Variability in minimal genomes: Analysis of tandem repeats in the microsporidia Encephalitozoon intestinalis Ana Galván a,b , Angela Magnet a , Fernando Izquierdo a , Soledad Fenoy a , Nuno Henriques-Gil c , Carmen del Aguila a,⇑ a Laboratorio de Parasitología, Facultad de Farmacia, Universidad San Pablo CEU, Urbanización Montepríncipe, CP 28668, Boadilla del Monte, Madrid, Spain b Escuela de Microbiología, Grupo de Parasitología, Universidad de Antioquia, Calle 67 No. 53-108, Medellín, Colombia c Laboratorio de Genética, Facultad de Medicina, Universidad San Pablo CEU, Urbanización Montepríncipe, CP 28668, Boadilla del Monte, Madrid, Spain article info Article history: Received 11 April 2013 Received in revised form 23 July 2013 Accepted 24 July 2013 Available online 2 August 2013 Keywords: Microsporidia Encephalitozoon intestinalis Genetic variability Genotype abstract Microsporidia are ubiquitous fungi with genomes that have undergone a strong reduction to the extreme cases of Encephalitozoon cuniculi and Encephalitozoon intestinalis. Genetic variability within species of the Encephalitozoon genus has been reported, with most of the studies based on the internal transcribed spacer (ITS) of the rDNA. However, in contrast to the picture of E. cuniculi and Encephalitozoon hellem, where different strains have been identified, no genetic variability has yet been observed in E. intestinalis. We have analysed tandem repeats included in putative coding sequences which could be used as poly- morphic markers in E. intestinalis. Eight candidate loci (M2, M2A, M3, M5, M7, M7A, M8 and PTP1) were established and 9 E. intestinalis cultured strains from North America, South America and Europe were analysed. M2, M7 and PTP1 nucleotide sequences were identical among the different strains and the Gen- Bank sequence. In contrast, we observed variants in 4 markers (M2A, M3, M7A and M8) which did not correspond to their respective reference sequences. The most noticeable finding was that with the M5 marker two genotypes were defined among the different strains studied, demonstrating genotypic vari- ability of E. intestinalis. Although the diversity described is certainly not high, which can be explained by a lower chance of genetic variability in its minimal genome, we have demonstrated that polymorphisms actually exist in E. intestinalis. Epidemiological studies using this genetic marker should now be con- ducted to elucidate the genetic variability in E. intestinalis and improve our knowledge of the epidemiol- ogy of this microsporidia. Ó 2013 Elsevier B.V. All rights reserved. 1. Introduction Special interest has been paid to minimal genomes in the last few years. Concerning prokaryotes, they have a practical applica- tion for the purpose of accomplishing synthetic forms of life (Gil et al., 2004; Shuler et al., 2012). Yet the idea of minimal genomes also addresses more fundamental questions about the essence of life itself (Juhas et al., 2011). However, when considering which genes are absolutely necessary for survival, there are some arbi- trary concepts involved, as most organisms (heterotrophs) use a wide variety of products generated by others. This is particularly the case of parasites with a strong dependence on the host metabolism. Among Eukaryotes, microsporidia have the most extreme gen- ome reductions. Their fungal nature is now accepted and they are considered as the earliest-diverging clade of sequenced fungi (Capella-Gutierrez et al., 2012). There are approximately 160 gen- era with more than 1300 species (Keeling, 2009; Lee et al., 2010; Peyretaillade et al., 2011), which parasite a wide range of verte- brate and invertebrate hosts (Didier, 2005; Didier and Weiss, 2008; Keeling, 2009). Eight genera and 14 species have been asso- ciated with human infections, among which Enterocytozoon bien- eusi and Encephalitozoon intestinalis are the most frequently reported, followed by Encephalitozoon cuniculi and Encephalitozoon hellem (Didier, 2005; Didier and Weiss, 2006, 2008). These oppor- tunistic pathogens cause a variety of systemic and nonsystemic diseases, with chronic diarrhea as the most common clinical man- ifestation (Didier, 2005; Didier and Weiss, 2006, 2008). Human microsporidia are recognized as emerging pathogens in immuno- compromised people, HIV/AIDS patients and organ transplant recipients among others, and they are also increasingly described in immunocompetent people (Abreu-Acosta et al., 2005; Didier, 2005; Didier and Weiss, 2008; Lopez-Velez et al., 1999; Lores et al., 2002). These species have also been identified in water sources as well as in wild, domestic and food-producing farm 1567-1348/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.meegid.2013.07.024 ⇑ Corresponding author. Tel.: +34 91 372 4770 - 4784; fax: +34 91 351 04 96. E-mail addresses: agalvandiaz@yahoo.com (A. Galván), angela.magnetdavila@ ceu.es (A. Magnet), ferizqui@ceu.es (F. Izquierdo), sfenrod@ceu.es (S. Fenoy), nhengil@ceu.es (N. Henriques-Gil), cagupue@ceu.es (C. del Aguila). Infection, Genetics and Evolution 20 (2013) 26–33 Contents lists available at ScienceDirect Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid