Medical Mycology, 2014, 52, 149–155doi: 10.1093/mmy/myt001

Advance Access Publication Date: 12 December 2013Review Article

Review Article

Genomics in Coccidioides: Insights into

evolution, ecology, and pathogenesis

Emily Whiston∗ and John W. Taylor

University of California–Berkeley, Department of Plant and Microbial Biology, Berkeley, California, USA

*To whom correspondence should be addressed. Emily Whiston, UC Berkeley, Dept. Plant and Microbial Biology, 321Koshland Hall, Berkeley CA 94720, USA. Tel: +1 (510) 642-8441; Fax: +1 (510) 642-4995; E-mail: whiston@berkeley.edu

Received 22 July 2013; Revised 18 September 2013; Accepted 7 October 2013

Abstract

Coccidioides immitis and C. posadasii, the causative agents of the mammalian dis-ease coccidioidomycosis, are dimorphic fungal pathogens distributed throughout desert-like environments in North and South America. Coccidioides spp. are members of theOnygenales, a diverse group of pathogenic and nonpathogenic fungi. Recently, fullgenomes have been published for Coccidioides and a number of other Onygenalesspecies. Phylogenomic comparisons and additional studies in Coccidioides populationgenomics and gene expression have shed light on the ecology and pathogenesis of Coc-cidioides and the other medically important species in this clade. Observed patterns ofgene family expansion/contraction and evidence of gene flow have provided insight tothe evolution of Coccidioides and greatly broadened our understanding of the diversityand sources of genetic variation found in fungi. In the future, expansion of the number ofsequenced isolates from all populations will allow deeper insight into the evolutionaryprocesses that have shaped this unique human pathogen. In addition, deep sequencingof isolates from a single Coccidioides population and pairing of those data with pheno-type information on growth and pathogenicity for genome-wide association analysis willallow researchers to find genes responsible for any phenotype, virulence included, thatshows variation in the population.

Key words: Coccidioides, genomics, next-generation sequencing, evolution, dimorphic fungal pathogen.

Introduction

Coccidioides immitis and C. posadasii are the causativeagents of the human disease, coccidioidomycosis (com-monly known as Valley fever or San Joaquin Valley fever).These dimorphic pathogens are associated with small mam-mals [1] and grow as typical fungal hyphae in the environ-ment and as endosporulating spherules in the mammalianhost. Asexual reproduction in hyphae produces arthroconi-dia, which can be inhaled by mammals and cause a pri-mary pulmonary infection. In the host, arthroconidia en-large to become spherules, in which endospores develop.

When released from mature spherules, endospores dissemi-nate in the host and enlarge to harbor the next generation ofspherules, which produce their own endospores in a contin-uing, complex cycle (Fig. 1). Although sexual reproductionhas not been observed, it is inferred to occur in both speciesbased on population genetic studies [2,3] of the balanceddistribution of mating type alleles (idiomorphs) [4,5] andon evidence of gene flow between the two Coccidioidesspecies [6].

In addition to small mammals, humans also inhale thespores and, although epidemiologic data are incomplete, at

C© The Author 2013. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

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Figure 1. Coccidioides spp. life cycle (adapted from [7]).

least 150c000 people annually are infected with Coccid-ioides spp. in the United States, 40% of whom developmild to severe pulmonary symptoms [8]. The majorityof infections resolve without medical intervention, but in1%–6% of diagnosed Coccidioides infections, endosporesfrom the lungs disseminate to other soft tissues and causesecondary infections. This disseminated disease is poten-tially fatal in healthy adults, although this more seriousform of infection is more common in children, the elderly,and immune-compromised patients [8–10]. The incidenceof coccidioidomycosis is rising in the American Southwest,where an almost 10-fold increase in reported disease wasnoted between 1998 and 2011, although these numbersare influenced by population growth, increased awareness,higher rates of testing, and improved reporting [10].

