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Environmental Microbiology Reports (2015)
doi:10.1111/1758-2229.12321
Recombination detection in Aspergillus fumigatus through single nucleotide polymorphisms typing Joana Teixeira,1,2 António Amorim,1,2,3 Ricardo Araujo1,2* 1 IPATIMUP, Institute of Molecular Pathology and Immunology of the University of Porto, Porto, Portugal 2 Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal 3 Faculty of Sciences, University of Porto, Porto, Portugal Summary The first evidence of sexual reproduction in Aspergillus fumigatus was reported in 2009. Nevertheless, it remains difficult to understand how A. fumigatus is able to reproduce through this mode in its natural environment and how frequently this occurs. The aim of this study was to analyse single nucleotide polymorphisms (SNPs) in a set of environmental and clinical isolates of A. fumigatus to detect signatures of recombination. A group of closely related Portuguese A. fumigatus isolates was characterized by mating type and the genetic diversity of the intergenic regions, microsatellites and multilocus sequence typing (MLST) genes. A group of 19 SNPs, organized in nine association groups and inherited in blocks, was identified and compared. Several variations on the association panel were detected on reference isolates of A. fumigatus AF293 and A1163, the sequence types available at MLST database and six clinical and environmental Portuguese isolates. About one to four haplotype disruptions were observed per isolate. Considering clinical and environmental isolates, sexual reproduction seems to occur more frequently than previously admitted in A. fumigatus. In this study, a practical SNP approach is proposed for detection of recombination events in larger collections of A. fumigatus. Introduction Aspergillus fumigatus is a saprophytic fungus with worldwide distribution. It is commonly found in soil rich in organic material, where it is involved in nutrient recycling Received 13 November, 2014; revised 27 June, 2015; accepted 10 July, 2015. *For correspondence. E-mail [email protected]; Tel. (+351) 225570700; Fax (+351) 225570799.
© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd
(Latgé, 2001; Tobal and Balieiro, 2014). This mould produces thousands of small airborne conidia with the ability to survive in different environmental conditions, including the human bronchioles and alveoli (Araujo et al., 2006; Tobal and Balieiro, 2014). Aspergillus fumigatus conidia are generally eliminated by innate immune response but these conidia can also cause severe and sometimes fatal infections and disseminated aspergillosis in immunocompromised patients (Perlroth et al., 2007; Araujo et al., 2008). For a long time, A. fumigatus was considered to reproduce strictly asexually, since its sexual cycle had never been observed. The genetic diversity of A. fumigatus was characterized as extremely low in relation to its close relatives, Neosartorya fischeri and Neosartorya spinosa (Rydholm et al., 2006). Nevertheless, molecular evidence supported the presence of sexual reproduction (Paoletti et al., 2005). Genome analysis has revealed several loci implicated in sexual development, namely the genes involved in mating processes, pheromone signalling, fruit body development and meiosis (Paoletti et al., 2005; Schurko et al., 2009). In fungi, sex commonly involves mating type (MAT) idiomorphs. Complementary MAT1-1 and MAT1-2 forms were recognized in A. fumigatus isolates worldwide presenting nearly equal frequencies (Paoletti et al., 2005; Ene and Bennett, 2014). The presence of a large number of TEs in the A. fumigatus genome can be interpreted as an additional sign for sexuality in this species (Fedorova et al., 2008). Since sexual reproduction controls the proliferation and accumulation of deleterious TEs, the presence of these elements along the A. fumigatus genome supports the idea that the fungus has mechanisms related to sex to eliminate the adverse effects of the TEs (Neuveglise et al., 1996; Fedorova et al., 2008; Ni et al., 2011). In 2009, the fully functional sexual reproductive cycle was documented for the first time (O’Gorman et al., 2009). The sex of this heterothallic fungus was induced under particularly fastidious environmental conditions leading to the formation of cleistothecia and ascospores. Aspergillus fumigatus teleomorph was classified as belonging to the genus Neosartorya and named Neosartorya fumigata O’Gorman, Fuller & Dyler sp. nov (O’Gorman et al., 2009). Follow-up studies showed that clinical isolates of this fungus could mate in less stringent conditions (Sugui et al., 2011). The patterns of linkage
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J. Teixeira, A. Amorim and R. Araujo
disequilibrium that reflect the occurrence of chromosomal recombination can also provide important information about the occurrence of sexuality. When asexual reproduction occurs, chromosomes are inherited in blocks without disruption and consequently some of the nucleotides are inherited with strong linkage disequilibrium, only diminished by recurrent mutation. On the other hand, if recombination occurs, the association between such nucleotides can be disrupted (McCarthy, 2005). A study of a Dutch A. fumigatus population showed that this fungus may reproduce asexually and sexually in nature based on the information revealed by linkage disequilibrium analyses of some markers (Klaassen et al., 2012). The genetic analysis of Candida glabrata, an opportunistic and haploid fungal pathogen similar to A. fumigatus, showed that this yeast has a similar ability to reproduce by asexual and sexual modes (Lott et al., 2010). On the other hand, there are fungal species, such as Penicillium marneffei, with occasional footprints of sexual reproduction but exhibiting patterns of extreme clonality and being genetically and spatially restricted (Henk et al., 2012; Ene and Bennett, 2014). Even considering the previous evidence, it remains difficult to understand how frequently A. fumigatus can reproduce by sexual mode. Therefore, the aim of this study was to analyse a set of environmental and clinical isolates of A. fumigatus in order to detect signs of recombination by employing genomic markers such as single nucleotide polymorphisms (SNPs). A group of strongly associated nucleotides was investigated in order to find some markers that support the occurrence of recombination through disruption of nucleotide associations. The proposed SNPs can be capable of detecting a fraction of recombination events that happen in A. fumigatus populations. The selected nucleotide positions were targeted by two minisequencing multiplexes, thus providing a simple and efficient strategy for analysis of those genomic positions in collections of A. fumigatus.
