Concerted evolution of repeat units within the gene encoding an immunomodulating parasitic cell surface glycoprotein in the human pathogenic fungi Coccidioides immitis and C. posadasii Hanna Johannesson, Jeffrey P. Townsend & John W. Taylor UC Berkeley, Dept. of Plant and Microbial Biology C. p. A B A 4 3 2 1 0 SOWgp repeat units evolve in concert 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0 1 2 3 4 C. immitis C. posadasii 0 0.01 0.02 0.03 0.04 0.05 0.06 0 1 2 C. immitis C. posadasii Different internal repeats within species (A) are more genetically similar than corresponding repeats between species (B) Concerted evolution by unequal crossing over adaptive significance The dimorphic life cycle of Coccidioides Saprobic life cycle in desert soil Parasitic life cycle in mammals vegetative mycelia germination air-dispersed arthroconidia isotropic growth segmentation endospore differentiation spherule endospore release host death C. i. Repeats included Mean pair-wise genetic distance (SD) A B All repeats Internal repeats C. immitis C. posadasii 0.074 (0.055) 0.064 (0.037) 0.065 (0.036) 0.045 (0.018) 0.018 (0.012) 0.047 (0.023) Corresponding internal repeats of the two species do not group together in a phylogeny. Instead, the internal repeats group by species. Modulation of host immune response with SOWgp Endosporulating spherule immunodominant SOWgp plasma membrane spherule inner wall spherule outer wall complex Mep1 SOWgp peptides released Model of modulation metalloproteinase (Mep1) enzyme is secreted from the fungus at endosporulation Mep1 digests SOWgp from surface of spherule and endospores immunodominant SOWgp peptides released induction of strong non-protective immune response endospores stripped of SOWgp evade detection by the host Modulation of host response with SOWgp Result: Polymorphism in SOWgp repeat number between strains - all strains of both species have either 3, 4 or 5 repeats Positive correlation between genetic distance and physical distance in the primary sequence: Spherule outer wall glycoprotein (SOWgp) gene Patient antibody-reactive domain aa# 1 20 60 82 127 174 221 315 268 356 398 422 N-terminal non-repeat sequence C-terminal non-repeat sequence Signal peptide Propeptide Proline/aspartate-rich repeats GPI anchor signal Cleavage site (Hung et al. 2002. Infection and immunity 70: 3443-3456) A high diversity of repeat sequence types - D( = 1-Sp ) : 0.83 (C. immitis) and 0.94 (C. posadasii) i 2 All repeats included Edge repeats excluded p3503.4(5) p3272.4(5) p1049.3(5) p2103.3(5) p1040.3(4) i1705.1(5) i2012.1(4) i2018.1(4) i2102.1(4) i2271.1(4) i2275.1(4) i2278.1(3) i3505.1(5) p3490.1(4) p1038.1(5) p2377.1(3) p2378.1(5) p2379.1(5) p3272.1(5) p3503.3(5) p2345.3(5) p2346.3(5) p2347.3(5) p2348.3(5) p2103.1(5) p1039.1(5) p1040.1(4) p1049.1(5) p1444.1(4) p2345.1(5) p2346.1(5) p2347.1(5) p2348.1(5) p3503.1(5) p1038.3(5) p2378.3(5) p2379.3(5) p3272.3(5) p2347.4(5) p1039.3(5) p1038.4(5) p1039.4(5) p1049.4(5) p1444.3(4) p2103.4(5) p2345.4(5) p2346.4(5) p2348.4(5) p2378.4(5) p2379.4(5) p3490.3(4) i1705.4(5) i2018.3(4) i2102.3(4) i2271.3(4) p3490.4(4) p1038.5(5) p2345.5(5) p2346.5(5) p2347.5(5) p2348.5(5) p2377.3(3) p2378.5(5) p2379.5(5) p3272.5(5) p3503.5(5) p1039.5(5) p1040.4(4) p1049.5(5) p1444.4(4) p2103.5(5) i1705.5(5) i2012.4(4) i2018.4(4) i2102.4(4) i2271.4(4) i2275.4(4) i2278.3(3) i3505.5(5) i2278.2(3) i2275.3(4) i1705.3(5) i2012.3(4) i1705.2(5) i2012.2(4) i2018.2(4) i2102.2(4) i2271.2(4) i2275.2(4) i3505.2(5) i3505.3(5) i3505.4(5) p3503.2(5) p2345.2(5) p2346.2(5) p2347.2(5) p2348.2(5) p2103.2(5) p1039.2(5) p1040.2(4) p1049.2(5) p1038.2(5) p1444.2(4) p2377.2(3 p2378.2(5) p2379.2(5) p3272.2(5) p3490.2(4) 73 97 76 56 51 77 56 C. posadasii - edge repeats C. immitis - edge repeats C. immitis internal repeats Repeats are named as follows: i=immitis, p=posadasii; RMSCC- strain number; repeat x out of (y) Estimation of population sizes and rates of