Coccidioides is found associated with small mammalsin desert-like environments in the New World. C. immitisis found throughout central and southern California andin northern Mexico, and C. posadasii is found through-out Arizona, Texas, Mexico, and parts of South America[11–16]. Recent evidence suggests that the range of Coccid-ioides spp. extends as far north as Washington state, whereseveral cases were reported in 2010–2011, with no patienthistory of recent travel [17]. There are no known pheno-typic differences in pathogenicity and the spherule growthphase between the two species. In the mycelial growth form,subtle differences in salt and thermal tolerance have beenobserved [6,11,18].

Coccidioides is a member of the Onygenales, which in-cludes nonpathogenic species, the dermatophyte pathogens(Arthroderma, Microsporum, and Trichophyton), and theother primary mammalian thermally dimorphic fungalpathogens (Fig. 2). Coccidioides excepted, the other ther-mally dimorphic fungal pathogens, Histoplasma, Blasto-

Figure 2. Distance phylogenetic tree. Onygenales in gray box. S,spherule-forming dimorphic fungal pathogens; D, dermatophytic fun-gal pathogens; Y, yeast-forming dimorphic fungal pathogens. Bayesiantree generated [19] from 100 randomly selected single-copy orthologs(OrthoMCL [20]) using the following genomes available from the BroadInstitute (http://www.broadinstitute.org/scientific-community/data): As-pergillus nidulans, A. fumigatus, Blastomces dermatiditis ER-3, Coccid-ioides immitis RS, C. posadasii Silveira, Histoplasma capsulatum NAm1,Microsporum gypseum CBS 118893, N. crassa OR74A/NC12, Paracoc-cidioides lutzii Pb01 (formerly P. basiliensis [21]), Trichophyton rubrumCBS 118892, and Uncinocarpus reesii.

myces, and Paracoccidioides, grow in a budding yeast mor-phology in the host environment (referred to throughout thetext as yeast-forming dimorphic pathogens [YFDP]). Notonly are there major morphological differences betweenspherules and yeast, there are also significant differences inimmune response to invading cells. Unlike yeast cells, whichare phagocytosed by host immune cells, mature spherulesare too large (60 to >100 µm in diameter) for phagocytosis[8].

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A previous article on Coccidioides focused on early pop-ulation genetics research that identified C. immitis andC. posadasii as separate species, identified geographic pop-ulations, and established the occurrence of recombinationin nature [22]. Recently, full genomes have been publishedfor Coccidioides and the other Onygenales species shownin Fig. 2. In addition to phylogenomic comparisons, stud-ies in Coccidioides population genomics and gene expres-sion have also recently been published. As with many otherorganisms, genome sequencing and comparative analyseshave shed considerable light on the ecology, evolution,and pathogenesis of Coccidioides and the other medicallyimportant species in this clade.

Comparative genomics of Coccidioides

and the Eurotiomycetes

The first Coccidioides full genomes, C. immitis isolate RSand C. posadasii isolate c735, were sequenced by the BroadInstitute and the J. Craig Venter Institute (JCVI, then the In-stitute for Genome Research), respectively. A 2009 phyloge-nomics study compared these two genomes with the otherOnygenales (Eurotiomycetes) that had been sequenced atthe time: Uncinocarpus reesii (nonpathogenic), Paracoccid-ioides brasiliensis (YFDP), three isolates of Histoplasmacapsulatum (YFDP), Blastomyces dermatitidis (YFDP), aswell as members of the Eurotiales (Eurotiomycetes) clade(eight species of Aspergillus and Penicillium marneffei)and representatives from the Sordariomycetes (Fusariumgraminearum and Sclerotinia sclerotiorum) [23]. This studylooked for evidence of natural selection in the form of genefamily expansion/contraction, individual gene gain/loss,and an excess of nonsynonymous nucleotide substitu-tions relative to synonymous substitutions (dn/ds) betweenC. immitis and C. posadasii.