Results and discussion Selection of SNPs Based on the assumption that associated nucleotides may be disrupted when sexual reproduction happens (McCarthy, 2005), a strategy for the detection of recombination in A. fumigatus was designed. An initial set of 30 environmental isolates collected from air and water samples at S. João Hospital, Porto, Portugal, was used for the identification and selection of associated SNPs. This group comprised closely related A. fumigatus isolates that were characterized as MAT1-1 (Paoletti et al., 2005). The genetic diversity was also characterized based on polymorphisms located in intergenic regions (Rydholm
Table 1. Polymorphic associated positions identified in Aspergillus fumigatus. Linkage
Chromosome/gene
Position
Nucleotides
1
Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr
17 198 193 362 2 19 366 233 327 445 425 564 113 792 103 221 199 72 805
A/G G/A C/T C/T A/T C/T A/C C/T A/G C/T C/T T/C T/C C/G A/deletion C/G T/C A/deletion A/T
2 3 4 5 6 7 8 9
1/Lipase 2/Zinc transporter 3/Catalase 3/Annexin 4/Superoxide dismutase 3/Annexin 5/Rodlet A 2/Zinc transporter 5/Rodlet A 1/β-1,3-glucanosyltransferase 1/Lipase 3/Catalase 4/Mating type protein 3/Catalase 6/STR_M8 3/Annexin 4/Superoxide dismutase 2/Zinc transporter 3/Catalase
et al., 2006), microsatellites (Araujo et al., 2009) and multilocus sequence typing (MLST) genes (Bain et al., 2007; Caramalho et al., 2013). The first step of the proposed SNP strategy was the selection of the polymorphic positions by sequencing analysis of the MLST genes (seven genes: annexin, beta-1,3-glucanosyltransferase, catalase, lipase, MAT protein, superoxide dismutase, zinc transporter; Bain et al., 2007), the rodlet A gene commonly used for molecular identification of Aspergillus spp. (Serrano et al., 2011) and the genomic region near microsatellite M8 that contains a SNP (STR_M8; Araujo et al., 2009). The polymorphic positions were documented and compared; the SNP locus was considered to be polymorphic if the frequency of the minor allele was at least 0.05 in the initial population (Maïga-Ascofaré et al., 2010). Two nucleotides were considered as associated when differing simultaneously in A. fumigatus isolates, that is when isolates were typed at two positions, for example AT or CC, and evidence for disruption was simultaneously observed in both positions. More than 80 associated SNPs were initially identified in the sequenced genes. Some of these SNPs were excluded due to their proximity, since SNPs very close to each other tend to be inherited together even in the presence of recombination (Hinds et al., 2005; Seng and Seng, 2008). A final set of 19 SNPs structured in nine groups – eight pairs and one trio of SNPs was selected (Table 1; Fig. S1). The selected SNPs were located in six different chromosomes (exception of chromosomes 7 and 8); four SNPs were located in the catalase gene, followed by annexin and zinc transporter genes with three SNPs. The catalase gene was previously reported on genetic diversity studies of A. fumigatus as the gene accumulating more polymorphisms (Bain et al., 2007; Caramalho
© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology Reports
Recombination signs in A. fumigatus
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Table 2. List of genomic alterations in Aspergillus fumigatus strains; disruption of association between nucleotides is shaded in grey – details of linkage groups are in Table 1. Association group
Reference AF293 Reference 1163 ST1 ST2 ST3 ST4 ST5 ST6 ST7 ST8 ST9 ST10 ST11 ST12 ST13 ST14 ST15 ST16 ST17 ST18 ST19 ST20 ST21 ST22 ST23 ST24 ST25 ST26 ST27 S. João Hospital 1 S. João Hospital 2 S. João Hospital 3 S. João Hospital 4 S. João Hospital 5 S. João Hospital 6
1 AGC or GAT
2 CT or TA
3 CA or TC
4 CA or TG
5 TC or CT
6 TT or CC
7 CA or Gdel
8 CT or GC
9 AA or delT
AAC GAC ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ AGT ✓ ✓ ✓ AAT ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓
CA CA ✓ ✓ ✓ ✓ ✓ ✓ ✓ CA ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ CA CA ✓ ✓ ✓ CA ✓ ✓ ✓ ✓ ✓ ✓ ✓ CA ✓ CA CA
✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ TA ✓ ✓ ✓ ✓
TA ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ CG ✓ ✓ ✓ ✓ ✓
✓ CC ✓ ✓ ✓ ✓ ✓ TT ✓ CC ✓ TT ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ TT ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓
✓ CT TC CT ✓ ✓ ✓ TC ✓ ✓ ✓ ✓ ✓ ✓ ✓ TC CT ✓ CT ✓ ✓ ✓ TC CT ✓ ✓
✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ GA GA ✓ ✓
✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ GT ✓ ✓ ✓ CC ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓
✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ delA ✓ ✓
