CHEMTRAILS/MORGELLON? Analysis of Respiratory Fungal Pathogens

This 2005 study may amplify the findings of forensic aerosol scientist, Cliff Carnicom. Chlamydospore Formation during Hyphal Growth in Cryptococcus neoformans Link: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1265899 Eukaryot Cell. 2005 October; 4(10): 1746–1754. doi: 10.1128/EC.4.10.1746-1754.2005. PMCID: PMC1265899 Copyright © 2005, American Society for Microbiology Xiaorong Lin and Joseph Heitman* Department of Molecular Genetics and Microbiology, Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710 *Corresponding author. Mailing address: Room 322 CARL Building, Box 3546, Research Drive, Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27710. Phone: (919) 684-2824. Fax: (919) 684-5458. E-mail: heitm001@duke.edu. Received June 17, 2005; Accepted July 26, 2005. Abstract Cryptococcus neoformans, a basidiomycetous fungal pathogen, infects hosts through inhalation and can cause fatal meningoencephalitis in individuals if untreated. This fungus undergoes a dimorphic transition from yeast to filamentous growth during mating and monokaryotic fruiting, which leads to the production of hyphae and airborne infectious basidiospores. Here we characterized a novel morphological feature associated with the filamentous stages of the life cycle of C. neoformans which resembles resting or survival structures known as chlamydospores in other fungi. The C. neoformans chlamydospore-like structure is rich in glycogen, suggesting that it might have a role as an energy store. However, characterization of mutants with decreased or increased levels of glycogen production showed that glycogen levels have little effect on filamentous growth, sporulation, or chlamydospore formation. These results suggest that the formation of chlamydospores is independent of glycogen accumulation level. We also show that chlamydospore formation does not require successful sporulation and that the presence of chlamydospores is not sufficient for sporulation. Although the biological functions of chlamydospores remain to be established for this pathogenic fungus, their formation appears to be an integral part of the filamentation process, suggesting that they could be necessary to support sexual sporulation under adverse conditions and thereby facilitate the production of infectious basidiospores or long-term survival propagules in harsh environments. Chlamydospores are produced by many fungi and represent enlarged, thick-walled vegetative cells with varied forms and condensed cytoplasm that form within hyphae or at hyphal tips. Despite poor cytological descriptions or documentation of their mode of generation, chlamydospores have been observed in three major clades of the fungal kingdom. For example, the basidiomycete black ink mushroom Coprinus cinereus (28) and Cryptococcus laurentii (30), the ascomycete nematode-trapping fungus Duddingtonia flagrans (20, 41), and zygomycete mucorales, such as Rhizopus schipperae (4, 55), have all been shown to produce chlamydospores. Even the fungus-like oomycete plant pathogens Phytophthora cinnamomi and Phytophthora parasitica produce chlamydospores. Biological functions ascribed to these chlamydospores differ between species. For example, desiccation-resistant chlamydospores of P. cinnamomi are produced within plant roots during drought and are transported in root fragments or soil, germinating to cause infections when warm, moist conditions are encountered. When chlamydospores of the nematode-trapping fungus D. flagrans are fed to domesticated animals, they can survive passage through the alimentary tract and reduce the number of parasitic nematode larvae that develop from eggs in the feces, thus preventing clinical disease (20, 41). In addition, the chlamydospore developmental phase of Aspergillus parasiticus has been associated with increased aflatoxin production, while chlamydospores of Fusarium species are the principal means of long-term survival during unfavorable periods in the soil and play an important role as the primary inocula infecting plants (1, 14). Chlamydospores have also been observed in human fungal pathogens such as Candida albicans, Paracoccidioides brasiliensis, Histoplasma capsulatum, and Blastomyces dermatitidis (19). Although the appearance of chlamydospores in C. albicans serves as a diagnostic test, these structures are rarely found in infected patients or animals and their pathological or biological functions are as yet unclear (11, 13, 36). The environmental cues for chlamydospore formation for various fungi are usually species specific and include nutrients (6, 27, 40), osmolarity, light (25, 29), pH (44), temperature (40), air (6), drug treatment (13), and plant stimulants (26). Because chlamydospores are not known to be formed in the model yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, relatively less is known about the molecular mechanisms controlling chlamydospore formation, with the exception of information gleaned from certain strains of Candida species (2, 25, 39, 46, 47). Thus, although chlamydospores are produced by a broad assortment of fungi, their mechanisms of formation, biological functions, and molecular regulation remain enigmatic. Here we describe the first identification of chlamydospores in the basidiomycetous pathogen Cryptococcus neoformans, the most common cause of fungal meningoencephalitis in humans and which is capable of infecting both immunocompromised and apparently healthy hosts (10). Although C. neoformans exists in a single-celled yeast form during infection and in culture, this dimorphic fungus can switch from yeast to filamentous growth during mating and monokaryotic fruiting (3, 22, 31, 34, 53), which eventually leads to production of airborne meiotic basidiospores, thought to be involved in dispersal and infection. C. neoformans strains can be readily isolated from the environment, particularly from soil contaminated with aged pigeon guano (10). Due to their microscopic nature, structures that promote C. neoformans survival in the environment are not known and could be sexual basidiospores, mitotic yeast cells, filaments, or other unknown structures. While