Genetic and virulence variation in an environmental population of the opportunistic pathogen...

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Genetic and virulence variation in an environmentalpopulation of the opportunistic pathogenAspergillus fumigatus

Fadwa Alshareef and Geoffrey D. Robson

Correspondence

Geoffrey D. Robson

[email protected]

Received 19 August 2013

Accepted 22 January 2014

Faculty of Life Sciences, Michael Smith Building, University of Manchester,Manchester M16 8QW, UK

Environmental populations of the opportunistic pathogen Aspergillus fumigatus have been shown

to be genotypically diverse and to contain a range of isolates with varying pathogenic potential. In

this study, we combined two RAPD primers to investigate the genetic diversity of environmental

isolates from Manchester collected monthly over 1 year alongside Dublin environmental isolates

and clinical isolates from patients. RAPD analysis revealed a diverse genotype, but with three

major clinical isolate clusters. When the pathogenicity of clinical and Dublin isolates was

compared with a random selection of Manchester isolates in a Galleria mellonella larvae model, as

a group, clinical isolates were significantly more pathogenic than environmental isolates.

Moreover, when relative pathogenicity of individual isolates was compared, clinical isolates were

the most pathogenic, Dublin isolates were the least pathogenic and Manchester isolates showed

a range in pathogenicity. Overall, this suggests that the environmental population is genetically

diverse, displaying a range in pathogenicity, and that the most pathogenic strains from the

environment are selected during patient infection.

INTRODUCTION

Aspergillus fumigatus is an opportunistic fungal patho-gen that can cause aspergillosis in immunocompromisedpatients, such as those with neutropenia or on immuno-suppressive therapy following solid organ transplantation(Abad et al., 2010; Ben-Ami et al., 2010; Dagenais & Keller,2009; Latge, 1999; Rementeria et al., 2005). The principalroute of infection is through the inhalation of airbornespores, which due to their small spore size are able to reachthe alveoli (Dagenais & Keller, 2009; Ibrahim-Granet et al.,2003; Latge, 1999; O’Gorman, 2011; Philippe et al., 2003).While other aspergilli can cause opportunistic infections(such as Aspergillus terreus and Aspergillus flavus), A.fumigatus accounts for ~90 % of all cases despite airbornespore numbers only accounting for ,1 % of all Aspergillusspores (Rementeria et al., 2005). A number of studieshave shown a high degree of genetic variation within theA. fumigatus population, and whilst some studies haveindicated potential geographical subgroups, comparisonsbetween clinical isolates and environmental isolates havelargely been unable to distinguish these populations(Araujo et al., 2010; Aufauvre-Brown et al., 1992; Balajeeet al., 2008; Chazalet et al., 1998; Denning et al., 1990;Duarte-Escalante et al., 2009; Leenders et al., 1999; Menottiet al., 2005). Moreover, epidemiological studies havelargely failed to match genotypes in infected patients withgenotypes found in hospitals, in part due to the high levelof genetic diversity in the environmental populations

(Araujo et al., 2010; Bart-Delabesse et al., 1999; Chazaletet al., 1998; Guinea et al., 2011; Leenders et al., 1999; deValk et al., 2007), and similar findings have been reportedin avian populations (Arne et al., 2011; Lair-Fulleringeret al., 2003; Olias et al., 2011). Whilst genotyping of isolatesfrom some individual patients has revealed infection withmultiple isolates, most patients appear to be colonized byonly one genotype despite exposure to a genetically diversepopulation (Alvarez-Perez et al., 2010; Bart-Delabesse et al.,1999; Chazalet et al., 1998; Guinea et al., 2011; Menottiet al., 2005; Tang et al., 1994; de Valk et al., 2007). This hasled to the suggestion that the lung environment selects for astrain or strains from the environment that are the mostadapted and therefore pathogenic (Cimon et al., 2001;Guinea et al., 2011; de Valk et al., 2007).

