Presumptive Identification of the Fungal Communities in Cystic Fibrosis Patients' Sputum Using Amplicon Length Heterogeneity Polymerase Chain Reaction

This thesis would not have been possible without the aid of my family, friends, laboratory members, and professors. First and foremost, I would like to thank Dr. Kalai Mathee for allowing me to enter her lab in August 2007 and enabling to embark on this journey. This experience has transformed me into more mature scientist, teaching me how to ask the right questions and the process needed to solve them. I would also like to acknowledge Dr. Lisa Schneper. She has helped me throughout the whole process, by graciously giving me input at every step of the way. I would like to express gratitude to Dr. Jennifer Richards for all her input in writing the thesis. She has been a great teacher and being in her class has been a pleasure. Moreover, I would like to thank all the committee members for their constructive criticism throughout the process. When I entered the lab in August, there was one person who literally was by my side, Melissa Doud. Without your input and guidance I would not have even been able to do these experiments. I would also like to thank you and Dr. Light for allowing me to meet some cystic fibrosis patients. It has allowed me to put a face on the disease, and help the patients' fight. For a period before I had entered the lab, Ms. Doud had an apprentice, who started the fungal aspect of the project, Caroline Veronese. Her initial work has enabled me to prefect the protocols and complete the ITS 1 region.One very unique aspect about Dr. Mathee's lab is the camaraderie. I would like to thank all the lab members for the good times in and out of the lab. These individuals have been able to make smile and laugh in parties and lab meetings. I would like to individually thank Balachandar Dananjeyan, Deepak Balasubramanian, and V arinderpal Singh Pannu for all the PCR help and Natalie Maricic for the laughs and being a great classmate. Last, but not least, I would like to acknowledge my family and friends for their support and keeping me sane: Cecilia, my mother, Mohammad, my father, Amir, my older brother, Billal, my younger brother, Ouday Akkari and Stephanie De Bedout, my best friends.

Avaliação da atividade antimicrobiana das defensinas recombinantes de ervilha (drr230a) e café (cd1) produzidas em Pichia pastoris

The inefficiency of chemical pesticides to control phytopathogenic fungi in agriculture and the frequent incidence of human diseases caused by bacteria which are resistant to antibiotics lead to the search for alternative antimicrobial compounds. In this context, plant defensins are a promising tool for the control of both plant and human pathogenic agents. Plant defensins are cationic peptides of about 50 amino acid residues, rich in cysteine and whose tridimensional structure is considerably conserved among different plant species. These antimicrobial molecules represent an important innate component from plant defense response against pathogens and are expressed in various plant tissues, such as leaves, tubers, flowers, pods and seeds. The present work aimed at the evaluation of the antimicrobial activity of two plant defensins against different phytopathogenic fungi and pathogenic bacteria to humans. The defensin Drr230a, whose gene was isolated from pea (Pisum sativum), and the defensin CD1,whose gene was identified within coffee (Coffea arabica) transcriptome, were subcloned in yeast expression vector and expressed in Pichia pastoris. The gene cd1 was subcloned as two different recombinant forms: CD1tC, containing a six-histidine sequence (6xHis) at the peptide C-terminal region and CD1tN, containing 6xHis coding sequence at the N-terminal region. In the case of the defensin Drr230a, the 6xHis coding sequence was inserted only at the N-terminal region. Assays of the antimicrobial activity of the purified recombinant proteins rDrr230a and rCD1 against Phakopsora pachyrhizi, causal agent of soybean Asian rust, were performed to analyze the in vitro spore germination inhibition and disease severity caused by the fungus in planta. Both recombinant defensins were able to inhibit P. pachyrhizi uredospore germination, with no difference between the antimicrobial action of either CD1tC or CD1tN. Moreover, rDrr230a and rCD1 drastically reduced severity of soybean Asian rust, as demonstrated by in planta assays. In spite of the fact that rCD1 was not able to inhibit proliferation of the human pathogenic bacteria Staplylococcus aureus and Klebsiella pneumoniae, rCD1 was able to inhibit growth of the phytopathogenic fungus Fusarium tucumaniae, that causes soybean sudden death syndrome. The obtained results show that these plant defensins are useful candidates to be used in plant genetic engineering programs to control agriculture impacting fungal diseases.