The primary finding in this study was the discovery ofgene family contractions and expansions in the Onygenales.Throughout the order, contractions were seen in gene fam-ilies that produce enzymes that metabolize plant cell walls,for example, cellulase, tannase, cutinase, pectic lyase, andpectinesterase. In Coccidioides spp. and in U. reesii, thesecontractions were accompanied by expansions in gene fami-lies that produce enzymes that digest animal protein, for ex-ample, proteases and keratinases [23]. These observationsled to the hypothesis that the Onygenales clade, includ-ing its nonpathogenic members, had undergone a lifestyleswitch away from primarily plant food sources and thatsome species, including Coccidioides and U. reesii, hadadapted to dead animal food sources [23]. Similar genefamily patterns have since been observed in the Onygenalesdermatophyte species, which cause ringworm and athlete’sfoot [24].

Gene family expansions in protease gene families in-cluded the subtilases [23]. A similar expansion in the sub-tilase gene family has been observed in the Hypocreales,which includes pathogens of invertebrate animals [25]. Al-though subtilases have been shown to play a role in der-matophytic infections [26], not all animal pathogens sharethis gene family expansion, for example, Histoplasma, Blas-tomyces, and Paracoccidioides spp. show no elevation in thenumber of proteases [23,24]. Thus, the independent expan-sions observed in the Onygenales and Hypocreales may beunrelated to pathogenicity. Rather, the subtilase gene familymay be important for the use of vertebrate and invertebrateanimals as a food source—whether dead or alive [25].

Experimental studies of the growth of U. reesii on plantand animal food sources were included in a 2011 study thatfocused on comparative genomics of Paracoccidioides spp.[27]. This study found that U. reesii growth is restricted oncarbohydrates and considerably improved on proteins [27],providing evidence that the gene family changes observed inCoccidioides and other Onygenales have a true impact onthe growth and ecology of fungi in this clade. The emerginghypothesis is that Coccidioides species are associated withanimals in both the parasitic and environmental phases oftheir life cycle. This continual association with animals im-plies that their patchy distribution in soil is due to the patchydistribution of the carcasses of dead animals in which thefungus, when released from the high temperature and el-evated carbon dioxide concentration that favor spheruleformation, reverts to hyphal growth and begins to producearthroconidia [28].

In addition to gene family expansion and contrac-tion, the phylogenomics study [23] discovered 291 lineage-specific genes that were found in both C. immitis and C.posadasii but not in any other species. Variable percentagesof lineage-specific genes have been reported in other fungi,ranging from <1% of genes in Aspergillus spp. [29], to 8%in Saccharomyces spp. [29], to 24% in Neurospora crassa[30]. Given the conservative approach of the Coccidioidesphylogenomics study and the different methods of annota-tion practiced at the Broad Institute (C. immitis RS) andJCVI (C. posadasii c735), this lineage-specific gene set islikely underestimated. When C. immitis RS and C. posadasiiisolate Silveira (also sequenced by the Broad Institute) arecompared with their nearest relatives and all GenBank se-quences, more than 800 lineage-specific genes are observedbased on gene orthology (E. Whiston and J. Taylor, unpub-lished data). Comparing this larger lineage-specific gene setto a recent Coccidioides transcriptional profiling study [31],lineage-specific genes are found to be enriched for genes up-regulated in the parasitic spherule growth phase (Table 1).This result implies that genes unique to Coccidioides are ofsignificant importance to the spherule growth cycle, which

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Table 1. Percent of differentially regulated genes (all genes

vs. lineage-specific genes) [23,31].

Differential regulation All genes Lineage-specific(%) genes (%) [23]

Upregulated in hyphae [31] 9.0 3.0Upregulated in spherules [27] 13.5 29.0

is unique to Coccidioides. Those interested in the details ofrecent transcriptional profiling studies should consult Whis-ton et al. and Viriyakosol et al. [31,32].

The phylogenomics study also reported evidence ofpositive selection in 57 individual genes as judged bydn/ds analysis between C. immitis and C. posadasii [23].These genes were enriched for metabolic processes andS-adenosylmethionine-dependent methyltransferase activ-ity [23]. Comparing these genes under positive selectionto the results of the transcriptional profiling study, 11 wereupregulated in the spherule phase and 13 were upregulatedin the hyphal phase [31]. The genes under positive selec-tion that were also upregulated in the hyphal growth phasemight reflect adaptation to environmental features apartfrom the living mammal. These differences could includephysical factors such as environmental temperature or soilchemistry as well as biological factors such as features ofmammal carcasses, competing microbes, or even parasitesthat differ in the allopatric ranges of the two Coccidioidesspecies [31]. Genes under positive selection that were up-regulated in the spherule growth phase, on the other hand,may be related to adaptation to differences in the livingmammal populations [31].