et al., 2013). Based on the selected genomic positions, a wild-type SNP profile was defined and posteriorly used to compare with other A. fumigatus isolates (Tables 1 and 2). In silico analysis of the associated nucleotides Sequencing results were deposited on an in-house database and the polymorphic positions were identified. The nucleotide information from the 27 sequence types (STs) available at MLST database (http://www.mlst.net/ databases/) was added to the database, as well as the genomic information of two reference A. fumigatus isolates, AF293 and A1163. The complete genome sequences of these two isolates were accessible at ENSEMBL (http://fungi.ensembl.org/index.html). BioEdit sequence alignment editor (available at http://www .mbio.ncsu.edu/bioedit/bioedit.html) and Geneious™ Pro 5.3.4 (Biomatters) were employed for gene alignment and detection of the polymorphic positions. Reference isolates
TC ✓ ✓ ✓ ✓ ✓ ✓ ✓
and 13 STs showed several variations on the associated SNP patterns (Table 2), in contrast with the wild-type genetic sequences initially defined. About one to four disruptions per isolate were observed, corresponding from 10% to 70% of the association panel. The highest number of disruptions was detected in AF293 and A1163, ST17 and ST22. These isolates were identified in the UK and USA more than 6 years ago; therefore, it is not surprising that these isolates are genetically very distinct from the initial group of Portuguese isolates. It is known that several mechanisms, such as genetic recombination and mutation, are responsible for genetic variations observed between populations. High genetic variation in A. fumigatus isolates was reported due to natural selection (Duarte-Escalante et al., 2009) and recombination (Klaassen et al., 2012). Mutation mechanisms can explain sporadic alterations of genome structure, but not all the disruptions reported above. Fungal isolates present a mutation rate of around 10−10 per locus per generation, meaning one mutation for every
© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology Reports
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J. Teixeira, A. Amorim and R. Araujo
Table 3. MULTILOCUS (version 1.3b) linkage disequilibrium analysis in Aspergillus fumigatus populations (1000 randomizations were tested). N°. of isolates
Populations Initial population of closely related isolates Second group of Portuguese isolates Total Portuguese isolates (initial and second groups) Set of 27 sequence types available at MLST database (http://www.mlst.net/databases/) Recombinant isolates (13 sequence types plus reference strains af293 and a1163 plus six isolates from this study) Six isolates from this study with disrupted SNP linkage Total isolates studied (Portuguese plus online)
N°. of haplotypes
Ia
rBarD a
30 50 80 27 21
15 30 40 17 20
2.44 2.03a 1.95a 1.29a 0.72b
0.15a 0.11a 0.11a 0.11a 0.04b
6 109
6 55
1.08c 1.81a
0.07c 0.10a
a. p < 0.001. b. p = 0.15. c. p = 0.31. Ia = index of association.
1010 locus per generation (Johnston, 2003). Moreover, new adaptive allelic combinations are produced more rapidly by recombination than by mutation, since the occurrence of successive mutations is necessary to origin a novel genotype (Milgroom, 1996; Billiard et al., 2012). Thus, it is more likely that the large number of SNP disruptions observed in this study is caused by recombination than by mutation, and the selected SNPs can in fact be used for detection of recombination events in A. fumigatus. Characterization of clinical and environmental isolates of A. fumigatus A second set of 50 clinical and environmental isolates of A. fumigatus from the Porto region was used in this study. This group of isolates was characterized (based on MAT type, intergenic, microsatellite and MLST diversity) as described above for the initial group of isolates. The selected nucleotide positions were targeted by two minisequencing multiplexes (Fig. S1), providing a simple and efficient strategy for analysis of those genomic positions. When our second collection of isolates was analysed, few disruptions were also noted. A total of two clinical and four environmental isolates showed alterations in the associated panel of SNPs (Table 2; Fig. S1). Curiously, one of the clinical isolates with SNP alterations was identified in a single cystic fibrosis patient with long (over 7 years) chronic colonization by A. fumigatus (Amorim et al., 2010). Larger populations of A. fumigatus analysed on this work exhibited significant values of index of association (between 1.29 and 2.24; Table 3), mainly Portuguese isolates and isolates whose information was stored at the MLST database. Curiously the highest index (Ia = 2.44, p < 0.001; Table 3) was found on the group of closely related isolates collected in a limited timeframe in Porto region. Inversely, low and not significant index of association and rBarD values were found in the set of Portuguese and online isolates with SNP disruptions.