Animal models are used widely to determine microbialpathogenicity, but their use is constrained by high costsand ethical considerations. As a result, a number of insectmodels have been developed as potential alternatives forstudying the pathogenicity of microbes and the efficacy ofantimicrobial agents (Desbois & Coote, 2012; Mak et al.,2010; Papaioannou et al., 2013; Thomas et al., 2013) as theycontain functional and structural features similar to theinnate immune system of mammals (Arvanitis et al., 2013;Browne et al., 2013). The greater wax moth larva (Galleriamellonella), in particular, has been used successfully tostudy pathogenicity for a range of microbial pathogens,including Acinetobacter baumannii, Enterococcus faecium,

Microbiology (2014), 160, 742–751 DOI 10.1099/mic.0.072520-0

742 072520 G 2014 The Authors Printed in Great Britain


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Escherichia coli, Legionella pneumophila, Listeria monocyto-genes, Pseudomonas aeruginosa, Staphylococcus aureus,Streptococcus pneumoniae, Candida albicans, Candidatropicalis, Cryptococcus neoformans, A. flavus, A. fumigatusand Fusarium spp. (Andrejko et al., 2009; Coleman et al.,2011; Desbois & Coote, 2012; Dunphy et al., 1986; Evans &Rozen, 2012; Fallon et al., 2011; Fuchs et al., 2010; Hardinget al., 2012; Harrison et al., 2006; Joyce & Gahan, 2010;Lebreton et al., 2011; Leuko & Raivio, 2012; Mesa-Arangoet al., 2013; Miyata et al., 2003; Mylonakis et al.,2005; Mukherjee et al., 2010; Peleg et al., 2009; Scully &Bidochka, 2009; Soukup et al., 2012). Moreover, virulencein these larvae has in many instances been shown to besimilar to that in mammals, and to involve similarpathogenicity and virulence determinants (Brennan et al.,2002; Cook & McArthur, 2013; Dunphy et al., 2003; Fuchs& Mylonakis, 2006; Jander et al., 2000; Mukherjee et al.,2010; Wand et al., 2011), including for A. fumigatus (Amichet al., 2013; Jackson et al., 2009; Li et al., 2011; O’Hanlonet al., 2011; Slater et al., 2011; Soukup et al., 2012).

In this study, we investigated the genetic variation in

airborne environmental A. fumigatus strains isolated over a

period of 12 months from the outdoor environment in

Manchester, Dublin environmental strains isolated from

the outdoor environment at one sample time and clinical

isolates using two RAPD primers. In addition, we also

compared the relative pathogenicity of environmental and

clinical isolates in a Galleria larva model, and report

that clinical isolates were at the most pathogenic end of

the spectrum whilst environmental isolates were found

throughout the spectrum from high to low, suggesting

clinical isolates represent the most pathogenic strains

present in the environment.

METHODS

Strains and maintenance. Clinical isolates were obtained from the

A. fumigatus culture collection, Wythenshaw Hospital, Manchester,

UK. Manchester environmental isolates were collected monthly over a

period of 12 months from the air at the University of Manchester

campus, between March 2009 and February 2010 (Alshareef &

Robson, 2014), and 60 strains were selected randomly for use in this

study. Dublin environmental isolates were kindly supplied by H.

Fuller (University of Dublin, Dublin, Ireland) and were isolated on a

single day from a single site at the Belfield Campus, University College

Dublin, Ireland. Isolates were grown on potato dextrose agar (PDA;

ForMedium) in tissue culture flasks and incubated at 37 uC until

confluent growth was obtained. Spores were harvested by gentle

agitation of the mycelial surface with a sterile loop with 20 ml 0.05 %

(v/v) Tween 20, filtered through four layers of lens tissue (Whatman)

to remove phialides and spore aggregates, and washed two times in

sterile water by centrifugation at 1500 g and resuspending the spores

in 20 ml sterile water. Spores were enumerated using a modified

Neubauer haemocytometer, and viability determined by serial

dilution and c.f.u. determination on PDA plates after 48 h incubation

at 37 uC. All strains had a spore viability .95 %. Spores were kept for

up to 1 week at 4 uC; for long-term storage, spore suspensions were

mixed with an equal volume of sterile glycerol and stored at 280 uC.

For DNA extraction, mycelium was grown in 50 ml potato dextrose

broth in 250 ml conical flasks for 48 h at 37 uC in an orbital shaker

(200 r.p.m.).