Evidence of natural selection has also been reported forone of the gene families found to have expanded in Coccid-ioides spp., the M35 metalloprotease family [23,33]. Here,positive selection was inferred to have acted on severalamino acid sites between gene duplications, which also indi-cated possible functional differentiation [33]. Measurementof expression levels of the 9 M35 domain-containing genesin C. immitis isolate RS showed that three were upregulatedin the pathogenic spherule phase and one was upregulatedin the hyphal phase [31].

Population genomics in Coccidioides

A recent population genomics study in Coccidioides com-pared full genomes for 10 isolates each of C. immitis andC. posadasii. It reported evidence of positive selection andgene flow due to hybridization and subsequent selection be-tween Coccidioides populations and species and identifiedhighly conserved vaccine candidates [6]. Genes under posi-tive selection were identified by the neutrality index, whichcompares the ratio of nonsynonymous to synonymous nu-

cleotide substitutions within populations (polymorphism)to those between populations (divergence). Where an ex-cess of nonsynonymous substitutions is seen in divergence,selection is inferred. The authors found that, of the genesshowing the strongest evidence of natural selection, 80%did not have any predicted functional domains or homologyto proteins with known function in Neurospora or Saccha-romyces. In contrast, 60% of predicted Coccidioides geneshave no predicted function [6]. Taken with the observa-tions that lineage-specific genes and genes upregulated inthe spherule phase are enriched for genes with no predicteddomains or function [31], these findings imply that controlof the spherule growth phase relies on a very different setof genes than those important to hyphal or yeast morpho-logical growth in model organisms. Experimental evidencealso suggests that spherule formation in Coccidioides re-lies on genes that are different from those in yeast forma-tion in the other mammalian dimorphic fungal pathogens.In P. brasiliensis, 4-hydroxyphenylpyruvate dioxygenase(4-HPPD) has been shown to play a role in conidia-to-yeast transformation [34]. However, a recent study showedthat inhibition of 4-HPPD has the opposite effect in Coc-cidioides: it prevents mycelial growth [32].

Comparative population genomics can be used to iden-tify vaccine candidates that are highly conserved betweenthe two species, so that the vaccine will protect against asmuch of the pathogen population as possible. Comparingthe 20 genomes to find genes that lacked codon-changing,single-nucleotide polymorphisms (SNPs), the authors iden-tified vaccine candidates with highly conserved amino acidsequences that would protect against both C. immitis andC. posadasii [6]. One set of vaccine candidates was iden-tified by predicting T-cell epitopes; 12 genes that encodeproteins that have multiple predicted epitopes and a highdegree of conservation within and between Coccidioidesspecies were identified [6]. The authors also assessed con-servation in a previously identified set of vaccine candidates,the eight proline-rich proteins (Prp) [35,36], and found avarying degree of T-cell epitopes and nonsynonymous SNPs[6]. Of the eight previously identified Prp genes, Prp1 andPrp2 have been tested both as single-target and combina-tion vaccines [35,37]. The Prp1/Prp2 combination vaccineprovided better immunity than either single-target vaccinebut did not provide sterilizing immunity in mice [35].

A recent study used population SNP data to assess con-servation of another set of vaccine candidates [38]. Thesecandidate proteins were identified from cell wall compo-nents that were seroreactive in patients with coccidioidomy-cosis [38]. A combination vaccine of five highly conservedpredicted epitopes from three seroreactive candidate pro-teins conferred partial protection against Coccidioides inmice [38].