These values were calculated using MULTILOCUS 1.3b (Agapow and Burt, 2001). Index of association and rBarD give information of linkage disequilibrium allowing the comparison of individuals that share the same allele at one locus and evaluating the possibility of sharing the same allele at other loci. These values are equal to zero if there is no linkage disequilibrium and this value increases when strong linkage disequilibrium is found. An index of association of or close to zero suggests panmixia and no mating restrictions on the populations. The diversity of reproductive modes and mating systems observed in fungi is fascinating. The reproductive strategy is a key issue for understanding population genetics of microbial organisms, since patterns of inheritance significantly impinge on the majority of evolutionary and ecological processes. Sexual reproduction of A. fumigatus was firstly reported in 2009 (O’Gorman et al., 2009) and since then other research groups could replicate and simplify such strategy in the laboratory and successfully cross clinical and environmental isolates (Szewczyk and Krappmann, 2010; Sugui et al., 2011). In fact there are novel and mounting pieces of evidence supporting that sexual reproduction is not restricted to any particular source. No large differences have been reported until now comparing the genetic diversity (Araujo et al., 2009; 2010), susceptibility (Guinea et al., 2005; Araujo et al., 2007) and virulence (Aufauvre-Brown et al., 1998) of clinical versus environmental isolates of A. fumigatus, and it is possible that in reproduction strategies both groups follow similar paths as well. Some studies already reported a similar fraction of both MATs in sets of clinical and environmental isolates of A. fumigatus (Paoletti et al., 2005; Ene and Bennett, 2014). The present work proposes an innovative strategy that can facilitate fungal population analyses (Caramalho et al., 2013; Eusebio et al., 2013). In this study, a set of associated SNPs was identified and compared in clinical and environmental isolates of A. fumigatus and it was possible to observe some
© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology Reports
Recombination signs in A. fumigatus genomic alterations most likely explained by recombination. The frequency of SNP disruptions caused by recombination is proportional to the genomic distance between markers being maximal when they are located in distinct chromosomes, as observed in the group of markers selected for testing our hypothesis. The proposed SNPs were capable of detecting a fraction of recombination events happening in A. fumigatus population of both clinical and environmental isolates. Sexual reproduction may in fact be more regular than currently assumed in A. fumigatus populations, occurring in both clinical and environmental sets of isolates. The diversity of reproduction systems in A. fumigatus is certainly on the origin of the huge genetic diversity regularly reported worldwide (Chazalet et al., 1998; Balajee et al., 2007; Araujo et al., 2009; 2012; Klaassen et al., 2012). The present study proposed a set of new SNP markers capable of identifying signatures of recombination through the disruption of genomically associated nucleotides. It remains a challenge to unveil the conditions triggering the sexual cycle of A. fumigatus in nature.
Acknowledgements RA was supported by Fundação para a Ciência e a Tecnologia (FCT) Ciência 2007 and by the European Social Fund. IPATIMUP integrates the i3S Research Unit, which is partially supported by FCT, the Portuguese Foundation for Science and Technology. The authors declare no conflict of interest.
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Johnston, M.O. (2003) Mutations and new variation: overview. eLS: 1–10. Klaassen, C.H.W., Gibbons, J.G., Fedorova, N.D., Meis, J.F., and Rokas, A. (2012) Evidence for genetic differentiation and variable recombination rates among Dutch populations of the opportunistic human pathogen Aspergillus fumigatus. Mol Ecol 21: 57–70. Latgé, J.-P. (2001) The pathobiology of Aspergillus fumigatus. Trends Microbiol 9: 382–389. Lott, T.J., Frade, J.P., and Lockhart, S.R. (2010) Multilocus sequence type analysis reveals both clonality and recombination in populations of Candida glabrata bloodstream isolates from U.S. surveillance studies. Eukaryot Cell 9: 619–625. McCarthy, J. (2005) Life, diversity and the pursuit of haplotypes. Nat Biotechnol 23: 1376–1377. Maïga-Ascofaré, O., Le Bras, J., Mazmouz, R., Renard, E., Falcão, S., Broussier, E., et al. (2010) Adaptive differentiation of Plasmodium falciparum populations inferred from single-nucleotide polymorphisms (SNPs) conferring drug resistance and from neutral SNP. J Infect Dis 202: 1095– 1103. Milgroom, M.G. (1996) Recombination and the multilocus structure of fungal populations. Annu Rev Phytopathol 34: 457–477. Neuveglise, C., Sarfati, J., Latge, J.-P., and Paris, S. (1996) Afut1, a retrotransposon-like element from Aspergillus fumigatus. Nucleic Acids Res 24: 1428–1434. Ni, M., Feretzaki, M., Sun, S., Wang, X., and Heitman, J. (2011) Sex in fungi. Annu Rev Genet 45: 405–430. O’Gorman, C.M., Fuller, H.T., and Dyer, P.S. (2009) Discovery of a sexual cycle in the opportunistic fungal pathogen Aspergillus fumigatus. Nature 457: 471–474. Paoletti, M., Rydholm, C., Schwier, E.U., Anderson, M.J., Szakacs, G., Lutzoni, F., et al. (2005) Evidence for sexuality in the opportunistic fungal pathogen Aspergillus fumigatus. Curr Biol 15: 1242–1248. Perlroth, J., Choi, B., and Spellberg, B. (2007) Nosocomial fungal infections: epidemiology, diagnosis, and treatment. Med Mycol 45: 321–346.