Virulence determination in Galleria larvae. Galleria larvae (Live

Food Direct) were stored in wood shavings in the dark at 4 uC for

up to 10 days and transferred to room temperature for 2 h before

use. Galleria larvae in groups of 30 were inoculated with 10 ml A.

fumigatus spore suspension into the haemocoel through the last

right or left proleg, using a Hamilton 1 ml gas-tight syringe (Fisher

Scientific) with a 10 ml repeating dispenser attachment (Jaytee

Biosciences). Spore concentrations of 16108 to 16103 ml21

were used to infect larvae, which were incubated at 37 uC in the

dark in Petri dishes (10 larvae per plate) and the mortality

monitored daily for 7 days. Mortality was assessed by lack of

movement in response to stimulation and by discoloration

(melanization) of the cuticle.

The survival of the Galleria larvae was analysed using the Mann–

Whitney U test (Stats Direct) with P,0.05 considered significant, and

differences in the survival rates of larvae inoculated with different

strains were compared. A non-parametric estimate of the mean

survival time for Galleria larvae was obtained as the area under the

Kaplan–Meier estimate of the survival curve.

DNA extraction. Mycelium was harvested on muslin cloth, washed

briefly with sterile water, and ground to a fine powder in liquid

nitrogen using a mortar and pestle. Ground mycelium was added to

50 ml Falcon tubes (on ice) to the 10 ml mark and an equal volume

of pre-heated DNA extraction buffer [0.7 M NaCl, 1 M Na2SO3,

0.1 M Tris/HCl (pH 7.5), 0.05 M EDTA, 1 % (w/v) SDS, 65 uC] was

added and mixed thoroughly with a pipette tip, and incubated

at 65 uC for 30 min to terminate nuclease activity and induce

protein denaturation. A NanoDrop 1000 spectrophotometer

(Thermo Scientific) was used to measure DNA concentrations in

samples.

RAPD. In total, 106 isolates were analysed using RAPD. The primers

used for RAPD were R108 (59-AGTGCACACC-39), UBC90 (59-GG-

GGGTTAGG-39), R151 (59-GCTGTAGTGT-39) and RC08 (59-AG-

GATGTCGAA-39) (Eurofins) as these have been used to successfully

fingerprint A. fumigatus populations in the past (O’Gorman et al.,

2009). The samples were run on 2.5 % (w/v) agarose gels in 40 mM

Tris/acetate EDTA buffer (pH 8.0). Only primers R108 and C08 were

used to compare all isolates, and RAPD bands included in the analysis

were scored as either absent (0) or present (1). The data from all

primers were pooled for each isolate and a pair’s similarity matrix was

calculated with Jaccard’s coefficient with the program FREETREE v.

0.9.1.50. A bootstrapped dendrogram with 1000 resamplings was

produced with NJPlot.

RESULTS

RAPD fingerprinting revealed genetic diversity inthe environmental population and clustering ofclinical isolates

Four primers were used initially for RAPD fingerprinting(R108, RC08, R151 and UBC90) with seven A. fumigatusisolates, and fingerprinting was repeated at least two timesto evaluate reproducibility. The four primers produced arange of RAPD bands, from 0.4 to 6 kb. All four primersgave several strong bands that were reproducible, alongwith many fainter bands (data not shown). Of the fourprimers tested, R108 and RC08 gave the most reproducible

Variation in an environmental population of A. fumigatus

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F. Alshareef and G. D. Robson

744 Microbiology 160


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results and had the greatest discriminatory power (0.992and 0.868, respectively) (Bikandi et al., 2004) in a pilotstudy, and were selected for fingerprinting the remainingisolates. Only the strongest and reproducible bands wereincluded in the analysis, and fainter and variable bandswere excluded. To increase discrimination, RAPD datafrom both primers were pooled prior to analysis. As thenumber of isolates exceeded the maximum number of wellson a gel, strain AF293 was run on every gel as an internalcontrol.

RAPD analysis of the Manchester environmental isolatesshowed a high genetic diversity in the population (Fig. 1).With few exceptions, isolates collected at the same timepoints could be distinguished from each other, indicatingmultiple sources of airborne spores. In addition, com-parison of Manchester isolates collected monthly for 12months indicated that the source of airborne spores variedat each time point. When environmental isolates wereanalysed along with clinical isolates, clinical isolatesdisplayed a degree of clustering with 21 of the 25 clinicalisolates forming three clusters accounting for 12, 5 and 4isolates, although, with the exception of isolates 22178 and22636 and isolates 17871 and 16258, they could bedistinguished from each other.

