Cutaneous Lagenidium deciduum infection in a patient with relapsed acute myeloid leukemia.

BACKGROUND: Lagenidium deciduum is an oomycete that can cause infections in mammals that present similarly to pythiosis and mucormycosis. Most of the existing case reports have occurred in canines and have been fatal. In animals, medical therapy has not been successful, so surgical excision is the mainstay of treatment. Lagenidium sp. infections in humans are rare. There is only one case of a human Lagenidium sp. infection in the literature, and it presented as an ocular infection. The human ocular infection was resistant to medical therapy and required a penetrating keratoplasty for cure. Additional reports of effective therapy are needed to guide management of this emerging pathogen. We present the first case of a cutaneous Lagenidium deciduum infection in a human patient, which is also the first documented case of a Lagenidium deciduum infection in an immunocompromised host of any species. CASE PRESENTATION: An 18-year-old female with relapsed acute myeloid leukemia, awaiting a haploidentical stem cell transplant, presented with erythematous cutaneous lesions on her left hip and bilateral buttocks that enlarged and blackened over several days. About 1 week later, boil-like lesions appeared on her bilateral buttocks. The skin lesions were initially presumed to be bacterial in origin, so the patient was treated with clindamycin and cefepime with little improvement. Upon further investigation, fungal cultures and skin biopsies revealed aseptate hyphae, so the patient was switched to isavuconazole and amphotericin B due to concern for mucormycosis. Phenotypic characterization and DNA sequencing were performed by the Fungus Testing Laboratory, University of Texas Health Science Center at San Antonio, which identified the causal fungal organism as Lagenidium deciduum. All of her cutaneous lesions were surgically excised, and the patient was treated with micafungin, terbinafine, doxycycline, and azithromycin. Micafungin and terbinafine were continued until she achieved engraftment post-transplant. CONCLUSIONS: We report the first successful treatment of a human Lagenidium infection in an immunocompromised host through a combination of aggressive surgical excision and prolonged antifungal therapy during the prolonged neutropenia associated with allogeneic stem cell transplant. Prompt diagnosis and management may prevent disseminated oomycosis.

Glycoside hydrolases family 20 (GH20) represent putative virulence factors that are shared by animal pathogenic oomycetes, but are absent in phytopathogens.

BACKGROUND: Although interest in animal pathogenic oomycetes is increasing, the molecular basis mediating oomycete-animal relationships remains virtually unknown. Crinkler (CRN) genes, which have been traditionally associated with the cytotoxic activity displayed by plant pathogenic oomycetes, were recently detected in transcriptome sequences from the entomopathogenic oomycete Lagenidium giganteum, suggesting that these genes may represent virulence factors conserved in both animal and plant pathogenic oomycetes. In order to further characterize the L. giganteum pathogenome, an on-going genomic survey was mined to reveal novel putative virulence factors, including canonical oomycete effectors Crinkler 13 (CRN13) orthologs. These novel sequences provided a basis to initiate gene expression analyses and determine if the proposed L. giganteum virulence factors are differentially expressed in the presence of mosquito larvae (Aedes aegypti). RESULTS: Sequence analyses revealed that L. giganteum express CRN13 transcripts. The predicted proteins, like other L. giganteum CRNs, contained a conserved LYLA motif at the N terminal, but did not display signal peptides. In contrast, other potential virulence factors, such as Glycoside Hydrolases family 20 (hexosaminidase) and 37 (trehalase) proteins (GH20 and GH37), contained identifiable signal peptides. Genome mining demonstrated that GH20 genes are absent from phytopathogenic oomycete genomes, and that the L. giganteum GH20 sequence is the only reported peronosporalean GH20 gene. All other oomycete GH20 homologs were retrieved from animal pathogenic, saprolegnialean genomes. Furthermore, phylogenetic analyses demonstrated that saprolegnialean and peronosporalean GH20 protein sequences clustered in unrelated clades. The saprolegnialean GH20 sequences appeared as a strongly supported, monophyletic group nested within an arthropod-specific clade, suggesting that this gene was acquired via a lateral gene transfer event from an insect or crustacean genome. In contrast, the L. giganteum GH20 protein sequence appeared as a sister taxon to a plant-specific clade that included exochitinases with demonstrated insecticidal activities. Finally, gene expression analyses demonstrated that the L. giganteum GH20 gene expression level is significantly modulated in the presence of mosquito larvae. In agreement with the protein secretion predictions, CRN transcripts did not show any differential expression. CONCLUSIONS: These results identified GH20 enzymes, and not CRNs, as potential pathogenicity factors shared by all animal pathogenic oomycetes.