Rydholm, C., Szakacs, G., and Lutzoni, F. (2006) Low genetic variation and no detectable population structure in Aspergillus fumigatus compared to closely related Neosartorya species. Eukaryot Cell 5: 650–657. Schurko, A.M., Neiman, M., and Logsdon, J.M., Jr (2009) Signs of sex: what we know and how we know it. Trends Ecol Evol 24: 208–217. Seng, K.C., and Seng, C.K. (2008) The success of the genome-wide association approach: a brief story of a long struggle. Eur J Hum Genet 16: 554–564. Serrano, R., Gusmao, L., Amorim, A., and Araujo, R. (2011) Rapid identification of Aspergillus fumigatus within the section Fumigati. BMC Microbiol 11: 82. Sugui, J.A., Losada, L., Wang, W., Varga, J., Ngamskulrungroj, P., Abu-Asab, M., et al. (2011) Identification and characterization of an Aspergillus fumigatus ‘supermater’ pair. MBio 2: e00234–11. Szewczyk, E., and Krappmann, S. (2010) Conserved regulators of mating are essential for Aspergillus fumigatus cleistothecium formation. Eukaryot Cell 9: 774– 783. Tobal, J.M., and Balieiro, M. (2014) Role of carbonic anhydrases in pathogenic micro-organisms: a focus on Aspergillus fumigatus. J Med Microbiol 63: 15–27.
Supporting information Additional Supporting Information may be found in the online version of this article at the publisher’s website: Fig. S1. Minisequencing multiplexes for characterization of associated nucleotides. A. List of markers used in SNaPshots 1 and 2 (minisequencing reactions) and genomic information; B. Position of each marker on the automated electropherogram; and C. Representative example of SNaPshot profiles (strain S. João Hospital 5) (peaks: orange – ladder; blue – guanine; black – cytosine; green – adenine; red – thymine). Table S1. Primers employed for gene amplification. Appendix S1. Experimental procedures.
© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology Reports
Environmental Microbiology Reports (2015)
doi:10.1111/1758-2229.12321
Recombination detection in Aspergillus fumigatus through single nucleotide polymorphisms typing Joana Teixeira,1,2 António Amorim,1,2,3 Ricardo Araujo1,2* 1 IPATIMUP, Institute of Molecular Pathology and Immunology of the University of Porto, Porto, Portugal 2 Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal 3 Faculty of Sciences, University of Porto, Porto, Portugal Summary The first evidence of sexual reproduction in Aspergillus fumigatus was reported in 2009. Nevertheless, it remains difficult to understand how A. fumigatus is able to reproduce through this mode in its natural environment and how frequently this occurs. The aim of this study was to analyse single nucleotide polymorphisms (SNPs) in a set of environmental and clinical isolates of A. fumigatus to detect signatures of recombination. A group of closely related Portuguese A. fumigatus isolates was characterized by mating type and the genetic diversity of the intergenic regions, microsatellites and multilocus sequence typing (MLST) genes. A group of 19 SNPs, organized in nine association groups and inherited in blocks, was identified and compared. Several variations on the association panel were detected on reference isolates of A. fumigatus AF293 and A1163, the sequence types available at MLST database and six clinical and environmental Portuguese isolates. About one to four haplotype disruptions were observed per isolate. Considering clinical and environmental isolates, sexual reproduction seems to occur more frequently than previously admitted in A. fumigatus. In this study, a practical SNP approach is proposed for detection of recombination events in larger collections of A. fumigatus. Introduction Aspergillus fumigatus is a saprophytic fungus with worldwide distribution. It is commonly found in soil rich in organic material, where it is involved in nutrient recycling Received 13 November, 2014; revised 27 June, 2015; accepted 10 July, 2015. *For correspondence. E-mail [email protected]; Tel. (+351) 225570700; Fax (+351) 225570799.