Virulence testing in Galleria larvae demonstrateda broad range in pathogenicity in theenvironmental population

The virulence of ten randomly selected Manchesterenvironmental isolates, ten Dublin environmental isolates,ten clinical isolates, and sequenced strains AF293 andAF1163 was determined in Galleria larvae inoculated with16103 to 16106 spores per larva by monitoring survivalover 7 days. Initial spore inoculum size had a large effect onGalleria larvae survival in all strains, with all strainsshowing the greatest survival after 7 days with 16103

spores and lowest survival with 16106 spores (data notshown). The greatest discrimination in survival betweenthe different strains was seen at an inoculum level of16105 spores per larva (Fig. 2). In order to compare therelative pathogenicity of the Manchester, Dublin andsequenced strains, survival over 7 days for each sporeinoculum for each group was combined and subjected toMann–Whitney and Kaplan–Meier analyses (Fig. 3). Theclinical isolates were significantly more pathogenic than theDublin and Manchester isolates (P,0.0001) at the fourdifferent doses. Moreover, the Manchester isolates weresignificantly more pathogenic than the Dublin isolates (106

and 105, P,0.0001; 104, P50.0135; 103, P50.0043). Thiswas also apparent when the survival of each group 4 daysafter inoculation was compared at initial spore concentra-tions from 16103 to 16106 spores per larva, confirmingthat the greater pathogenicity of the clinical isolates wasacross the spectrum of spore inoculum concentrations (Fig.4). When the percentage survival values after 4 days foreach individual strain at an inoculum of 16105 spores perlarva were compared, the clinical isolates clustered togetheras the most pathogenic, the Dublin isolates the leastpathogenic and the Manchester isolates showed a broadspectrum of pathogenicity (Fig. 5).

DISCUSSION

A variety of molecular typing methods have beendeveloped over the past 20 years to investigate geneticdiversity in the population of a wide variety of fungalspecies. Many of these techniques have also been used tostudy genetic diversity in the A. fumigatus population. Themethods include RAPD (Anderson et al., 1996; Aufauvre-Brown et al., 1992; Mondon et al., 1995; O’Gorman et al.,2009), RFLP (Spreadbury et al., 1990), restriction enzymeanalysis (Denning et al., 1990), amplified fragment lengthpolymorphism (Vos et al., 1995) and sequence-specificDNA primer analysis (Mondon et al., 1997), and morerecently microsatellite length polymorphism (Balajee et al.,2008; Bart-Delabesse et al., 1998; de Valk et al., 2007),MLST (Bain et al., 2007) and variable number of shorttandem repeat typing (Vanhee et al., 2009). These methodshave also demonstrated that there is a large geneticdiversity within the A. fumigatus population (Balajeeet al., 2008; Chazalet et al., 1998; Leenders et al., 1999; deValk et al., 2009; Vanhee et al., 2009; O’Gorman et al.,2009) and have largely been used to investigate theepidemiology of isolates following clinical outbreaks ofdisease in hospitals (Alvarez-Perez et al., 2010; Bart-Delabesse et al., 1999; Vanhee et al., 2010; Guinea et al.,2011), as well as to study genotypic diversity of strains inavian disease (Arne et al., 2011; Lair-Fulleringer et al., 2003;Olias et al., 2011). The recent discovery of a functionalsexual cycle in A. fumigatus probably accounts for thegenetic diversity observed in the population (O’Gormanet al., 2009), and much of this diversity appears to beconcentrated in discrete genomic islands within the DNAthat encode for proteins involved in heterokaryon incom-patibility and cell-wall-associated proteins that may beinvolved in virulence (Fedorova et al., 2008). In addition,whilst the macromorphology of A. fumigatus strains

Fig. 1. Dendrogram analysis of RAPD fingerprinting of A. fumigatus isolates (March 2009–February 2010) using combinedRAPD primers R108 and RC08. The strains are Manchester airborne isolates (month of isolation+code number) (indicated bythe absence of grey/black boxes on the right), clinical isolates (code number) (indicated by black boxes on the right), Dublinairborne isolates (RB+code number) (indicated by grey boxes on the right), and sequenced strains AF1163 and AF293(indicated by circles on the right). Bar, 0.1 (10 % genetic difference). Values at nodes represent the percentage bootstrapsupport (based on 1000 resamplings).




