Indentification And Investigation Of The Pathogenesis Of Pallidocercospora Crystallina-induced Cutaneous Phaeohyphomycosis

Background: Phaeohyphomycosis(PHM)covers cutaneous,subcutaneous and systemic infections caused by a diverse group of darkly pigmented fungi which can be distinguished from other fungi infection by the presence of septate pigmented hyphae or yeast in tissue.Dematiaceous fungi refer to a group of spores or filamentious which cantains characteristic melanin in their cell wall.Surveys for fungi spores in soil or associated plants routinely detect these moulds.The distribution of dematiaceous fungi is prevalent worldwide.Currently,over 150 species of fungi from more than 70 genus were identified in the etiology of phaeohyphomycosis.The immune background of infected host can be divided into two categories.One category of host immune status is the immune compentent individuals.Howerver,in a recent decade,increasing number of otherwise healthy individuals were suffered from PHM due to inherited CARD9 mutation.The other host immune is the immune imcompetent status which can be caused by immune suppressive drugs or chemotherapy drugs.A 35 years old woman who had a 16-years history of dark red plaque on her face was finally diagnosed as PHM.A dark fungus was isolated from the excised tissue which the accurate identification of the fungi at the species level was impossible by morphological methods due to its nonsporulating on different culture media.DNA sequences of the ITS1-5.8S-ITS2 regions and several evolutionary conservative regions were amplified by PCR.Sequencing results were compared using BLAST and Pallidocercospora crystallina(its sexual morph is known as Mycosphaerella crystallina)was indetified.P.crystallina,a phytopathogenic fungus,usually causes leaf and fruit spots.The P.crystallina infection had not been documented as human pathogen in the literature.The growth characteristicand the pathogenicity of P.crystallina were largely unknown.Therefore,the underlysing mechanism of P.crystallina infection was investigated.Objietive: In order to characterize this novel pathogen,morphology,growth charactstic,whole genome sequencing and in vivo immune response in mice were explored.Whole-exon sequencing were carried out in her family.Finally,the expression and function of CARD9 mutation were investigated.Methods: 1.A 16-years history of subcutaneous PHM was diagnoised due to P.crystallina infection.The skin biopsy was done and funguswas isolated from the excised lesion.DNA sequences of the ITS1-5.8S-ITS2 regions,18 Sr DNA,actin,elongation factor were amplified by PCR.Sequencing results were compared using BLAST.2.The growth charactistic of P.crystallina was observed at different culture condition.The structure of P.crystallina was observed through microscope and scanning electron microscope.3.To investigate the growth charactistic of P.crystallina at different culture conditions.4.Total genomic DNA of cultured P.crystallina was extracted.Purified DNA was used to construct Illumina standard shotgun library and then was sequenced using the Illumina Hi Seq platform.5.P.crystallina-induced PHM mice model was extablished in C57BL/6,BALB/c,and ICR mice.The pathgenecity of P.crystallina was explored in immune conpentent hosts in vivo.6.P.crystallina-induced PHM mice model was extablished in nude mice.The pathgenecity of P.crystallina was explored in immune supressive hosts in vivo.7.Whole-exome sequencing of the patient and her parents was done and analyzied.8.To explore the expression and function of detected mutation from whole-exon sequencing.Results: 1.A 35-year-old woman reported that 16 years earlier,she had noticed a papule formation on her left face after piercing by a poplar branch.The papules slowly increased in size and were resistant to conventional treatments including cryotherapy and antibiotics.The skin biopsy specimen showed exuberant epidermal pseudoepitheliomatous hyperplasia.In dermis,there was a heavy mixed infiltrate.Notably,deep pigmented septate hyphae and ovoid-shaped yeast were observed from stratum corneum to dermis infiltrate.DNA sequences of the ITS1-5.8S-ITS2 regions,18 S r DNA,actin,elongation factor were amplified by PCR.Sequencing results were compared using BLAST search,which indicated the Pallidocercospora crystallina with a similarity of 100%.2.Velvety and black colony was cultured from the excised skin tissue on SDA,PDA,OA or CMA after 2 