Gene Disruption in Coccidioides Using Hygromycin or Phleomycin Resistance Markers
The following transformation protocol is based on homologous recombination that occurs between a gene disruption or gene replacement construct and a target gene of Coccidioides. The DNA constructs employed contain either the gene that encodes for hygromyc
- PDF / 444,896 Bytes
- 17 Pages / 504.57 x 720 pts Page_size
- 98 Downloads / 206 Views
. Introduction Coccidioides spp. are the causative agent of a human respiratory disease known as coccidioidomycosis or San Joaquin Valley Fever. Two species of Coccidioides have been reported on the basis of molecular and biogeographical differences: Coccidioides immitis is found primarily in the San Joaquin Valley of California, while C. posadasii is widespread throughout endemic regions in the Americas (1).
Alexandra C. Brand and Donna M. MacCallum (eds.), Host-Fungus Interactions: Methods and Protocols, Methods in Molecular Biology, vol. 845, DOI 10.1007/978-1-61779-539-8_9, © Springer Science+Business Media, LLC 2012
131
132
C.-Y. Hung et al.
In spite of the genetic diversity revealed by comparative genomic sequence analyses of these two species (2), laboratory animal studies have shown no significant difference in either the virulence or growth and development of the organisms. The pathogen is a diphasic fungus that produces mycelia and air-dispersed spores (arthroconidia) when grown on a simple glucose-yeast extract (GYE) agar medium and a complex spherule-endospore cycle when cultured in a defined glucose/salts medium (3). Inhalation of airborne spores by a mammalian host is followed by development of an elaborate parasitic cycle in lung tissue, which is unique amongst the medically important fungi. The parasitic cycle is initiated by conversion of the tiny barrel-shaped arthroconidia (ca. 2 × 6 μm) into multinucleate round cells that grow isotropically to produce large spherules (60 to >100 μm in diameter). The content of a mature spherule undergoes a complex process of differentiation to yield an average of 200–300 endospores in vivo, each with an initial diameter of approximately 4–6 μm. Endospores that are released from the maternal spherule and survive within the host undergo isotropic growth and give rise to a second generation of endosporulating spherules (4). Coccidioides is a formidable opportunistic human pathogen which can cause disease in immunocompromised people, as well as a primary pathogen that can infect healthy individuals who reside and work in or visit the endemic areas. About 60% of primary pulmonary infections with Coccidioides are asymptomatic, evident only by skin test reactivity (5). In the remaining 40%, symptomatic infection can occur in various manifestations: acute pneumonia, chronic progressive pneumonia, appearance of pulmonary nodules and cavities, extrapulmonary non-meningeal disease, or meningitis (6). Coccidioidomycosis has also been reported to be a frequent laboratory-acquired, respiratory disease (7). Plate cultures of the saprobic phase of Coccidioides grown in laboratory incubators produce large numbers of dry spores which can easily contaminate the environment if not handled properly. Live cultures of Coccidioides spp. must be maintained in a facility with biological safety level 3 (BSL3) containment because of the highly infectious nature of Coccidioides arthroconidia (8). Both the saprobic and parasitic phases of the two Coccidioides species are haploid (9). Coc
Data Loading...