Medical mycology and fungal immunology : new research perspectives addressing a major world health challenge

Peer reviewed

Pollen and spore counts of sediment core GeoB12624

In tropical eastern Africa, vegetation distribution is largely controlled by regional hydrology, which has varied over the past 20 000 years. Therefore, accurate reconstructions of past vegetation and hydrological changes are crucial for a better understanding of climate variability in the tropical southeastern African region. We present high-resolution pollen records from a marine sediment core recovered offshore of the Rufiji River delta. Our data document significant shifts in pollen assemblages during the last deglaciation, identifying, through changes in both upland and lowland vegetation, specific responses of plant communities to atmospheric (precipitation) and coastal (coastal dynamics and sea-level changes) alterations. Specifically, arid conditions reflected by a maximum pollen representation of dry and open vegetation occurred during the Northern Hemisphere cold Heinrich event 1 (H1), suggesting that the expansion of drier upland vegetation was synchronous with cold Northern Hemisphere conditions. This arid period is followed by an interval in which forest and humid woodlands expanded, indicating a hydrologic shift towards more humid conditions. Droughts during H1 and the shift to humid conditions around 14.8 kyr BP in the uplands are consistent with latitudinal shifts of the intertropical convergence zone (ITCZ) driven by high-latitude Northern Hemisphere climatic fluctuations. Additionally, our results show that the lowland vegetation, consisting of well-developed salt marshes and mangroves in a successional pattern typical for vegetation occurring in intertidal habitats, has responded mainly to local coastal dynamics related to marine inundation frequencies and soil salinity in the Rufiji Delta as well as to the local moisture availability. Lowland vegetation shows a substantial expansion of mangrove trees after ~ 14.8 kyr BP, suggesting an increased moisture availability and river runoff in the coastal area. The results of this study highlight the decoupled climatic and environmental processes to which the vegetation in the uplands and the Rufiji Delta has responded during the last deglaciation.

Water-, pH- and temperature relations of germination for the extreme xerophiles Xeromyces bisporus (FRR 0025), Aspergillus penicillioides (JH06THJ), and Eurtotium halophilicum (FRR 2471)

Water activity, temperature and pH are determinants for biotic activity of cellular systems, biosphere function and, indeed, for all life processes. This study was carried out at high concentrations of glycerol, which concurrently reduces water activity and acts as a stress protectant, to characterize the biophysical capabilities of the most extremely xerophilic organisms known. These were the fungal xerophiles: Xeromyces bisporus (FRR 0025), Aspergillus penicillioides (JH06THJ) and Eurotium halophilicum (FRR 2471). High-glycerol spores were produced and germination was determined using 38 media in the 0.995–0.637 water activity range, 33 media in the 2.80–9.80 pH range and 10 incubation temperatures, from 2 to 50°C. Water activity was modified by supplementing media with glycerol+sucrose, glycerol+NaCl and glycerol+NaCl+sucrose which are known to be biologically permissive for X. bisporus, A. penicillioides and E. halophilicum respectively. The windows and rates for spore germination were quantified for water activity, pH and temperature; symmetry/asymmetry of the germination profiles were then determined in relation to supra- and sub-optimal conditions; and pH- and temperature optima for extreme xerophilicity were quantified. The windows for spore germination were ~1 to 0.637 water activity, pH 2.80–9.80 and > 10 and < 44°C, depending on strain. Germination profiles in relation to water activity and temperature were asymmetrical because conditions known to entropically disorder cellular macromolecules, i.e. supra-optimal water activity and high temperatures, were severely inhibitory. Implications of these processes were considered in relation to the in-situ ecology of extreme conditions and environments; the study also raises a number of unanswered questions which suggest the need for new lines of experimentation.

Monitoring Infection Risk for Air and Soil Borne Fungal Plant Pathogens using Antibody and DNA Techniques and Mathematical Models describing Environmental Parameters

Economic losses resulting from disease development can be reduced by accurate and early detection of plant pathogens. Early detection can provide the grower with useful information on optimal crop rotation patterns, varietal selections, appropriate control measures, harvest date and post harvest handling. Classical methods for the isolation of pathogens are commonly used only after disease symptoms. This frequently results in a delay in application of control measures at potentially important periods in crop production. This paper describes the application of both antibody and DNA based systems to monitor infection risk of air and soil borne fungal pathogens and the use of this information with mathematical models describing risk of disease associated with environmental parameters.

Genetic Modification of Disease Resistance: Fungal and Oomycete Pathogens

Development of recombinant DNA technology allowed scientists to manipulate plant genomes, making it possible to study genes and exploit them to modify novel agronomic traits. Here, we review the current and future potential of genetic modification (GM) strategies used to increase the resistance of plants to oomycete and fungal pathogens. Numerous resistance genes (R-genes) have been cloned, and under laboratory conditions, transgenic plants have given promising results against some important plant pathogens. However, only a few have so far been deployed as commercial crop plants.GMof plants to disrupt pathogenicity, such as by inhibiting or degrading pathogenicity factors, especially by necrotrophic pathogens, has also been exploited. The potential to engineer plants for the production of antimicrobial peptides or to modify defense-signaling pathways have been successfully demonstrated under laboratory conditions. The most promising current technology is genome editing, which allows researchers to edit DNA sequences directly in their endogenous environment. The potential of this approach is discussed in detail and examples where broad-spectrum resistance has been achieved are given.