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(Latgé, 2001; Tobal and Balieiro, 2014). This mould produces thousands of small airborne conidia with the ability to survive in different environmental conditions, including the human bronchioles and alveoli (Araujo et al., 2006; Tobal and Balieiro, 2014). Aspergillus fumigatus conidia are generally eliminated by innate immune response but these conidia can also cause severe and sometimes fatal infections and disseminated aspergillosis in immunocompromised patients (Perlroth et al., 2007; Araujo et al., 2008). For a long time, A. fumigatus was considered to reproduce strictly asexually, since its sexual cycle had never been observed. The genetic diversity of A. fumigatus was characterized as extremely low in relation to its close relatives, Neosartorya fischeri and Neosartorya spinosa (Rydholm et al., 2006). Nevertheless, molecular evidence supported the presence of sexual reproduction (Paoletti et al., 2005). Genome analysis has revealed several loci implicated in sexual development, namely the genes involved in mating processes, pheromone signalling, fruit body development and meiosis (Paoletti et al., 2005; Schurko et al., 2009). In fungi, sex commonly involves mating type (MAT) idiomorphs. Complementary MAT1-1 and MAT1-2 forms were recognized in A. fumigatus isolates worldwide presenting nearly equal frequencies (Paoletti et al., 2005; Ene and Bennett, 2014). The presence of a large number of TEs in the A. fumigatus genome can be interpreted as an additional sign for sexuality in this species (Fedorova et al., 2008). Since sexual reproduction controls the proliferation and accumulation of deleterious TEs, the presence of these elements along the A. fumigatus genome supports the idea that the fungus has mechanisms related to sex to eliminate the adverse effects of the TEs (Neuveglise et al., 1996; Fedorova et al., 2008; Ni et al., 2011). In 2009, the fully functional sexual reproductive cycle was documented for the first time (O’Gorman et al., 2009). The sex of this heterothallic fungus was induced under particularly fastidious environmental conditions leading to the formation of cleistothecia and ascospores. Aspergillus fumigatus teleomorph was classified as belonging to the genus Neosartorya and named Neosartorya fumigata O’Gorman, Fuller & Dyler sp. nov (O’Gorman et al., 2009). Follow-up studies showed that clinical isolates of this fungus could mate in less stringent conditions (Sugui et al., 2011). The patterns of linkage
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disequilibrium that reflect the occurrence of chromosomal recombination can also provide important information about the occurrence of sexuality. When asexual reproduction occurs, chromosomes are inherited in blocks without disruption and consequently some of the nucleotides are inherited with strong linkage disequilibrium, only diminished by recurrent mutation. On the other hand, if recombination occurs, the association between such nucleotides can be disrupted (McCarthy, 2005). A study of a Dutch A. fumigatus population showed that this fungus may reproduce asexually and sexually in nature based on the information revealed by linkage disequilibrium analyses of some markers (Klaassen et al., 2012). The genetic analysis of Candida glabrata, an opportunistic and haploid fungal pathogen similar to A. fumigatus, showed that this yeast has a similar ability to reproduce by asexual and sexual modes (Lott et al., 2010). On the other hand, there are fungal species, such as Penicillium marneffei, with occasional footprints of sexual reproduction but exhibiting patterns of extreme clonality and being genetically and spatially restricted (Henk et al., 2012; Ene and Bennett, 2014). Even considering the previous evidence, it remains difficult to understand how frequently A. fumigatus can reproduce by sexual mode. Therefore, the aim of this study was to analyse a set of environmental and clinical isolates of A. fumigatus in order to detect signs of recombination by employing genomic markers such as single nucleotide polymorphisms (SNPs). A group of strongly associated nucleotides was investigated in order to find some markers that support the occurrence of recombination through disruption of nucleotide associations. The proposed SNPs can be capable of detecting a fraction of recombination events that happen in A. fumigatus populations. The selected nucleotide positions were targeted by two minisequencing multiplexes, thus providing a simple and efficient strategy for analysis of those genomic positions in collections of A. fumigatus.
Results and discussion Selection of SNPs Based on the assumption that associated nucleotides may be disrupted when sexual reproduction happens (McCarthy, 2005), a strategy for the detection of recombination in A. fumigatus was designed. An initial set of 30 environmental isolates collected from air and water samples at S. João Hospital, Porto, Portugal, was used for the identification and selection of associated SNPs. This group comprised closely related A. fumigatus isolates that were characterized as MAT1-1 (Paoletti et al., 2005). The genetic diversity was also characterized based on polymorphisms located in intergenic regions (Rydholm
Table 1. Polymorphic associated positions identified in Aspergillus fumigatus. Linkage
Chromosome/gene
Position
Nucleotides
1
Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr
17 198 193 362 2 19 366 233 327 445 425 564 113 792 103 221 199 72 805
A/G G/A C/T C/T A/T C/T A/C C/T A/G C/T C/T T/C T/C C/G A/deletion C/G T/C A/deletion A/T
2 3 4 5 6 7 8 9
1/Lipase 2/Zinc transporter 3/Catalase 3/Annexin 4/Superoxide dismutase 3/Annexin 5/Rodlet A 2/Zinc transporter 5/Rodlet A 1/β-1,3-glucanosyltransferase 1/Lipase 3/Catalase 4/Mating type protein 3/Catalase 6/STR_M8 3/Annexin 4/Superoxide dismutase 2/Zinc transporter 3/Catalase