many soilborne fungi produce chlamydospores as long-lived survival structures under hostile environmental conditions (15, 44), there has been no previous report of such structures in C. neoformans. Here we describe the formation of a morphological structure associated with the filamentous growth of C. neoformans that is strikingly similar to chlamydospores produced by other fungi, providing the first documentation of such structures in this organism. These data reveal a novel growth option in the life cycle of C. neoformans and provide a robust and genetically tractable model for studying the morphogenesis and molecular basis of the development and function of these cellular structures. Discussion A clearer understanding of the reproductive modes and survival structures of fungal pathogens in the environment is of great importance with regard to their ecology and epidemiology. Budding, conidiation, sporulation, fragmentation of hyphae, and conversion of hyphal elements into chlamydospores are common modes of reproduction. In some soilborne fungi, chlamydospores have been documented to have a role as survival structures. This study is the first report of C. neoformans chlamydospores, which are produced behind the active hyphal growth zone during filamentous growth, and it elucidates a novel stage of the life cycle for this pathogen. Although chlamydospores have been observed in the related fungus Cryptococcus laurentii (30), it is, however, dangerous to extrapolate to other Cryptococcus species, as in the case in Candida species. Among all the Candida species, only the two most prevalent pathogens, Candida albicans and Candida dubliniensis, have been observed to produce chlamydospores, while other Candida species, such as Candida tropicalis, Candida glabrata, Candida parapsilosis, Candida krusei, Candida lusitaniae, Candida kefyr, and Candida guilliermondii, do not. Filaments of C. neoformans produce both intercalary and terminal chlamydospores, which are potentially fully functional (independent from the mycelium) and physiologically active, as they are capable of generating new branches and yeast cells. Although we found that chlamydospores in C. neoformans are enriched in glycogen, mutants with altered glycogen levels still form chlamydospores, suggesting the possibility for the storage of other materials and regulatory mechanisms independent of glycogen accumulation. Whether the energy stored in the chlamydospores is for their own survival and reproduction or to support proficient basidiospore production and/or maturation is an important unanswered question. Since we were unable to identify any single mutation that specifically blocks chlamydospore formation, the exact nature of the process that leads to chlamydospore production and the biological function of the structures remains to be defined. The inability of our genetic screen to separate the formation of chlamydospores from filamentous growth suggests that chlamydospore production could be an integral part of hyphal growth in C. neoformans in response to harsh environments. Basidiospores are proposed to be the propagules for C. neoformans dispersal and infection, and it is likely that basidiospores are also long-term survival structures in nature. However, in many other fungi, chlamydospores serve this role. Our identification of chlamydospores in C. neoformans suggests the interesting possibility of an overlooked role for these structures in the popular model of C. neoformans survival and propagation. It is possible that these two different reproduction modes of C. neoformans coexist in nature and serve independent biological roles, or alternatively, there may be a key connection between the formation of chlamydospores and the production of basidiospores. While we have shown that the formation of chlamydospores is apparently independent of the production of basidiospores, given that the tps1 mutant is blocked in sporulation during mating but can still form chlamydospores, this does not mean that sporulation is independent of chlamydospore formation. The availability of large-scale screens of insertional mutants or of a genome-wide deletion mutation collection in C. neoformans may yield insight into the relationship between these two processes and provide a model for chlamydospore production in related fungi. During our small-scale screen of insertion mutants, we did notice an inverse relationship between blastospore and chlamydospore production by vegetative hyphae, suggesting that there may be a balance between the formation of these two reproductive forms. We also observed that robust blastospore formation is usually associated with suppressed hyphae, poor aerial hyphal production, and sporulation, while chlamydospore formation is associated with better aerial hyphae production and sporulation. The different developmental pathways to produce blastospores or chlamydospores might reflect the choice that the hyphae make, either to maintain vegetative growth and multiply rapidly or to enter terminal growth, leading to the production of aerial hyphae and basidiospores. The balance between the three reproduction forms (blastospores, chlamydospores, and basidiospores) may be dependent on genetic background, developmental stages, and environmental cues that require further clarification and may yield new clues to the nature and formation of infectious C. neoformans propagules. Acknowledgments This work was supported by NIAID R01 grant AI50113 to J.H. J.H. was a Burroughs Welcome Fund Scholar in Molecular Pathogenic Mycology and an Investigator of the Howard Hughes Medical Institute. We thank Yong-Sun Bahn for strains; John Perfect, Alex Idnurm, James Fraser, Weihua Fan, and Felicia Walton for critical reading; and Arturo Casadevall, June Kwon-Chung, and Andrew Alspaugh for comments. This 2005 study may amplify the findings of forensic aerosol scientist, Cliff Carnicom. A few of the many relavent links are available at: http://carnicom.com/bio11.htm http://www.carnicom.com/culture3.htm Yeast, Candida Albicans and suppressed immune system vulnerability in humans. http://www.marconews.com/news/2009/apr/21/ask-pharmacist-candida-causes-dozens-disorders/