Fungal and Oomycete Diseases

Fungal and oomycete pathogens are the causal agents of many important plant diseases. They affect crops that are staple foods for humans and livestock and are responsible for significant economic losses every year. This in turn generates a global social impact. Although fungi and oomycetes evolved separately, they share similar strategies and weaponry to attack plants. Here we review the challenges to global food security posed by these pathogens, current technologies used for detection and diagnostics, the latest understanding of pathogens' strategies to colonize plants, and current and future control measures. Genomic sequences of several important fungal and oomycete pathogens, as well as many crop plants, are now available and are helping to increase understanding of host–pathogen interactions. Recent developments in this field are discussed.

Fungal contamination of sandpits from recreational parks and schools: a potential risk for human health

Sandpits used by children are frequently visited by wild life which constitutes a source of fungal pathogens and allergenic fungi. This study aimed to take an unannounced snapshot of the urban levels of fungal contaminants in sands, using for this purpose two public recreational parks, three elementary schools and two kindergartens. All samples were from Lisbon and neighboring municipalities and were tested for fungi of clinical interest. Potentially pathogenic fungi were isolated from all samples besides one. Fusarium dimerum (32.4%) was found to be the dominant species in one park and Chrysonilia spp. in the other (46.6%). Fourteen different species and genera were detected and no dermatophytes were found. Of a total of 14 species and genera, the fungi most isolated from the samples of the elementary schools were Penicillium spp. (74%), Cladophialophora spp. (38%) and Cladosporium spp. (90%). Five dominant species and genera were isolated from the kindergartens. Penicillium spp. was the only genus isolated in one, though with remarkably high counts (32500 colony forming units per gram). In the other kindergarten Penicillium spp. were also the most abundant species, occupying 69% of all the fungi found. All of the samples exceeded the Maximum Recommended Value (MRV) for beach sand defined by Brandão et al. 2011, which are currently the only quantitative guidelines available for the same matrix. The fungi found confirm the potential risk of exposure of children to keratinophilic fungi and demonstrates that regular cleaning or replacing of sand needs to be implemented in order to minimize contamination.

Analysis of surfaces for characterization of fungal burden: does it matter?

Objectives: Mycological contamination of occupational environments can be a result of fungal spores’ dispersion in the air and on surfaces. Therefore, it is very important to assess it in both types of the samples. In the present study we assessed fungal contamination in the air and in the surface samples to show relevance of surfaces sampling in complementing the results obtained in the air samples. Material and Methods: In total, 42 settings were assessed by the analysis of air and surfaces samples. The settings were divided into settings with a high fungal load (7 poultry farms and 7 pig farms, 3 cork industries, 3 waste management plants, 2 wastewater treatment plants and 1 horse stable) and a low fungal load (10 hospital canteens, 8 college canteens and 1 maternity hospital). In addition to culture-based methods, molecular tools were also applied to detect fungal burden in the settings with a higher fungal load. Results: From the 218 sampling sites, 140 (64.2%) presented different species in the examined surfaces when compared with the species identified in the air. A positive association in the high fungal load settings was found between the presence of different species in the air and surfaces. Wastewater treatment plants constituted the setting with the highest number of different species between the air and surface. Conclusions: We observed that surfaces sampling and application of molecular tools showed the same efficacy of species detection in high fungal load settings, corroborating the fact that surface sampling is crucial for a correct and complete analysis of occupational scenarios.

Prevalence of Aspergillus section Fumigati in Portuguese slaughterhouses: a fungal and mycotoxin concern

Introduction - Within the Aspergillus genus, Aspergillus fumigatus species is one of the most ubiquitous saprophytic fungi and is considered the species with higher clinical relevance. The fungi belonging to the Fumigati section are the most common cause of invasive aspergillosis and a major source of infection related mortality in immunocompromised patients. One of the most abundant metabolites produced by Aspergillus fumigatus is the metabolite gliotoxin, which exhibits a diverse array of biologic effects on the immune system. Further, environments contaminated with A. fumigatus may be the cause or enhance respiratory problems in the workers of those specific settings. These species produce specific allergens and mycotoxins that could cause respiratory disorders. Aim of the study - The aim of the present work was to determine the prevalence of Aspergillus section Fumigati by cultural and molecular methods in poultry; swine and bovine; and large animal (bovine and horses) slaughterhouses.

Fungal contamination in coffee samples: a public health concern

Introduction - Fungi are natural coffee contaminants and under certain environmental conditions have the potential to produce toxins. Many studies revealed that the important toxigenic fungal genera (Aspergillus and Penicillium) are natural coffee contaminants, and are present from the field to storage. Aspergilli from the Circumdati and Nigri sections are known to produce high levels of ochratoxin A, a mycotoxin known as nephrotoxic for animals and humans. This work aimed to evaluate fungal distribution and also the prevalence of Aspergillus sections Fumigati, Flavi, Nigri and Circumdati from Coffea arabica (Arabica coffee) and Coffea canephora (Robusta coffee) green samples.