et al., 2006), microsatellites (Araujo et al., 2009) and multilocus sequence typing (MLST) genes (Bain et al., 2007; Caramalho et al., 2013). The first step of the proposed SNP strategy was the selection of the polymorphic positions by sequencing analysis of the MLST genes (seven genes: annexin, beta-1,3-glucanosyltransferase, catalase, lipase, MAT protein, superoxide dismutase, zinc transporter; Bain et al., 2007), the rodlet A gene commonly used for molecular identification of Aspergillus spp. (Serrano et al., 2011) and the genomic region near microsatellite M8 that contains a SNP (STR_M8; Araujo et al., 2009). The polymorphic positions were documented and compared; the SNP locus was considered to be polymorphic if the frequency of the minor allele was at least 0.05 in the initial population (Maïga-Ascofaré et al., 2010). Two nucleotides were considered as associated when differing simultaneously in A. fumigatus isolates, that is when isolates were typed at two positions, for example AT or CC, and evidence for disruption was simultaneously observed in both positions. More than 80 associated SNPs were initially identified in the sequenced genes. Some of these SNPs were excluded due to their proximity, since SNPs very close to each other tend to be inherited together even in the presence of recombination (Hinds et al., 2005; Seng and Seng, 2008). A final set of 19 SNPs structured in nine groups – eight pairs and one trio of SNPs was selected (Table 1; Fig. S1). The selected SNPs were located in six different chromosomes (exception of chromosomes 7 and 8); four SNPs were located in the catalase gene, followed by annexin and zinc transporter genes with three SNPs. The catalase gene was previously reported on genetic diversity studies of A. fumigatus as the gene accumulating more polymorphisms (Bain et al., 2007; Caramalho
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Recombination signs in A. fumigatus
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Table 2. List of genomic alterations in Aspergillus fumigatus strains; disruption of association between nucleotides is shaded in grey – details of linkage groups are in Table 1. Association group
Reference AF293 Reference 1163 ST1 ST2 ST3 ST4 ST5 ST6 ST7 ST8 ST9 ST10 ST11 ST12 ST13 ST14 ST15 ST16 ST17 ST18 ST19 ST20 ST21 ST22 ST23 ST24 ST25 ST26 ST27 S. João Hospital 1 S. João Hospital 2 S. João Hospital 3 S. João Hospital 4 S. João Hospital 5 S. João Hospital 6
1 AGC or GAT
2 CT or TA
3 CA or TC
4 CA or TG
5 TC or CT
6 TT or CC
7 CA or Gdel
8 CT or GC
9 AA or delT
AAC GAC ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ AGT ✓ ✓ ✓ AAT ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓
CA CA ✓ ✓ ✓ ✓ ✓ ✓ ✓ CA ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ CA CA ✓ ✓ ✓ CA ✓ ✓ ✓ ✓ ✓ ✓ ✓ CA ✓ CA CA
✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ TA ✓ ✓ ✓ ✓
TA ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ CG ✓ ✓ ✓ ✓ ✓
✓ CC ✓ ✓ ✓ ✓ ✓ TT ✓ CC ✓ TT ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ TT ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓
✓ CT TC CT ✓ ✓ ✓ TC ✓ ✓ ✓ ✓ ✓ ✓ ✓ TC CT ✓ CT ✓ ✓ ✓ TC CT ✓ ✓
✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ GA GA ✓ ✓
✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ GT ✓ ✓ ✓ CC ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓
✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ delA ✓ ✓
et al., 2013). Based on the selected genomic positions, a wild-type SNP profile was defined and posteriorly used to compare with other A. fumigatus isolates (Tables 1 and 2). In silico analysis of the associated nucleotides Sequencing results were deposited on an in-house database and the polymorphic positions were identified. The nucleotide information from the 27 sequence types (STs) available at MLST database (http://www.mlst.net/ databases/) was added to the database, as well as the genomic information of two reference A. fumigatus isolates, AF293 and A1163. The complete genome sequences of these two isolates were accessible at ENSEMBL (http://fungi.ensembl.org/index.html). BioEdit sequence alignment editor (available at http://www .mbio.ncsu.edu/bioedit/bioedit.html) and Geneious™ Pro 5.3.4 (Biomatters) were employed for gene alignment and detection of the polymorphic positions. Reference isolates
TC ✓ ✓ ✓ ✓ ✓ ✓ ✓
and 13 STs showed several variations on the associated SNP patterns (Table 2), in contrast with the wild-type genetic sequences initially defined. About one to four disruptions per isolate were observed, corresponding from 10% to 70% of the association panel. The highest number of disruptions was detected in AF293 and A1163, ST17 and ST22. These isolates were identified in the UK and USA more than 6 years ago; therefore, it is not surprising that these isolates are genetically very distinct from the initial group of Portuguese isolates. It is known that several mechanisms, such as genetic recombination and mutation, are responsible for genetic variations observed between populations. High genetic variation in A. fumigatus isolates was reported due to natural selection (Duarte-Escalante et al., 2009) and recombination (Klaassen et al., 2012). Mutation mechanisms can explain sporadic alterations of genome structure, but not all the disruptions reported above. Fungal isolates present a mutation rate of around 10−10 per locus per generation, meaning one mutation for every
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J. Teixeira, A. Amorim and R. Araujo
Table 3. MULTILOCUS (version 1.3b) linkage disequilibrium analysis in Aspergillus fumigatus populations (1000 randomizations were tested). N°. of isolates
Populations Initial population of closely related isolates Second group of Portuguese isolates Total Portuguese isolates (initial and second groups) Set of 27 sequence types available at MLST database (http://www.mlst.net/databases/) Recombinant isolates (13 sequence types plus reference strains af293 and a1163 plus six isolates from this study) Six isolates from this study with disrupted SNP linkage Total isolates studied (Portuguese plus online)
N°. of haplotypes
Ia
rBarD a
30 50 80 27 21
15 30 40 17 20
2.44 2.03a 1.95a 1.29a 0.72b
0.15a 0.11a 0.11a 0.11a 0.04b
6 109
6 55
1.08c 1.81a
0.07c 0.10a
a. p < 0.001. b. p = 0.15. c. p = 0.31. Ia = index of association.
