molecular and structural similarities. For example, IgE cross- reactivity has been demonstrated between helminth (e.g. filarial and Ascaris) tropomyosins and the tropomysoins of mites (Der p 10) [22,23] and cockroaches (Bla g 7) [24]. In addition, cross- reactivity between cockroach glutathione-S transferase (GST; Bla g 5) and filarial GST of Wuchereria bancrofti has also been shown to occur [25]. The extensive number of homologues found between helminth proteins and allergens has led to the speculation that these similarities may underlie the allergenicity of IgE-inducing proteins [26]. This elegant hypothesis proposes that Th2-dominated immune responses have evolved to control helminth infection, but, because of molecular mimicry, the host also may become hyperresponsive to innocuous environmental proteins (allergens) leading to clinically apparent allergic disease [26]. This suggests that the ‘‘allergenicity’’ of some common allergens is a bystander effect that evolved as a consequence of natural immune responses to helminth antigens. Remarkably, structural homology is indeed involved in the allergenicity of Der p 2, a major house dust mite allergen that belongs to the same protein family of humans (MD2 protein) [27] and helminths (ML proteins). The relative structural similarity of allergens with self-proteins have also been regarded as a factor underlying allergenicity [28,29] either by interfering with specific host immune pathways [27] or by failing to induce ‘‘strong Th1 polarization’’ [29]. Although multiple mechanisms of allergic sensitization have been proposed [6], molecular mimicry – either with proteins of microbial pathogens (particularly by helminths) or of humans– seems to be a general unifying hypothesis to explain allergenicity [26,28,29]. Therefore, having systematic analyses of parasites and other microorganisms that could mimic (at a molecular level) allergens could allow for insight into potential mechanisms by which allergencity is conferred. To this end, we performed a compre- hensive study of 499 molecularly-defined allergens and their structural relationship to predicted proteins of prototypical pathogens from which whole genome data were available so as to search for ‘‘allergen homologues’’ among the microbial genera. While our initial goal was to examine solely the helminth/allergy interface (e.g. Brugia malayi, Loa loa, Wuchereria bancrofti and Schistosoma mansoni), we broadened the analysis to include four protozoan, four bacterial and three fungal genomes in large part to understand the distinction between Th2-inducing organisms/ proteins and those derived from organisms that more consistently induce Type-1 and Type-17 immune responses. This analysis enabled us to address if allergenicity is related to: a) structural similarities to helminth (Th2-inducing) or fungal (Th17-inducing) organisms that have many allergenic proteins; b) structural dissimilarities to proteins of protozoan or bacterial (Th1 inducing) organisms; or c) structural relatedness to human proteins. Our results suggest that immunogenicitybased on structural differences with human proteins is a major force driving the immune response to allergens. Results Extensive Molecular Similarity between Common Allergens and Parasite Proteins We created a list of molecularly well-defined allergens covering 499 allergens catalogued in the AllFam database [3] that included 145/180 of the allergen families. This list was used to perform in silico searches for homologues across 15 pathogen genomes including four helminths (Brugia malayi, Loa loa, Wuchereria bancrofti and Schistosoma mansoni), four protozoans (Leishmania major, Tolerance to Self and Allergenicity Trypanosoma cruzi, Plasmodium falciparum and Toxoplasma gondii), four bacteria (Escherichia coli, Staphylococcus aureus, Mycobacterium tubercu- losis and Listeria monocytogenis) and three fungi (Candida albicans, Histoplasma capsulatum and Aspergillus fumigatus). A protein was considered homologous to the allergen if the expected value (e- value, that measures the likelihood of the amino acid match had occurred by chance) between them was less then 1026, regardless the amino acid identity level. If the pair was not considered homologous (e-value equal to or greater than 1026) the amino acid identity between them was arbitrarily set to zero. We found a relatively large number of allergens having homologues among the many microbial organisms studied (Figure 1 and Table S1). Across the four helminth genera, the median number of encoded allergens with homologues was 202/ 499, representing 40% of the 499 allergens. This level of homology was similar to that found among the fungi studied (Figure 1A). Allergens had significantly fewer homologues (p,0.0001 by Fisher’s exact test) in protozoan genera (160/499; 32%) and even fewer in the bacteria group (71/499; 14%). This same pattern could be recognized in the number of allergen families (ALLFAM) involved (Figure 1B). Interestingly, if an allergen had a homologue in one genus of a certain phyla, it was also likely to have homologues in the other genera of the same phyla, with rare exceptions (Table S1 and Table S2). When there were homologues between the allergens and the proteins of the pathogenic organisms (except for the bacteria), for most of them (,85% of the allergens) the amino acid identity levels were typically above 30% (Figure 1C). A majority of these showed an identity level between 25% and 45%. In contrast, the majority of the bacterial/ allergen homologues had amino acid identity levels below 30% (Figure 1D). The median amino acid identity level between allergens and their respective homologous microbial proteins was 37% for helminths, 34% for fungi, 32% for protozoa and only 28% for bacteria (Figure 1D). We were thus able to confirm previous findings suggesting allergens are rarely homologous to bacterial proteins [30] probably because of big phylogenic distance. Because bacteria had fewer homologues and lower levels of homology when compared to the other microbes, this group was excluded from the subsequent analyses. Allergens Conserved with Microbial Proteins Display Lower Levels of Allergenicity Among the 312 allergens displaying homologues in helminths, protozoa and fungi (the HPF group) of the 499 studied, we found 180 to be shared among the three organism groups analyzed (Figure 2A). In contrast, no homologues among any of the genera studied were found for 37% of the allergens (187/499) (Figure 2A) representing 43 of the 145 AllFams included. To evaluate the relationship between conservation of sequence and allergenicity, we collected the IgE prevalences for those allergens for which such information was available (Table S1) either from the literature or from the World Health Organization and the International Union of Immunology Societies (www.allergen.org) databases for system- atic allergen nomenclature (a total of 357/499 allergens). We used the standard definition of a major allergen, that being molecularly defined substances from a complex mixture (extract) that induces IgE responses in .50% of patients allergic to that complex material source, i.e. allergen extract [31]. We found that 40% of those allergens that were highly conserved across the phyla were major allergens (blue bar of Figure 2B). In contrast, of the allergens that had no homologues in these 3 microbial phyla, 75% were major allergens (left black bar of Figure 2B) (p,0.0001 by Fisher’s exact test). These data suggest that there is a negative relationship between the level of conservation and the level of allergenicity. To PLoS ONE | www.plosone.org 2 July 2012 | Volume 7 | Issue 7 | e40552