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appears to be largely similar within the population, themicromorphology (such as conidia and vesicle size andshape), pathogenicity and enzyme secretion have beenfound to be variable between isolates (Alp & Arikan,2008; Aufauvre-Brown et al., 1998; Ben-Ami et al., 2010;Birch et al., 2004; Chamilos et al., 2007, 2010; Mondonet al., 1995, 1996; Olias et al., 2011; Rinyu et al., 1995).Limited studies on enzyme secretion have demonstrated arange of expression levels and isoenzymes from differentisolates (Alp & Arikan, 2008; Birch et al., 2004; Blancoet al., 2002; Lin et al., 1995; Rinyu et al., 1995; Rodriguezet al., 1996).

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Fig. 2. Percentage survival of wax moth larvae inoculated withvarious strains of A. fumigatus over 7 days. Thirty Galleria larvaewere inoculated with isolates of A. fumigatus at 1�105 c.f.u. perlarva and incubated at 37 6C for 7 days. The number of survivingGalleria larvae was determined daily. Controls were inoculatedwith sterile 0.05 % (v/v) Tween 20 (�). (a) Manchester envi-ronmental strains: Mar13 (D), Mar15 (e), Mar20 ($), Mar32 (&),Apr12 (m), Apr15 (¤), May1 (grey circle), Jun4 (grey square),Jun5 (grey triangle) and Jul1 (grey diamond). (b) Dublinenvironmental strains: RB11 (D), RB12 (e), RB13 ($), RB14(&), RB15 (m), RB16 (¤), RB17 (grey circle), RB18 (greysquare), RB19 (grey triangle) and RB20 (grey diamond). (c)Clinical strains 17871 (D), 15562 (e), 177796 ($), 16258 (&),16795 (m), 15819 (¤), 17406 (grey circle), 17768 (grey square),17882 (grey triangle) and 16916 (grey diamond). Sequencedstrains AF293 (#) and AF1163 (h) were included as internalcontrols.

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Fig. 3. Mann–Whitney analysis of the mean survival of G.

mellonella inoculated with Manchester environmental (black bars),Dublin environmental (white bars) and clinical (grey bars) strains ofA. fumigatus at spore concentrations of 1�103, 1�104, 1�105 and1�106 spores per larva after 7 days. Controls were inoculated withsterile 0.05 % (v/v) Tween 20 and retained 100 % viability over 7days. Error bars represent SEM.

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Fig. 4. Kaplan–Meier approximation of the mean survival time of G.

mellonella inoculated with A. fumigatus clinical (&), Manchesterenvironmental (h), Dublin environmental ($) and AF293 andAF1163 (#) strains over 7 days following inoculation with1�105 c.f.u. per larva. Controls were inoculated with sterile0.05 % (v/v) Tween 20 (�).




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In our study, RAPD typing also demonstrated a largedegree of genetic variability within the airborne populationof A. fumigatus collected monthly over a period of 12months from the same site in Manchester (Fig. 1).Moreover, isolates typed from the same time point showedseveral genotypes, indicating a mixture of sources con-tributed to the airborne population at any one time. Thiscontrasted with the small number of isolates from Dublin,which were also collected at a single time point from asingle site (H. Fuller, personal communication), that withone exception showed identical genotypes with bothprimers (Fig. 1), indicating that the majority of airbornespores at the time of sampling may have arisen from a singlesource. However, other isolates collected from Dublin atdifferent time points also showed a range of phenotypes,indicating variable sources over time (O’Gorman et al.,2009). That a range of genotypes was found at each monthlysampling point in this study is not unexpected as A.fumigatus spores are dispersed readily in the atmospheredue to their small size and samples were taken in the openoutdoor environment. Other studies that have typed A.fumigatus spores collected from the environment havealso shown a high degree of genotypic diversity in thepopulation, including the enclosed environments of hospi-tals where it might have been expected that the number ofcontributing sources would have been smaller (Araujo et al.,2010; Debeaupuis et al., 1997; Guinea et al., 2011; Menottiet al., 2005).