weeks of incubation.Direct microscopic examination of cultured fungi showed filamentous septate hyphae with pale brown thickened wall,but without sporulating.Furthermore,transmission electron microscope revealed that connected hyphae were vertically or horizontally fractured without producing spores.3.The fungi were seed into SDA or PDA with or without actidione supplement for two weeks under at 28°C,35°C or 37°C.Results indicated thata“volcanic islands”-like black colony was gradually formed at 28°C,while only a small(0.4cm in diameter)black colony was formed at 35°C.Importantly,the fungi were unable to grow at 37°C.In addition,the fungi were unable to grow in SDA or PDA with actidione at 28°C during an incubation period of 2 weeks.4.After sequencing,raw reads were trimmed and filtered using Trimmomatic-0.36.De novo assembly of short read sequences were performed using SPAdes genome assembler(v.3.5.0),multi-kmer values were 77,91 and 127.The assembled 36958195 bp genome encodes 15499 putative coding genes.The genome was further mapped to the Eukaryotic Clusters of Orthologs(KOG)database,KEGG pathway database and the Pathogen-Host Interactions database to further characterize the putative proteins.5.Subcutaneously injected P.crystallina suspension can induced different degree of footpad swelling in C57BL/6,BALB/c,and ICR mice.However,footage swelling gradually decreased over the 3 to 4 weeks.No mortality due to subcutaneously injected P.crystallina suspension was observed.In addition,no mortality was documented when BALB/c,C57BL/6 and ICR mice were intraperitoneally infected with P.crystallina.Four weeks after infection,histological examination revealed no sign of infection.6.Subcutaneously injected P.crystallina suspension can induced amilded degree of footpad swelling innude mice comparing to immune competent mice.However,footage swelling gradually decreased over the 3 weeks.No mortality due to subcutaneously injected P.crystallina suspension was observed.In addition,no mortality was documented when nude mice were intraperitoneally infected with P.crystallina.7.Whole-exome sequencing of the patient and her parents was done and detected a homozygous missense mutation in CARD9(c.1118 G>C or p.R373P),which may lead to the suspectibility of the patient to P.crystallina infection.8.The m RNA and protein expression were similar among cells carrying homozygous(C/C),heterozygous(G/C)and wild type(G/G)CARD9 alleles.Comparing to heterozygous(G/C)and wild type(G/G)neutrophils,however,the anti-P.crystallina ability of neutrophils was significantly reduced in homozygous(C/C)mutation.Conclusion: Our research suggested that P.crystallina can lead to the PHM in human beings.Morphology and growth charactrizatics P.crystallina reveled that this fungus can not induced to produce spores in different culture conditions.Therefore,the identification through morphology is impossibile for P.crystallina.The core temperature of human beings can inhibit the growth of P.crystallina which may partially explain the localized lesion in our patient during the 16-years historyof infection.The whole-genome sequencing of the P.crystallina was mapped to several database.The results indicated lower pathogenicity of P.crystallina.P.crystallina-induced PHM mice model indicated that P.crystallina was unable to cause disseminated infection in immune competent or immune suppressive mice.Importantly,a novel homozygous missense mutation in CARD9(c.1118 G>C or p.R373P)was indentified through the whole-exon sequencing of the patient and her patients,whichmay confer her susceptibility to P.crystallina infection.Moreover,the m RNA and proteinexpression were similar among cells carrying homozygous(C/C),heterozygous(G/C)and wild type(G/G)CARD9 alleles.Comparing to heterozygous(G/C)and wild type(G/G)neutrophils,however,the anti-P.crystallina ability of neutrophils was significantly reduced in homozygous(C/C)mutation.The results indicated that,comparing to heterozygous or wild type neutrophils,the anti-P.crystallina ability was significantly reduced of the patient's neutrophils.In conclusion,the pathogenicity of P.crystallina was unable to cause infection in immune compentent hosts.Inherited CARD9 mutation maybe the underlying cause of otherwise healthy individuals who were suffered from invasive fungal infections.Physicians should be aware of the possibility of CARD9 mutation even in seemingly healthy patients with unexplained PHM.