homogenization and recombination Conclusion: SOWgp consists of tandemly repeated proline rich domains. The repeats evolve in concert, by the mechanism of unequal crossing over. Concerted evolution is most prominent in C. immitis, probably because it has a smaller population size. We suggest that the adaptive significance of concerted evolution in repeated domains of SOWgp is for the pathogen to maintain multipe identical antigen-binding sites. The pathogen is thought to mislead the hosts’ immune system by stripping off SOWgp from the outer cell wall of released endospores at sporulation. The non-protective immune response to released SOWgp results in exacerbation of the disease, and provides protection to the pathogen against phagocytic and fungicidal activities of the host innate immune system. By having a repetitive structure of SOWgp, consisting of numerous identical epitopes, the non-protective response of the immune system is enhanced and the pathogen is able to efficiently divert the efforts of the immune system. All repeats Internal repeats b C. i. C. p. C. i. C. p. f 0.78 0.30 0.61 0.25 C 1 0.020 0.0071 0.091 0.026 C 2 0.014 0.0022 0.077 0.0075 λ 4.3x10 -11 2.6x10 -11 1.1x10 -10 5.3x10 -11 β 6.8x10 -8 6.5x10 -9 2.8x10 -8 7.6x10 -9 N 9.3x10 7 7.9x10 8 2.0x10 8 9.9x10 8 Under the assumption of an allelic mutation rate (v) of 7.2 x 10-10, based on the nucleotide mutation rate of N. crassa and the number of neutrally evolving sites of 16 (the total number of polymorphic sites within species) We applied an infinite-alleles model for the concerted evolution of a multigene family (Ohta, T., Theor. Popul. Biol. 23, 216-240) to our SOWgp data. This model predicts the equilibrium values of f, the observed average pairwise identity of corresponding repeats on separate chromosomes; C1, the observed average pairwise identity of all repeats within chromosomes; C2, the observed average pairwise identity of all non-corresponding repeats between chromosomes. The model predicts these statistics given values for v, the allelic mutation rate; N, the effective population size; β, the rate at which equal interchromosomal crossover occurs between repeated domains; and λ, the rate at which a repeat domain is converted to be identical to another of the (n - 1) homologous domains. Observed identity between repeat units higher in C. immitis than in C. posadasii Accordingly, we found a higher rate of homogenization and recombination, and a smaller population size, in C. immitis. This model has previoulsy been used to estimate rates of homogenization and recombination between genes in the MHC complex (Ohta, T. 1982. PNAS 79: 3251-4). We found much lower rates of homogenization and recombination in SOWgp, which is likely a result of investigating within gene dynamics of concerted evolution, in a system without gene conversion . Coccidioidomycosis Etiological agents: Coccidioides immitis and C. posadasii The disease is epidemic: 400-4000 cases/year reported Coccidioides is endemic to semiarid soils of the New World (The model of immunomodulation proposed by Hung et al. 2002. Infection and immunity 70: 3443-3456) Distance in the primary sequence Distance in the primary sequence Genetic distance Genetic distance Method: the average genetic distance between repeat units at different distances in the array were compared distance in sequence: genetic distance: Commons License. F1000 Po nder Creative Commons License. F1000 Posters: Use Pe e Permitted under Creative Commons License. F1000 Posters: Use Permitted under 0 Posters: Use Permitted under Creative Commons License. F1000 Posters: Use Permitted under Creative Com ers: Use Permitted under Creative Commons License. F1000 Posters: Use Permitted under Creative Commons Licens mitted under Creative Commons License. F1000 Posters: Use Permitted under Creative Com reative Commons License. F1000 Posters: Use Permitted unde ons License. F1000 Posters: Use Pe