Genomics Analysis And Molecular Diagnosis On Novel And Rare Invasive Fungal Pathogen

Invasive fungal infection(IFI)refers to the fungus which invades into the below of skin corneum,mucosa,deep tissue or viscera organs.Under a certain condition,the IFI could spread to and infect the whole body,including the deep tissue infection and fungemia.In the recent 30 years,the morbidity and death rate caused by IFI is increasing rapidly,as the consequence of a long-term application of significant dose of Broad spectrum antibiotics and antineoplastic agents,as well as the intervening of various catheter in body,the raising of immune dysfunction patients,such as malignant tumor and HIVs,and organ transplantation and invasive medical operations.Simultaneously,the new and burst cases of fungal pathogen occur continuously.Contrast to the boom of IFI cases,the studies on it are still weakly.Researches on the genome sequencing,molecular diagnosis,and medicine developing lag behind those on virus and bacteria.Due to the study on new and rare fungal pathogen by means of genome sequencing and molecular diagnosis,this research focus on the construction of whole-genome data,evolution of closely related species,anagenesis of pathogenesis-related gene,presumption of pathogenesis mechanics,prediction of molecular diagnosis marker and so on.This study will lay the foundations for further prevention,diagnosis and therapy.In this study,the combination of the second-generation and third-generation sequencing platform were implemented to construct an efficient method to match new and rare IFI genome de novo as well as deeply analysis on pathogenic factor.As to new finding IFI Penicillium capsulatum and Emmonsia beijingensis nov.,under the conditions of lacking reference genome,the whole-genome data was sequenced for the first time,and after de novo assembly,a high completeness genomic map was acquired following by an analysis on the virulence factor of these fungi and their closely related species.Since 2011,the second-generation sequencing technology developed rapidly as the representative of Illumina Hiseq and Miseq.With the cost and the sequencing time cutting down,there is coming a revolution of genomics research.But the shortage of the second-generation sequencing,such as the short reading length,bring great difficulty to the following bioinformatics analysis work,such as matching,assembling and annotating.Especially in the case of eukaryotes with more repeated sequences,the de novo assembly will be more difficult.In contrast,the advantage of Pacbio RSII,as the representative of third-generation sequencing,is that it has long reading length,and could cross repeated zone successfully.This could reduce not only the number of contig generated by sequencing,but also the following genome assembly work.But the error of third-generation sequencing could be greater than the former two generations,the error rate of single base could even be 10-15%.Generally,both the second-generation and the third-generation sequencing have the advantages and shortages.Thus choosing the appropriate sequencing platform should be under the reasonable evaluation,so that to complete certain species genome de novo sequencing.As the combination of second-generation and third-generation sequencing platform was implement in this study,the problem of genome de novo assembly on new and rare invasive fungi was solved and great achievements was acquired after the further analysis of the genetic information.Penicillium capsulatum belongs to Eurotiales,Penicillium,which has been used in industrial papermaking.There hadn't been reports on its pathogenicity ever.Penicillium fungi play important role in human life,with barely pathogenicity.The case in this study is a rare IFI caused by Penicillium capsulatum.In 2013,Chinese scientist discovered the Penicillium capsulatum infected coenobium in the lung of a Type II diabetes patient.With the combination of second-generation Illumina Mi Seq platform and third-generation Pacbio RSII platform,the whole-genomic map was drawn,and the systematic evolution was analyzed.Besides,the classification of evolutionary relationships,which has more disputes now,has also been solved successfully.The achievements could provide new vision to the molecular evolution.As a result,the virulence factors can be grouped into seven main functional aspects: 1)thermo-tolerance,2)cell wall composition and maintenance,3)resistance to the immune response,4)toxins,5)nutrient uptake during invasive growth,6)signaling,metabolic regulation and response to stress conditions,and 7)allergens.All the groups of genes were analyzed carefully and the reference solution was proposed to research the pathogenicity of a new invasive fungus.Emmonsia is a kind of dimorphic fungi,which exists as hypha in environment,while exists as thin wall yeast balloon cell in mammalian tissus.The hosts of Emmonsia are mainly rodents.And it is rarely occurred that the Emmonsia invades human and leads to deep infection.But in recent two years,New England Journal of Medicine continuously reported that HIV patients in South Africa were deeply invaded by Emmonsia which has aroused