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To evolutionary biologists, the most interesting resultfrom the 2010 Coccidioides population genomics study wasevidence of the movement of genomic regions between pop-ulations and species of C. immitis and C. posadasii [6].The movement of these regions is due to hybridizationand subsequent backcrosses of hybrid individuals with theparental populations, that is, introgressive hybridization.Genes from the donor species that confer a selective advan-tage to members of the recipient species then sweep throughthe recipient population. One introgressed region of partic-ular interest has a highly conserved boundary, which in-dicates that at least one gene near that boundary is un-der positive selection. In this case, two genes are locatedat the conserved boundary: a metalloprotease gene (mep4,CIMG_00508) and an unnamed gene (CIMG_00509) [6].In transcriptional analysis, mep4 was not expressed in ei-ther the hyphal or spherule growth phase [31]. However,the unnamed gene, CIMG_00509, was one of the 15 mostupregulated genes in the spherule phase [31]. CIMG_00509encodes a short peptide whose function is not yet known,although it is predicted to be secreted.

The Coccidioides population genomics study [6] was thefirst to show evidence of introgression between fungal pop-ulations and species. A more recent population genomicsstudy of N. crassa provided evidence of introgression be-tween previously unknown populations and was able to testhypotheses about the adaptive value of specific alleles to en-vironmental variation using gene deletion experiments [39].Taken together, these studies suggest that introgression infungi is an important source of genetic variation and is amechanism for adaptive evolution, perhaps as importantas the analogous lateral gene transfer in prokaryotic mi-crobes [40]. Further sequencing of additional isolates fromall Coccidioides spp. populations would shed additionallight on the evolutionary processes responsible for the de-velopment of Coccidioides’ lifestyle and unique pathogenicgrowth form as spherules.

Future directions

There are two obvious areas where additional genomic re-search can aid the effort to prevent and treat coccidioidomy-cosis. The first would be to develop a gene deletion libraryfor Coccidioides, as has been done for other pathogens suchas Cryptococcus neoformans [41] and Candida albicans[42]. If Neurospora is a guide, one could expect that 6000transformant strains would suffice to account for all genesthat can be deleted, that is, the nonessential genes [43]. Thiscollection could be used to assess the effect of gene deletionson any phenotype and also to test hypotheses about the roleof specific genes suspected of influencing a specific pheno-type.

The second area for future genomic research in Coccid-ioides would be to apply genome-wide association studies(GWAS) to Coccidioides, which would allow one to as-sociate genetic variation present in approximately 100 in-dividuals from a wild, recombining population with anyphenotype showing variation among the same individuals.In the past, when genome sequencing was extremely costly,it was desirable to sequence just the strain used to constructthe gene knock-out library, even though it then requiredphenotyping the thousands of single-gene deletions madein that strain. Now that next-generation sequencing hasbecome inexpensive, it is more attractive to phenotype ap-proximately 100 individuals for variations in growth andpathogenicity and then sequence their genomes. Using thisapproach, which can match any gene, essential or not, withphenotype, genes have been discovered for complex, mor-phological phenotypes in Neurospora using as few as 24individuals [44]. For the phenotype of pathogenicity in theplant pathogenic fungus, Heterobasidion annosum, geneshave been discovered using just 24 individuals [45]. Us-ing a related approach, comparative population genomics,genes responsible for adaptation to differing environmentaltemperatures have been found using as few as 40 individ-uals [39]. Sequencing both the genome and transcriptomewould, in addition, allow researchers deduce the biologi-cal function of unknown genes by comparing their expres-sion levels with those of genes in known pathways. To useGWAS, it is essential that the isolates come from a single,recombining population. Fortunately, Coccidioides specieshave been the subject of some of the most detailed pop-ulation studies of any fungus [46], making it possible toidentify randomly mating populations both for GWAS andfor interpopulation comparisons. With the recent delistingof Coccidioides spp. from the Select Agents and Toxins List[47], both the creation of gene disruption libraries and theapplication of GWAS have become more realistic goals.

Development of these two genetic and genomic tools—aGWAS population of 100 individuals from one populationand as few as 30 individuals from a second population anda collection of gene deletion strains—would greatly speedthe search for the genes responsible for real-world adaptivephenotypes and significantly improve efforts to prevent andtreat coccidioidomycosis. These tools would also inspire thedevelopment of similar tools for other human pathogenicfungi.

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