scholarly journals Isolation and Characterization of Cryptococcus neoformans Spores Reveal a Critical Role for Capsule Biosynthesis Genes in Spore Biogenesis

2009 ◽  
Vol 8 (4) ◽  
pp. 595-605 ◽  
Author(s):  
Michael R. Botts ◽  
Steven S. Giles ◽  
Marcellene A. Gates ◽  
Thomas R. Kozel ◽  
Christina M. Hull
Keyword(s):  
Cryptococcus Neoformans ◽  
Fungal Pathogens ◽  
Critical Role ◽  
Spore Formation ◽  
Yeast Cells ◽  
Isolation And Characterization ◽  
Specific Configuration ◽  
First Time

ABSTRACT Spores are essential particles for the survival of many organisms, both prokaryotic and eukaryotic. Among the eukaryotes, fungi have developed spores with superior resistance and dispersal properties. For the human fungal pathogens, however, relatively little is known about the role that spores play in dispersal and infection. Here we present the purification and characterization of spores from the environmental fungus Cryptococcus neoformans. For the first time, we purified spores to homogeneity and assessed their morphological, stress resistance, and surface properties. We found that spores are morphologically distinct from yeast cells and are covered with a thick spore coat. Spores are also more resistant to environmental stresses than yeast cells and display a spore-specific configuration of polysaccharides on their surfaces. Surprisingly, we found that the surface of the spore reacts with antibodies to the polysaccharide glucuronoxylomannan, the most abundant component of the polysaccharide capsule required for C. neoformans virulence. We explored the role of capsule polysaccharide in spore development by assessing spore formation in a series of acapsular strains and determined that capsule biosynthesis genes are required for proper sexual development and normal spore formation. Our findings suggest that C. neoformans spores may have an adapted cell surface that facilitates persistence in harsh environments and ultimately allows them to infect mammalian hosts.

scholarly journals Isolation and Characterization of Brenneria quercina, Causal Agent for Bark Canker and Drippy Nut of Quercus spp. in Spain

2003 ◽  
Vol 93 (4) ◽  
pp. 485-492 ◽  
Author(s):  
Elena G. Biosca ◽  
Raquel González ◽  
María José López-López ◽  
Santiago Soria ◽  
Carmina Montón ◽  
...  
Keyword(s):  
Fungal Pathogens ◽  
Reference Strain ◽  
Biochemical Characterization ◽  
Causal Agent ◽  
Enzyme Linked Immunosorbent Assay ◽  
Isolation And Characterization ◽  
Pathogenicity Tests ◽  
Different Origins ◽  
First Time

The drippy nut disease of oak was first described in California in 1967 and, since then, the causal agent has not been reported in any other area. This study describes for the first time in Europe the isolation of Brenneria (Erwinia) quercina from bark canker in addition to drippy bud and drippy nut in Quercus ilex and Q. pyrenaica. The bark canker and drippy bud symptoms were not previously described as caused by this bacterium. No fungal pathogens were associated with any of the symptoms. Physiological and biochemical characterization identified the pathogenic isolates from Spain as belonging to B. quercina, similar to the reference strain CFBP 1266. Fatty acid profiles of the Spanish isolates also were similar to the strain of B. quercina from California. Serological analysis by indirect immunofluorescence and enzyme-linked immunosorbent assay using polyclonal antisera against the reference strain of B. quercina and one Spanish oak isolate revealed some antigenic heterogeneity between isolates of different origins. Pathogenicity tests demonstrated that the Spanish isolates were able to reproduce internal symptoms of necrosis and acorn exudation in Q. ilex and Q. pyrenaica and suggest that B. quercina may be associated, among other causes, with the oak decline syndrome affecting Spanish oak forests.

scholarly journals Isolation and Characterization of Senescent Cryptococcus neoformans and Implications for Phenotypic Switching and Pathogenesis in Chronic Cryptococcosis

2009 ◽  
Vol 8 (6) ◽  
pp. 858-866 ◽  
Author(s):  
Neena Jain ◽  
Emily Cook ◽  
Immaculata Xess ◽  
Fahmi Hasan ◽  
Dietrich Fries ◽  
...  
Keyword(s):  
Mathematical Modeling ◽  
Cryptococcus Neoformans ◽  
Chronic Infection ◽  
Yeast Cells ◽  
Phenotypic Switching ◽  
Isolation And Characterization ◽  
Immunocompromised Hosts ◽  
Infection Experiments