Previous studies attempting to identify environmentalsources of patient infection in hospital environments havegenerally found only a low number of patient isolate

genotypes and only a small number could be matched withan environmental phenotype, probably due to insufficientenvironmental samples being collected to capture the highlevel of genotype diversity reported (Araujo et al., 2010;Guinea et al., 2011). In addition, in those studies whereenvironmental samples were taken at different time periodsor locations within the same building, in only a fewinstances was an identical genotype recovered, indicating ahigh degree of variability occurs at the same location overtime and at any one time in different locations (Araujoet al., 2010; Guinea et al., 2011). Many aspects of thepathogenesis of aspergillosis remain to be clarified, includ-ing the contribution of the place of acquisition, incubationperiod, virulence variation in different Aspergillus isolatesand the possible contribution of more than one clone inhuman infections (Alvarez-Perez et al., 2010). Interestingly,in our study there was some genetic clustering amongst theclinical isolates where 21 of the 25 isolates clustered intothree groups that also contained some environmentalisolates, suggesting that some traits were detected that wereshared amongst some isolates that may be associated with ahigher pathogenicity (Fig. 1).

Whilst many studies have used mammalian models toinvestigate the impact of specific gene function onpathogenicity (Askew, 2008; Rementeria et al., 2005), therehave been few studies that have compared the pathogeni-city of different isolates of A. fumigatus, and those that havereported a range of pathogenicity between isolates (Jacksonet al., 2009; Lionakis et al., 2005; Reeves et al., 2004).Invertebrate models of pathogenicity offer a cheaper andquicker alternative to animal models, enabling large

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Fig. 5. Percentage survival of Galleria larvae 4 days after inoculation with 1�105 spores per larva of various strains of A.

fumigatus: clinical (black bars), Manchester environmental (white bars) and Dublin environmental (grey bars). Controls wereinoculated with sterile 0.05 % (v/v) Tween 20.




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numbers of isolates to be compared (Chamilos et al., 2010;Kavanagh & Reeves, 2004). In A. fumigatus, these modelshave included Galleria larvae and Toll-deficient mutants ofDrosophila (Jackson et al., 2009; Lionakis et al., 2005;Reeves et al., 2004), and relative pathogenicity has beencorrelated with pathogenicity levels in mammalian models(Slater et al., 2011; Chamilos et al., 2010). In this presentstudy, a range in pathogenicity against Galleria larvae wasapparent in both clinical isolates and environmentalisolates (Fig. 2). Moreover, as a group, the clinical isolateswere significantly (P,0.005) more pathogenic than theenvironmental isolates (Fig. 3) with the exception of theclinical isolates and the Manchester environmental isolatesat an inoculum level of 16106 spores per larva. However,the environmental isolates showed an overlap in patho-genicity with the clinical strains (Fig. 5), suggesting thatclinical isolates may represent the most pathogenic of theenvironmental isolates and that the infection process inpatients may select for the most pathogenic strains presentin the environment. Selection for the most pathogenicstrains from the environment has been suggested pre-viously as an explanation for why patients are ofteninfected with only one strain and in those cases where co-infection is present, only two or three genotypes have beenobserved despite a highly diverse environmental popu-lation (Alvarez-Perez et al., 2010; Debeaupuis et al., 1997;Menotti et al., 2005; Guinea et al., 2011; de Valk et al.,2007; Tang et al., 1994). Moreover, in one study, whilstseveral genotypes were recovered from the respiratory tractof a patient, only one was recovered from invasive diseasefrom solid organs, suggesting further selection for invasion(de Valk et al., 2007). It is now clear that the pathogenicityof this opportunistic pathogen is polygenetic rather thanbeing dependent on any one virulence factor (Askew, 2008;Rhodes, 2006), with a broad range of factors beingimplicated as playing a role in the pathogenicity, includingextracellular hydrolases, nutrient acquisition and metabo-lism, toxin production, melanin and transcription factors,and secondary messenger pathways with broad genotypicand phenotypic effects (Amich et al., 2013; Amich &Krappmann, 2012; Jackson et al., 2009; Richie et al., 2009;Kim et al., 2013; Krappmann et al., 2004; Li et al., 2011;O’Hanlon et al., 2011; Schrettl & Haas, 2011; Soukup et al.,2012). Differing levels of expression of these factors in thepopulation may therefore give rise to the range inpathogenicity observed in the A. fumigatus population,and the ability to adapt and infect the lung. In addition,infection also depends on host factors that will vary indiffering individuals (Hartmann et al., 2011; Sales-Camposet al., 2013). Thus, selection in the lung of A. fumigatusfrom a diverse range of genotypes will be multifactorialdependent on the interplay between A. fumigatus and hostgenotype.

ACKNOWLEDGEMENTS

The authors would like to thank the government of Saudi Arabia for ascholarship to F. A.

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