1010 locus per generation (Johnston, 2003). Moreover, new adaptive allelic combinations are produced more rapidly by recombination than by mutation, since the occurrence of successive mutations is necessary to origin a novel genotype (Milgroom, 1996; Billiard et al., 2012). Thus, it is more likely that the large number of SNP disruptions observed in this study is caused by recombination than by mutation, and the selected SNPs can in fact be used for detection of recombination events in A. fumigatus. Characterization of clinical and environmental isolates of A. fumigatus A second set of 50 clinical and environmental isolates of A. fumigatus from the Porto region was used in this study. This group of isolates was characterized (based on MAT type, intergenic, microsatellite and MLST diversity) as described above for the initial group of isolates. The selected nucleotide positions were targeted by two minisequencing multiplexes (Fig. S1), providing a simple and efficient strategy for analysis of those genomic positions. When our second collection of isolates was analysed, few disruptions were also noted. A total of two clinical and four environmental isolates showed alterations in the associated panel of SNPs (Table 2; Fig. S1). Curiously, one of the clinical isolates with SNP alterations was identified in a single cystic fibrosis patient with long (over 7 years) chronic colonization by A. fumigatus (Amorim et al., 2010). Larger populations of A. fumigatus analysed on this work exhibited significant values of index of association (between 1.29 and 2.24; Table 3), mainly Portuguese isolates and isolates whose information was stored at the MLST database. Curiously the highest index (Ia = 2.44, p < 0.001; Table 3) was found on the group of closely related isolates collected in a limited timeframe in Porto region. Inversely, low and not significant index of association and rBarD values were found in the set of Portuguese and online isolates with SNP disruptions.
These values were calculated using MULTILOCUS 1.3b (Agapow and Burt, 2001). Index of association and rBarD give information of linkage disequilibrium allowing the comparison of individuals that share the same allele at one locus and evaluating the possibility of sharing the same allele at other loci. These values are equal to zero if there is no linkage disequilibrium and this value increases when strong linkage disequilibrium is found. An index of association of or close to zero suggests panmixia and no mating restrictions on the populations. The diversity of reproductive modes and mating systems observed in fungi is fascinating. The reproductive strategy is a key issue for understanding population genetics of microbial organisms, since patterns of inheritance significantly impinge on the majority of evolutionary and ecological processes. Sexual reproduction of A. fumigatus was firstly reported in 2009 (O’Gorman et al., 2009) and since then other research groups could replicate and simplify such strategy in the laboratory and successfully cross clinical and environmental isolates (Szewczyk and Krappmann, 2010; Sugui et al., 2011). In fact there are novel and mounting pieces of evidence supporting that sexual reproduction is not restricted to any particular source. No large differences have been reported until now comparing the genetic diversity (Araujo et al., 2009; 2010), susceptibility (Guinea et al., 2005; Araujo et al., 2007) and virulence (Aufauvre-Brown et al., 1998) of clinical versus environmental isolates of A. fumigatus, and it is possible that in reproduction strategies both groups follow similar paths as well. Some studies already reported a similar fraction of both MATs in sets of clinical and environmental isolates of A. fumigatus (Paoletti et al., 2005; Ene and Bennett, 2014). The present work proposes an innovative strategy that can facilitate fungal population analyses (Caramalho et al., 2013; Eusebio et al., 2013). In this study, a set of associated SNPs was identified and compared in clinical and environmental isolates of A. fumigatus and it was possible to observe some
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Recombination signs in A. fumigatus genomic alterations most likely explained by recombination. The frequency of SNP disruptions caused by recombination is proportional to the genomic distance between markers being maximal when they are located in distinct chromosomes, as observed in the group of markers selected for testing our hypothesis. The proposed SNPs were capable of detecting a fraction of recombination events happening in A. fumigatus population of both clinical and environmental isolates. Sexual reproduction may in fact be more regular than currently assumed in A. fumigatus populations, occurring in both clinical and environmental sets of isolates. The diversity of reproduction systems in A. fumigatus is certainly on the origin of the huge genetic diversity regularly reported worldwide (Chazalet et al., 1998; Balajee et al., 2007; Araujo et al., 2009; 2012; Klaassen et al., 2012). The present study proposed a set of new SNP markers capable of identifying signatures of recombination through the disruption of genomically associated nucleotides. It remains a challenge to unveil the conditions triggering the sexual cycle of A. fumigatus in nature.
Acknowledgements RA was supported by Fundação para a Ciência e a Tecnologia (FCT) Ciência 2007 and by the European Social Fund. IPATIMUP integrates the i3S Research Unit, which is partially supported by FCT, the Portuguese Foundation for Science and Technology. The authors declare no conflict of interest.
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Supporting information Additional Supporting Information may be found in the online version of this article at the publisher’s website: Fig. S1. Minisequencing multiplexes for characterization of associated nucleotides. A. List of markers used in SNaPshots 1 and 2 (minisequencing reactions) and genomic information; B. Position of each marker on the automated electropherogram; and C. Representative example of SNaPshot profiles (strain S. João Hospital 5) (peaks: orange – ladder; blue – guanine; black – cytosine; green – adenine; red – thymine). Table S1. Primers employed for gene amplification. Appendix S1. Experimental procedures.
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