ABSTRACT Although several virulence factors and associated genes have been identified, the mechanisms that allow Cryptococcus neoformans to adapt during chronic infection and to persist in immunocompromised hosts remain poorly understood. Characterization of senescent cells of C. neoformans demonstrated that these cells exhibit a significantly enlarged cell body and capsule but still cross the blood-brain barrier. C. neoformans cells with advanced generational age are also more resistant to phagocytosis and killing by antifungals, which could promote their selection during chronic disease in humans. Senescent cells of RC-2, a C. neoformans strain that undergoes phenotypic switching, manifest switching rates up to 11-fold higher than those of younger cells. Infection experiments with labeled cells suggest that senescent yeast cells can potentially accumulate in vivo. Mathematical modeling incorporating different switching rates demonstrates how increased switching rates promote the emergence of hypervirulent mucoid variants during chronic infection. Our findings introduce the intriguing concept that senescence in eukaryotic pathogens could be a mechanism of microevolution that may promote pathoadaptation and facilitate evasion of an evolving immune response.

scholarly journals The enigmatic role of fungal annexins: the case of Cryptococcus neoformans

2019 ◽  
Author(s):  
Maria Maryam ◽  
Man Shun Fu ◽  
Alexandre Alanio ◽  
Emma Camacho ◽  
Diego S. Goncalves ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Fungal Pathogens ◽  
Cell Function ◽  
Model Organism ◽  
Fungal Species ◽  
Yeast Cells ◽  
Dependent Manner ◽  
Membrane Repair

AbstractAnnexins are multifunctional proteins that bind to phospholipid membranes in a calcium-dependent manner. Annexins play a myriad of critical and well-characterized roles in mammals, ranging from membrane repair to vesicular secretion. The role of annexins in the kingdoms of bacteria, protozoa and fungi have been largely overlooked. The fact that there is no known homologue of annexins in the model organism may contribute to this gap in knowledge. However, annexins are found in most medically important fungal pathogens, with the notable exception of Candida albicans. In this study we evaluated the function of the one annexin gene in Cryptococcus neoformans, a causative agent of cryptococcosis. This gene CNAG_02415, is annotated in the C. neoformans genome as a target of calcineurin through its transcription factor Crz1, and we propose to update its name to cryptococcal annexin, AnnexinC1. C. neoformans strains deleted for AnnexinC1 revealed no difference in survival after exposure to various chemical stressor relative the wild type, as well as no major alteration in virulence or mating. The only alteration observed in strains deleted for AnnexinC1 was a small increase in the titan cells formation in vitro. The preservation of annexins in many different fungal species suggests an important function, and therefore the lack of a strong phenotype for annexin-deficient C. neoformans is suggestive of either redundant genes that can compensate for the absence of AnnexinC1 function or novel functions not revealed by standard assays of cell function and pathogenicity.ImportanceCryptococcus neoformans is the deadliest human fungal pathogen, causing almost 200,000 deaths each year. Treatment of this lethal infection is lengthy, and in some patients therapy is not curative and patients require lifelong therapy. Fundamental research in this yeast is needed so that we can understand mechanisms of infection and disease and ultimately devise better therapies. In this work we investigated a fungal representative of the annexin family of proteins, specifically in the context of virulence and mating. We find that the cryptococcal annexin does not seem to be involved in virulence or mating but affects generation of titan cells, enlarged yeast cells that are detected in the lungs of mammalian hosts. Our data provides new knowledge in an unexplored area of fungal biology.

Isolation and Characterization of Papillomavirus DNA from Nasal Inverting (Schneiderian) Papillomas

1987 ◽  
Vol 96 (2) ◽  
pp. 170-173 ◽  
Author(s):  
Don S. Respler ◽  
Anthony Jahn ◽  
Alan Pater ◽  
Mary M. Pater
Keyword(s):  
Restriction Enzyme Digestion ◽  
Definitive Evidence ◽  
Isolation And Characterization ◽  
Total Dna ◽  
Etiological Agents ◽  
First Time ◽  
Papillomavirus Dna ◽  
Base Pair Insertion

To elucidate the possible role of papillomaviruses as etiological agents in nasal inverting papillomas, DNA hybridization techniques were used. Total DNA from two nasal inverting papillomas was examined for the presence of DNA from human papillomavirus (HPV) types 6 and 11 under stringent conditions of hybridization. Both lesions contained DNA hybridizing with HPV 11. Restriction enzyme digestion and subsequent Southern blotting of the DNA samples revealed that one lesion contained viral DNA identical to HPV 11a. The DNA of the other lesion contained an extra 500 base pair insertion. These results provide definitive evidence for the first time for the association of HPV with nasal inverting papillomas.

