Defining Critical Genes During Spherule Remodeling and Endospore Development in the Fungal Pathogen, Coccidioides posadasii.

Coccidioides immitis and C. posadasii are soil dwelling dimorphic fungi found in North and South America. Inhalation of aerosolized asexual conidia can result in asymptomatic, acute, or chronic respiratory infection. In the United States there are approximately 350,000 new infections per year. The Coccidioides genus is the only known fungal pathogen to make specialized parasitic spherules, which contain endospores that are released into the host upon spherule rupture. The molecular determinants involved in this key step of infection remain largely elusive as 49% of genes are hypothetical with unknown function. An attenuated mutant strain C. posadasii Δcts2/Δard1/Δcts3 in which chitinase genes 2 and 3 were deleted was previously created for vaccine development. This strain does not complete endospore development, which prevents completion of the parasitic lifecycle. We sought to identify pathways active in the wild-type strain during spherule remodeling and endospore formation that have been affected by gene deletion in the mutant. We compared the transcriptome and volatile metabolome of the mutant Δcts2/Δard1/Δcts3 to the wild-type C735. First, the global transcriptome was compared for both isolates using RNA sequencing. The raw reads were aligned to the reference genome using TOPHAT2 and analyzed using the Cufflinks package. Genes of interest were screened in an in vivo model using NanoString technology. Using solid phase microextraction (SPME) and comprehensive two-dimensional gas chromatography - time-of-flight mass spectrometry (GC × GC-TOFMS) volatile organic compounds (VOCs) were collected and analyzed. Our RNA-Seq analyses reveal approximately 280 significantly differentially regulated transcripts that are either absent or show opposite expression patterns in the mutant compared to the parent strain. This suggests that these genes are tied to networks impacted by deletion and may be critical for endospore development and/or spherule rupture in the wild-type strain. Of these genes, 14 were specific to the Coccidioides genus. We also found that the wild-type and mutant strains differed significantly in their production versus consumption of metabolites, with the mutant displaying increased nutrient scavenging. Overall, our results provide the first targeted list of key genes that are active during endospore formation and demonstrate that this approach can define targets for functional assays in future studies.

Direct detection of Coccidioides from Arizona soils using CocciENV, a highly sensitive and specific real-time PCR assay.

Coccidioides immitis and Coccidioides posadasii are soil fungi endemic to desert regions of the southwestern United States, and the causative agents of valley fever, or coccidioidomycosis. Studies have shown that the distribution of Coccidioides in soils is sporadic and cannot be explained by soil characteristics alone, suggesting that biotic and other abiotic factors should be examined. However, tools to reliably and robustly screen the large number of soils needed to investigate these potential associations have not been available. Thus, we developed a real-time polymerase chain reaction (PCR) assay for testing environmental samples by modifying CocciDx, an assay validated for testing clinical specimens to facilitate coccidioidomycosis diagnosis. For this study, we collected soil samples from previously established locations of C. posadasii in Arizona and new locations in fall 2013 and spring 2014, and screened the extracted DNA with the new assay known as CocciEnv. To verify the presence of Coccidioides in soil using an alternate method, we employed next generation amplicon sequencing targeting the ITS2 region. Results show our modified assay, CocciEnv, is a rapid and robust method for detecting Coccidioides DNA in complex environmental samples. The ability to test a large number of soils for the presence of Coccidioides is a much-needed tool in the understanding of the ecology of the organism and epidemiology of the disease and will greatly improve our understanding of this human pathogen.

The population biology of coccidioides: epidemiologic implications for disease outbreaks.

Studies of field- and patient-derived isolates conducted over the past 75 years have provided a general picture of the population structure of Coccidioides, the cause of coccidioidomycosis. Premolecular studies provided a general outline of the geographical range, epidemiology and distribution of the fungus. Recent studies based on molecular markers have demonstrated that the genus is comprised of two genetically diverse, and genetically isolated, species: Coccidioides immitis and C. posadasii. Both species are composed of biogeographically distinct populations. Structure for two of these populations (C. immitis from central California, and C. posadasii from southern Arizona) indicates that frequent genetic recombination occurs within the entire geographic range of each population, even though sex has never been observed in the genus. Outbreaks of coccidioidomycosis are not the result of the spread of a single clonal isolate, but are caused by a diversity of genotypes. Although it is now possible to match patient isolates to populations, the lack of apparent structure within each population and the current paucity of environmental isolates limit map-based epidemiological approaches to understanding outbreaks. Therefore, a comprehensive database comprised of soil-derived isolates from across the biogeographic range of Coccidioides will improve the utility of this approach. Appropriate collection of environmental isolates will assist the investigation of remaining questions regarding the population biology of Coccidioides. The comparative genomics of representative genotypes from both species and all populations of Coccidioides will provide a thorough set of genetic markers in order to resolve the population genetics of this pathogenic fungus.

Quantifying Aphanomyces euteiches in Alfalfa with a Fluorescent Polymerase Chain Reaction Assay.

ABSTRACT A polymerase chain reaction (PCR) assay using a set of specific primers and a dual-labeled probe (TaqMan) was developed to quantify the amount of Aphanomyces euteiches DNA in alfalfa plants exhibiting varying levels of disease severity. The study included isolates of race 1 and race 2 of A. euteiches. The assay also discriminated between alfalfa populations for resistance based on analysis of DNA extracted from bulked plant samples. Analysis of individual plants and bulked plant samples of standard check populations with both pathogen isolates resulted in Spearman rank correlations between pathogen DNA content and disease severity index ratings that were greater than 0.75 and highly significant (P < 0.0005). In experiments with a race 1 isolate, the amount of pathogen DNA present in the resistant check WAPH-1 was significantly less than in the susceptible check Saranac. In experiments with a race 2 isolate, the amount of pathogen DNA in the resistant check WAPH-5 was significantly less than in either of the susceptible checks, Saranac and WAPH-1. Discrimination between commercial cultivars based on quantitative PCR analysis of bulked plant samples was similar to classification based on visual assessment of disease severity.

Factor analysis of the items of the State-Trait Anxiety Inventory.

The present investigation was designed to determine by factor analysis the nature of the items that comprise the A-State and A-Trait scales of the State-Trait Anxiety Inventory. Three factors were identified. Factor I was defined exclusively by items from the A-State scale. The underlying dimension tapped by the scale was interpreted as state anxiety (how one feels at a particular moment in time); support thus was provided for Spielberger's A-State concept. Items from the A-Trait scale, however, identified two separate factors, neither of which was clearly consonant with Spielberger's concept of A-Trait. Factor II appeared to tap state anxiety according to how the individual generally feels or a typical level of state anxiety as remembered over an indefinite period of time. Factor III was interpreted as a measure of neuroticism.

Interrelationships of perceptual modality, short-term memory and reading achievement.

Visual and auditory components of short-term memory and perception were used as predictors of vocabulary and comprehension components of reading for 72 children from Grades 2 to 5 in a low socio-economic rural school. All six variables were significantly intercorrelated (with the exception of visual short-term memory and auditory perception). When canonical correlation analysis was applied using the four scores measuring short-term memory and perception as predictors of the two reading scores, one was significant, and each variable made a significant contribution. Not only are short-term memory and perception a part of learning to read but both visual and auditory channels are important.