Lagenidium giganteum pathogenicity in mammals.

Infections of mammals by species in the phylum Oomycota taxonomically and molecularly similar to known Lagenidium giganteum strains have increased. During 2013-2014, we conducted a phylogenetic study of 21 mammalian Lagenidium isolates; we found that 11 cannot be differentiated from L. giganteum strains that the US Environmental Protection Agency approved for biological control of mosquitoes; these strains were later unregistered and are no longer available. L. giganteum strains pathogenic to mammals formed a strongly supported clade with the biological control isolates, and both types experimentally infected mosquito larvae. However, the strains from mammals grew well at 25°C and 37°C, whereas the biological control strains developed normally at 25°C but poorly at higher temperatures. The emergence of heat-tolerant strains of L. giganteum pathogenic to lower animals and humans is of environmental and public health concern.

Transcriptome analysis of the entomopathogenic oomycete Lagenidium giganteum reveals putative virulence factors.

A combination of 454 pyrosequencing and Sanger sequencing was used to sample and characterize the transcriptome of the entomopathogenic oomycete Lagenidium giganteum. More than 50,000 high-throughput reads were annotated through homology searches. Several selected reads served as seeds for the amplification and sequencing of full-length transcripts. Phylogenetic analyses inferred from full-length cellulose synthase alignments revealed that L giganteum is nested within the peronosporalean galaxy and as such appears to have evolved from a phytopathogenic ancestor. In agreement with the phylogeny reconstructions, full-length L. giganteum oomycete effector orthologs, corresponding to the cellulose-binding elicitor lectin (CBEL), crinkler (CRN), and elicitin proteins, were characterized by domain organizations similar to those of pathogenicity factors of plant-pathogenic oomycetes. Importantly, the L. giganteum effectors provide a basis for detailing the roles of canonical CRN, CBEL, and elicitin proteins in the infectious process of an oomycete known principally as an animal pathogen. Finally, phylogenetic analyses and genome mining identified members of glycoside hydrolase family 5 subfamily 27 (GH5_27) as putative virulence factors active on the host insect cuticle, based in part on the fact that GH5_27 genes are shared by entomopathogenic oomycetes and fungi but are underrepresented in nonentomopathogenic genomes. The genomic resources gathered from the L. giganteum transcriptome analysis strongly suggest that filamentous entomopathogens (oomycetes and fungi) exhibit convergent evolution: they have evolved independently from plant-associated microbes, have retained genes indicative of plant associations, and may share similar cores of virulence factors, such as GH5_27 enzymes, that are absent from the genomes of their plant-pathogenic relatives.

Severe meningoencephalitis secondary to calvarial invasion of Lagenidium giganteum forma caninum in a dog.

Background: The oomycete Lagenidiumgiganteum forma caninum is an uncommon cause of severe dermal and subcutaneous infections in dogs with possible vascular invasion and other fatal sequelae. Infection within the central nervous system of affected dogs has not been previously reported. Case Description: A 6-year-old spayed female mixed-breed dog was evaluated at a referral institution with a 2-month history of suspected fungal infection in the region of the right mandibular lymph node that was refractory to surgical resection and empiric medical therapy. Physical examination identified a 6-cm fluctuant subcutaneous mass caudoventral to the ramus of the right mandible and a second firm mass in the region of the right caudal maxilla. Lesional punch biopsies were submitted for fungal culture and polymerase chain reaction (PCR), which subsequently identified L. giganteum forma caninum infection. Initial treatment consisted of anti-inflammatory doses of prednisone and hyperbaric oxygen therapy. Four weeks following initial evaluation, the patient was presented with progressive neurological signs consistent with a forebrain lesion. Magnetic resonance imaging revealed soft-tissue, contrast-enhancing lesions ventral to the calvarium adjacent to the site of original surgical resection and throughout the brain. Humane euthanasia was elected, and postmortem examination was consistent with the extension of local disease from the right masseter muscle into the right ventral calvarium. Postmortem DNA sequencing confirmed the identity of the organism as L. giganteum forma caninum. Conclusion: This is the first reported case of intracranial lagenidiosis in the dog. PCR distinguished this species from other Lagenidium species and from oomycetes of other genera, such as Pythiuminsidiosum and Paralagenidium karlingii. Regional extension of cutaneous lagenidiosis should therefore be considered in cases with concurrent or spontaneous neurologic disease.