scholarly journals Isolation and characterization of a new cold-active protease from psychrotrophic bacteria of Western Himalayan glacial soil

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Saleem Farooq ◽  
Ruqeya Nazir ◽  
Shabir Ahmad Ganai ◽  
Bashir Ahmad Ganai
Keyword(s):  
Specific Activity ◽  
Temperature Range ◽  
Psychrotrophic Bacteria ◽  
Isolation And Characterization ◽  
Cold Active ◽  
Active Protease ◽  
Quantitative Screening ◽  
First Time ◽  
Low K

AbstractAs an approach to the exploration of cold-active enzymes, in this study, we isolated a cold-active protease produced by psychrotrophic bacteria from glacial soils of Thajwas Glacier, Himalayas. The isolated strain BO1, identified as Bacillus pumilus, grew well within a temperature range of 4–30 °C. After its qualitative and quantitative screening, the cold-active protease (Apr-BO1) was purified. The Apr-BO1 had a molecular mass of 38 kDa and showed maximum (37.02 U/mg) specific activity at 20 °C, with casein as substrate. It was stable and active between the temperature range of 5–35 °C and pH 6.0–12.0, with an optimum temperature of 20 °C at pH 9.0. The Apr-BO1 had low Km value of 1.0 mg/ml and Vmax 10.0 µmol/ml/min. Moreover, it displayed better tolerance to organic solvents, surfactants, metal ions and reducing agents than most alkaline proteases. The results exhibited that it effectively removed the stains even in a cold wash and could be considered a decent detergent additive. Furthermore, through protein modelling, the structure of this protease was generated from template, subtilisin E of Bacillus subtilis (PDB ID: 3WHI), and different methods checked its quality. For the first time, this study reported the protein sequence for psychrotrophic Apr-BO1 and brought forth its novelty among other cold-active proteases.

scholarly journals Characterization of a Novel Fructosyltransferase from Lactobacillus reuteri That Synthesizes High-Molecular-Weight Inulin and Inulin Oligosaccharides

2002 ◽  
Vol 68 (9) ◽  
pp. 4390-4398 ◽  
Author(s):  
S. A. F. T. van Hijum ◽  
G. H. van Geel-Schutten ◽  
H. Rahaoui ◽  
M. J. E. C. van der Maarel ◽  
L. Dijkhuizen
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Lactobacillus Reuteri ◽  
Bacterial Strains ◽  
Potential Health ◽  
Isolation And Characterization ◽  
A Cell ◽  
Encoding Gene ◽  
First Time

ABSTRACT Fructosyltransferase (FTF) enzymes produce fructose polymers (fructans) from sucrose. Here, we report the isolation and characterization of an FTF-encoding gene from Lactobacillus reuteri strain 121. A C-terminally truncated version of the ftf gene was successfully expressed in Escherichia coli. When incubated with sucrose, the purified recombinant FTF enzyme produced large amounts of fructo-oligosaccharides (FOS) with β-(2→1)-linked fructosyl units, plus a high-molecular-weight fructan polymer (>107) with β-(2→1) linkages (an inulin). FOS, but not inulin, was found in supernatants of L. reuteri strain 121 cultures grown on medium containing sucrose. Bacterial inulin production has been reported for only Streptococcus mutans strains. FOS production has been reported for a few bacterial strains. This paper reports the first-time isolation and molecular characterization of (i) a Lactobacillus ftf gene, (ii) an inulosucrase associated with a generally regarded as safe bacterium, (iii) an FTF enzyme synthesizing both a high molecular weight inulin and FOS, and (iv) an FTF protein containing a cell wall-anchoring LPXTG motif. The biological relevance and potential health benefits of an inulosucrase associated with an L. reuteri strain remain to be established.

scholarly journals The Effect of Octapeptide Repeats on Prion Folding and Misfolding

2021 ◽  
Vol 22 (4) ◽  
pp. 1800
Author(s):  
Kun-Hua Yu ◽  
Mei-Yu Huang ◽  
Yi-Ru Lee ◽  
Yu-Kie Lin ◽  
Hau-Ren Chen ◽  
...  
Keyword(s):  
Amyloid Fibrils ◽  
Age Of Onset ◽  
Prion Diseases ◽  
Critical Role ◽  
Threshold Effect ◽  
Central Feature ◽  
Terminal Domain ◽  
Amyloid Aggregates