great attention.In China,the first case was that Emmonsia invaded into lung which was discovered by Peking Union Medical College Hospital in 2005.The patient was assumed to be tuberculosis initially,but as the corresponding teatments were invalid,the pathogenic bacteria fetched from lung tissue were cultivated and identified as Emmonsia deep infected by following verification of morphology and multi-locus sequence.This new discovery was named as Emmonsia beijingensis nov.The whole-genome sequencing in this research is the first study on this new finding invasive Emmonsia.The combination of the second-generation Illumina MiSeq and the third-generation Pacbio RSII platforms is an effective measure.With the advantages union of deep sequencing in short sequence and crossing repeated region in long sequence,the result in high completeness and credibility was acquired,meanwhile the analysis on virulence factors of this new species was also processed based on the Pathogen-Host Interaction database(PHI).Besides,the comparison between Emmonsia beijingensis nov.and two stains of fungi reported in South Africa was taken,which focused on the differences of virulence factors.The result shows that Emmonsia beijingensis nov.has less virulence factors than those in South Africa.This is the first time to illustrate that why the Emmonsia invasive accidents were not booming in China like that in South Africa.Cryptococcal meningitis is a kind of serious central nervous system diseases,which could occur bad consequence and lead to high dead rates.Cryptococcus neoformans is the main pathogen.They normally infect the immunodeficiency people.But since 1999,the finding of human Cryptococcal booming in British Colombia,Vancouver Island of Canada,the surrounding area of Canada,and some area of United States showed that the main pathogen were not Cryptococcus neoformans but Cryptococcus gattii.Furthermore,Cryptococcus gattii could cause pneumonia and meningitis on people with normal immunity.Thereby,it is more and more concerned on Cryptococcus gattii all over the world.There is seldom research information about the pathogen mechanism of Cryptococcus gattii in China,much less reports on genome.The first report of Cryptococcus gattii in China was from the group of Liao Wanqing in 1980.The pathogen of Cryptococcus gattii used in this study is from Cryptococcal meningitis patient's cerebrospinal fluid isolated by the group of Liao Wan-qing.The work of deep sequencing and whole-genome assembling of this pathogen were implemented,as well as the virulence factors analysis based on PHI.Considering the characteristics of IFI,such as unconspicuous pathogen,atypical clinical manifestation,limited treatments,high mortality and disability rate,it is significant to improve prognosis by means of early distinctive diagnosis.The pathogen isolation of pathogenic fungi and phenotypic identification may provide key etiology evidence for clinical diagnose of IFI.Phenotypic identification is based on morphologic characters and biochemical reaction.But it will spent long period to cultivate fungi,moreover the restrict conditions for certain pathogens growing.There always find fungi contaminating from environments while it is hard to distinguish colonization or spread of the pathogen.And the false negative rate could be extremely high while cultivating the pathogens from patients which had been treated by anti-infective medicines.As to molecular diagnosis,it has efficient,accurate and promptly characters,and provides firm foundation for early diagnosis of invasive fungi.But former molecular diagnosis mainly depended on single or multi sequences PCR tests for identification,and it was normally identifying the fungi which could be cultivated successfully.This method is far more to satisfy the demand of clinical diagnosis.Due to the fact that fungi cultivating in clinical cases is always difficult,it could be a big challenge to diagnosis if it is difficult to identify the fungal morphology and clinical symptoms.And the patients could also be late for treatment in time.It is the first time that a new-generation sequencing technique is implemented in this research to direct sequencing diagnosis from samples to sequences for complicated case that the fungi are hard to cultivate.Furthermore,the method of single-cell sequencing was used to isolate pathogen and to identify.Finally,a reasonable and credible result was achieved and it could be efficient to guide clinical therapy.Besides,a new approach was provided to identify the fungal pathogen which is hard to be cultivated.In general,the technology of sequencing develops rapidly,and takes great prospect to medical science.The booming of invasive fungi cases implies that the more precise approach should be applied for recognizing,preventing,diagnosing and treating.Via the genome research and molecular diagnosis for new and rare invasive fungal pathogen,the utilization of combination of second-generation and third-generation sequencing in this study,could deepen the understanding of fungal pathogenic mechanism,and could provide new approach and prospective to promote the development of fungal infective diagnosis.