Phylogenetic analysis of Lacazia loboi places this previously uncharacterized pathogen within the dimorphic Onygenales

Abstract

Lacazia loboi is the last of the classical fungal pathogens to remain a taxonomic enigma, primarily because it has resisted cultivation and only causes cutaneous and subcutaneous infections in humans and dolphins in the New World tropics. To place it in the evolutionary tree of life, as has been done for the other enigmatic human pathogens Pneumocystis carinii and Rhinosporidium seeberi, we amplified its 18S small-subunit ribosomal DNA (SSU rDNA) and 600 bp of its chitin synthase-2 gene. Our phylogenetic analysis indicated that L. loboi is the sister taxon of the human dimorphic fungal pathogen Paracoccidioides brasiliensis and that both species belong with the other dimorphic fungal pathogens in the order Onygenales. The low nucleotide variation among three P. brasiliensis 18S SSU rDNA sequences contrasts with the surprising amount of nucleotide differences between the two sequences of L. loboi used in this study, suggesting that the nucleic acid epidemiology of this hydrophilic pathogen will be rewarding.

FIG. 1

The clinical features encountered in lobomycosis are of importance to differentiate between paracoccidioidomycosis and infections caused by L. loboi. (A) Characteristic para-keloidal nodules of lobomycosis (patient 1) used in this study to extract genomic DNA from its in vivo yeast forms. (B) Tissue section obtained from the patient in panel A. Note the uniform cell sizes and the tubules connecting the chains of L. loboi yeast cells, two important features of its in vivo morphology (Gomori-methenamine silver stain; magnification, ×800).

FIG. 2

Neighbor-joining tree of aligned 18S rDNA sequences of two L. loboi individuals (a, patient 1; b, patient 2), all 28 available Onygenales sequences, and four Eurotiales species and one Chaetothyriales (Capronia pilosella) as outgroups. Together, the two L. loboi sequences form the sister clade to another Latin American endemic human pathogen, P. brasiliensis. The distance between the two L. loboi isolates and the distance to its neighbor are remarkably large. Numbers above and below the branches are percentages of bootstrap-resampled data sets supporting the branch as obtained by neighbor-joining and parsimony analyses, respectively. The only dimorphic human pathogen not in the clade with L. loboi is C. immitis. There was no topological conflict between this neighbor-joining tree and the consensus of 312 most parsimonious trees found in a heuristic search with 1,000 random additions of taxa, in part because the parsimony consensus tree was more poorly resolved. The scale bar represents 0.005 nucleotide substitutions per nucleotide. The organisms used in this tree and the GenBank accession numbers of their 18S rDNA sequences are as follows: Aphanoascus mephitalis, AB015779; Arthroderma ciferrii, AB015769; Arthroderma incurvatum, AB015770; Ascocalvatia alveolata, AB015782; Aspergillus fumigatus, AB008401; Auxarthron compactum, AB015767; Auxarthron zuffianum, AZU29395; B. dermatiditis, X59420; B. dermatitidis, M55624 and M63096 (two strains); C. pilosella, U42473; E. parva (=Chrysosporium parvum), U29390; C. immitus, X58571; Ctenomyces serratus, U29391; Eremascus albus, M83258; Eupenicillium javanicum, U21298; Eurotium rubrum, U00970; Gymnascella aurantiaca, AB015772; Gymnoascoideus petalosporus, U29392; H. capsulatum var. capsulatum, X58572; H. capsulatum var. duboisii, Z75306; L. loboi a, AF238301; L. loboi b, AF255331; Malbranchea albolutea, L28063; Malbranchea aurantiaca, AB015786; Malbranchea dendritica, U29389; Malbranchea filamentosa, L28065; Malbranchea gypsea, L28066; Neosartorya fischeri, U21299; Onygena equina, U45442; P. brasiliensis, AF227151, AF238302, and AF241655; Pectinotrichum llanense, AB015783; Renispora flavissima, U29393, Rollandina hyalinospora, AB015775; Spiromastix warcupii, AB015768; Trichophyton rubrum, X58570; and Uncinocarpus reesii, U29394. The three B. dermatitidis sequences are identical except for one or two ambiguous nucleotide positions. The three P. brasiliensis sequences are identical except for two gaps and one nucleotide difference in sequence AF227151.

FIG. 3

Neighbor-joining tree of aligned CHS2 nucleotide sequences of one L. loboi strain, other dimorphic human pathogens, and two Eurotiales and two Chaetothyriales species as outgroup taxa. The L. loboi sequence is the sister taxon to P. brasiliensis and a member of the strongly supported clade of all dimorphic human pathogens except C. immitis. Changes in the parameters of maximum-likelihood multiple-hit correction affected the placement of C. immitis but not of the other taxa. Parsimony analysis (branch and bound) gave one tree with the same topology as the neighbor-joining tree. Numbers above and below the branches are percentages of bootstrap-resampled data sets supporting the branch as obtained by neighbor-joining and parsimony analyses, respectively. The scale bar represents 0.1 nucleotide substitution per nucleotide. The organisms used in this tree and the GenBank accession numbers of the CHS nucleotide sequences are as follows: B. dermatitidis M82943, C. immitis U60213, Emericella nidulans M82941, Exophiala jeanselmei M82945, H. capsulatum M82949, L. loboi AF238303, P. brasiliensis Y09231, Penicillium marneffei U60516, Phaeococcus exophiale M82953.