Misfolding of prion protein (PrP) into amyloid aggregates is the central feature of prion diseases. PrP has an amyloidogenic C-terminal domain with three α-helices and a flexible tail in the N-terminal domain in which multiple octapeptide repeats are present in most mammals. The role of the octapeptides in prion diseases has previously been underestimated because the octapeptides are not located in the amyloidogenic domain. Correlation between the number of octapeptide repeats and age of onset suggests the critical role of octapeptide repeats in prion diseases. In this study, we have investigated four PrP variants without any octapeptides and with 1, 5 and 8 octapeptide repeats. From the comparison of the protein structure and the thermal stability of these proteins, as well as the characterization of amyloids converted from these PrP variants, we found that octapeptide repeats affect both folding and misfolding of PrP creating amyloid fibrils with distinct structures. Deletion of octapeptides forms fewer twisted fibrils and weakens the cytotoxicity. Insertion of octapeptides enhances the formation of typical silk-like fibrils but it does not increase the cytotoxicity. There might be some threshold effect and increasing the number of peptides beyond a certain limit has no further effect on the cell viability, though the reasons are unclear at this stage. Overall, the results of this study elucidate the molecular mechanism of octapeptides at the onset of prion diseases.

scholarly journals Expression, Purification, and Characterization of (R)-Sulfolactate Dehydrogenase (ComC) from the Rumen MethanogenMethanobrevibacter milleraeSM9

Archaea ◽  
2017 ◽  
Vol 2017 ◽  
pp. 1-6
Author(s):  
Yanli Zhang ◽  
Linley R. Schofield ◽  
Carrie Sang ◽  
Debjit Dey ◽  
Ron S. Ronimus
Keyword(s):  
Biosynthetic Pathway ◽  
Critical Role ◽  
Reduction Reaction ◽  
Coenzyme M ◽  
Purification And Characterization ◽  
The Third ◽  
Optimal Activity ◽  
Methyl Coenzyme M Reductase

(R)-Sulfolactate dehydrogenase (EC 1.1.1.337), termed ComC, is a member of an NADH/NADPH-dependent oxidoreductase family of enzymes that catalyze the interconversion of 2-hydroxyacids into their corresponding 2-oxoacids. The ComC reaction is reversible and in the biosynthetic direction causes the conversion of (R)-sulfolactate to sulfopyruvate in the production of coenzyme M (2-mercaptoethanesulfonic acid). Coenzyme M is an essential cofactor required for the production of methane by the methyl-coenzyme M reductase complex. ComC catalyzes the third step in the first established biosynthetic pathway of coenzyme M and is also involved in methanopterin biosynthesis. In this study, ComC fromMethanobrevibacter milleraeSM9 was cloned and expressed inEscherichia coliand biochemically characterized. Sulfopyruvate was the preferred substrate using the reduction reaction, with 31% activity seen for oxaloacetate and 0.2% seen forα-ketoglutarate. Optimal activity was observed at pH 6.5. The apparentKMfor coenzyme (NADH) was 55.1 μM, and for sulfopyruvate, it was 196 μM (for sulfopyruvate theVmaxwas 93.9 μmol min−1 mg−1andkcatwas 62.8 s−1). The critical role of ComC in two separate cofactor pathways makes this enzyme a potential means of developing methanogen-specific inhibitors for controlling ruminant methane emissions which are increasingly being recognized as contributing to climate change.

Isolation and characterization of SRF accessory proteins

1993 ◽  
Vol 340 (1293) ◽  
pp. 325-332 ◽  
Keyword(s):  
Ternary Complex ◽  
Map Kinases ◽  
Serum Response Factor ◽  
Regulatory Element ◽  
Isolation And Characterization ◽  
Activation Of Transcription ◽  
Serum Response ◽  
Dna Binding Properties

Many genes which are regulated by growth factors contain a common regulatory element, the serum response element (SRE). Activation of transcription by the SRE involves a ternary complex formed between a ubiquitous factor, serum response factor (SRF), and a second protein, p62/TCF. We used a yeast genetic screen to isolate cDNAs encoding a protein, SAP-1, with the DNA binding properties of p62/TCF. The SAP-1 sequence contains three regions of homology to the previously uncharacterized Elk-1 protein, which also acts as an SRF accessory protein. Only two of these regions are required for cooperative interactions with SRF in the ternary complex. The third contains several conserved sites for the MAP kinases, whose activity is regulated in response to growth factor stimulation. We discuss the potential role of these proteins in regulation of the c-fos SRE.

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