Description of three novel Lagenidium (Oomycota) species causing infection in mammals.

BACKGROUND: Recent molecular phylogenetic analysis of Lagenidium strains recovered from subcutaneous lesions in cats, dogs, and a human with lagenidiosis resolved into four clades; one of them was Lagenidium giganteum, but three others were novel. AIMS: Due to the recent increase in L. giganteum infections from mammals, we studied 21 Lagenidium strains isolated from dogs and a human available in our collection. METHODS: Molecular phylogenetic studies and phenotypic characteristics were used to characterize the strains. RESULTS: We report the finding of three novel species, herein designated as Lagenidium ajelloi, sp. nov., Lagenidium albertoi sp. nov, and Lagenidium vilelae sp. nov. Their morphological and growth features are also presented. CONCLUSIONS: Our study revealed the presence of three novel Lagenidium species infecting mammals.

Molecular phylogeny and taxonomy of Lagenidium-like oomycetes pathogenic to mammals.

Over the past twenty years, infections caused by previously unrecognised oomycete pathogens with morphological and molecular similarities to known Lagenidium species have been observed with increasing frequency, primarily in dogs but also in cats and humans. Three of these pathogens were formally described as Lagenidium giganteum forma caninum, Lagenidium deciduum, and Paralagenidium karlingii in advance of published phylogenetic verification. Due to the complex nature of Lagenidium taxonomy alongside recent reports of mammalian pathogenic species, these taxa needed to be verified with due consideration of the available data for Lagenidium and its allied genera. This study does so through morphologic characterisation of the mammalian pathogenic species, and phylogenetic analyses. The six-gene phylogeny generally supports the most recent comprehensive classification of Lagenidium with a well-supported Lagenidium clade that includes the mammalian pathogens L. giganteum f. caninum and L. deciduum, and well-supported clades for which the names Myzocytiopsis and Salilagenidium can be applied. The genus Paralagenidium is phylogenetically unrelated to any of the main clades within the class Peronosporomycetes. Close relationships between pathogens of mammals and those of insects or nematodes were revealed. Further characterisation of Lagenidium-like taxa is needed to establish the risk of mammalian infection by pathogens of insects and nematodes.

Evolution of glutamine synthetase in heterokonts: evidence for endosymbiotic gene transfer and the early evolution of photosynthesis.

Although the endosymbiotic evolution of chloroplasts through primary and secondary associations is well established, the evolutionary timing and stability of the secondary endosymbiotic events is less well resolved. Heterokonts include both photosynthetic and nonphotosynthetic members and the nonphotosynthetic lineages branch basally in phylogenetic reconstructions. Molecular and morphological data indicate that heterokont chloroplasts evolved via a secondary endosymbiosis, involving a heterotrophic host cell and a photosynthetic ancestor of the red algae and this endosymbiotic event may have preceded the divergence of heterokonts and alveolates. If photosynthesis evolved early in this lineage, nuclear genomes of the nonphotosynthetic groups may contain genes that are not essential to photosynthesis but were derived from the endosymbiont genome through gene transfer. These genes offer the potential to trace the evolutionary history of chloroplast gains and losses within these lineages. Glutamine synthetase (GS) is essential for ammonium assimilation and glutamine biosynthesis in all organisms. Three paralogous gene families (GSI, GSII, and GSIII) have been identified and are broadly distributed among prokaryotic and eukaryotic lineages. In diatoms (Heterokonta), the nuclear-encoded chloroplast and cytosolic-localized GS isoforms are encoded by members of the GSII and GSIII family, respectively. Here, we explore the evolutionary history of GSII in both photosynthetic and nonphotosynthetic heterokonts, red algae, and other eukaryotes. GSII cDNA sequences were obtained from two species of oomycetes by polymerase chain reaction amplification. Additional GSII sequences from eukaryotes and bacteria were obtained from publicly available databases and genome projects. Bayesian inference and maximum likelihood phylogenetic analyses of GSII provided strong support for the monophyly of heterokonts, rhodophytes, chlorophytes, and plants and strong to moderate support for the Opisthokonts. Although the phylogeny is reflective of the unikont/bikont division of eukaryotes, we propose based on the robustness of the phylogenetic analyses that the heterokont GSII gene evolved via endosymbiotic gene transfer from the nucleus of the red-algal endosymbiont to the nucleus of the host. The lack of GSIII sequences in the oomycetes examined here further suggests that the GSIII gene that functions in the cytosol of photosynthetic heterokonts was replaced by the endosymbiont-derived GSII gene.