and swarming motility assays, H. pylori 43504 suspensions were prepared with the strain alone or with the strain in AMX, CLA, or TET at ½ MIC. Next, the media were incubated at 37 ºC, under microaerophilia. To assess biofilm formation in the presence of one of the antimicrobials at subinhibitory antimicrobial concentrations, bacterial suspensions (109 CFU/mL) were prepared in 2.5% FBS containing AMX, CLA, or TET at ½ MIC. After incubation for 10 days, H. pylori 43504 grown in medium containing AMX, CLA, or TET at ½ MIC presented greater swimming motility and lower swarming motility than the non-treated strain. H. pylori 43504 grown in medium containing AMX, CLA, or TET at ½ MIC showed stronger biofilm production than the non-treated strain. Our results showed that AMX, CLA, or TET at subinhibitory concentrations favors H. pylori 43504 swimming motility and biofilm formation after incubation for 3 days. This may have clinical consequences and make the microorganism difficult to eradicate

Proteomic Analysis Reveals That Iron Availability Alters the Metabolic Status of the Pathogenic Fungus Paracoccidioides brasiliensis

Paracoccidioides brasiliensis is a thermodimorphic fungus and the causative agent of paracoccidioidomycosis (PCM). The ability of P. brasiliensis to uptake nutrients is fundamental for growth, but a reduction in the availability of iron and other nutrients is a host defense mechanism many pathogenic fungi must overcome. Thus, fungal mechanisms that scavenge iron from host may contribute to P. brasiliensis virulence. In order to better understand how P. brasiliensis adapts to iron starvation in the host we compared the two-dimensional (2D) gel protein profile of yeast cells during iron starvation to that of iron rich condition. Protein spots were selected for comparative analysis based on the protein staining intensity as determined by image analysis. A total of 1752 protein spots were selected for comparison, and a total of 274 out of the 1752 protein spots were determined to have changed significantly in abundance due to iron depletion. Ninety six of the 274 proteins were grouped into the following functional categories; energy, metabolism, cell rescue, virulence, cell cycle, protein synthesis, protein fate, transcription, cellular communication, and cell fate. A correlation between protein and transcript levels was also discovered using quantitative RT-PCR analysis from RNA obtained from P. brasiliensis under iron restricting conditions and from yeast cells isolated from infected mouse spleens. In addition, western blot analysis and enzyme activity assays validated the differential regulation of proteins identified by 2-D gel analysis. We observed an increase in glycolytic pathway protein regulation while tricarboxylic acid cycle, glyoxylate and methylcitrate cycles, and electron transport chain proteins decreased in abundance under iron limiting conditions. These data suggest a remodeling of P. brasiliensis metabolism by prioritizing iron independent pathways

Comparative Genomic Analysis of Human Fungal Pathogens Causing Paracoccidioidomycosis

Paracoccidioides is a fungal pathogen and the cause of paracoccidioidomycosis, a health-threatening human systemic mycosis endemic to Latin America. Infection by Paracoccidioides, a dimorphic fungus in the order Onygenales, is coupled with a thermally regulated transition from a soil-dwelling filamentous form to a yeast-like pathogenic form. To better understand the genetic basis of growth and pathogenicity in Paracoccidioides, we sequenced the genomes of two strains of Paracoccidioides brasiliensis (Pb03 and Pb18) and one strain of Paracoccidioides lutzii (Pb01). These genomes range in size from 29.1 Mb to 32.9 Mb and encode 7,610 to 8,130 genes. To enable genetic studies, we mapped 94% of the P. brasiliensis Pb18 assembly onto five chromosomes. We characterized gene family content across Onygenales and related fungi, and within Paracoccidioides we found expansions of the fungal-specific kinase family FunK1. Additionally, the Onygenales have lost many genes involved in carbohydrate metabolism and fewer genes involved in protein metabolism, resulting in a higher ratio of proteases to carbohydrate active enzymes in the Onygenales than their relatives. To determine if gene content correlated with growth on different substrates, we screened the non-pathogenic onygenale Uncinocarpus reesii, which has orthologs for 91% of Paracoccidioides metabolic genes, for growth on 190 carbon sources. U. reesii showed growth on a limited range of carbohydrates, primarily basic plant sugars and cell wall components; this suggests that Onygenales, including dimorphic fungi, can degrade cellulosic plant material in the soil. In addition, U. reesii grew on gelatin and a wide range of dipeptides and amino acids, indicating a preference for proteinaceous growth substrates over carbohydrates, which may enable these fungi to also degrade animal biomass. These capabilities for degrading plant and animal substrates suggest a duality in lifestyle that could enable pathogenic species of Onygenales to transfer from soil to animal hosts.National Institute of Allergy and Infectious Diseases (U.S.)National Institutes of Health. Department of Health and Human Services (contract HHSN266200400001C)National Institutes of Health. Department of Health and Human Services(contract HHSN2722009000018C)Brazil. National Council for Scientific and Technological Developmen