Primary Research On The Function Of Genes Tcr1,IDO1 And IDO2 In Talaromyces Marneffei

Talaromyces marneffei,formerly known as Penicillium marneffei,is one of the temperature-dependent dimorphic fungus and also is an opportunistic pathogen,which mainly infects immunocompromised population.The pathogen endemic was mainly popular in Southeast Asia and south region of China and it can causes a fatal disseminated disease in patients,which called Penicilliosis marneffei and it has a poor prognosis.T.marneffei has severely impairment to human health,but its two-phase transition and pathogenesis remains unclear.Kynurenine pathway of Tryptophan metabolism plays an important role in the pathogen infection and the pathogen scavenging.Indoleamine 2,3-dioxygenase(IDO)is rate-limiting enzyme in kynurenine pathway.When IDO has a higher expression in host cells,it can inhibit the reproduction and the spread of pathogen in the host and reduce inflammation.In contrast,it can help the pathogen escape from innate immune cells and increase inflammation while IDO has a higher expression in pathogenic fungi.The high expression of IDO in various tumor tissues also can help the pathogen evade the elimination of host immune system.Therefore,we hypothesized that the kynurenine metabolic pathway of Tryptophan metabolism in T.marneffei may play a vital role in its pathogenic mechanism.Recently,mostly researchers have paid more attention to the dimorphic switching mechanism of T.marneffei,which is generally considered to be important to pathogenicity.Our research group has performed high-throughput ssRNA-seq to get the transcriptional profiles of 4 different growth stages of T.marneffei and found that the genes involved in the kynurenine metabolic pathway of Tryptophan metabolism were identified as up-regulated during the hyphal-to-yeast transition or in pathogenic yeast phase,such as Tcr1,IDO1 and IDO2 genes.The genes mentioned above are distinctly expressed during the two-phase transition.So we speculated that they may play an important role in dimorphic switching of T.marneffei.The primary research of the functions of these three genes can lay the foundation for the further study of the role of the tryptophan metabolism pathway in the pathogenic mechanism of T.marneffei,and also can provide clues for studying the dimorphic switching and pathogenic mechanisms of T.marneffei.Objective: To explore the function of Tcr1,IDO1 and IDO2 genes required for tryptophan metabolism pathway and highly expressed in pathogenic yeast may provide clues for the further research of the tryptophan metabolism pathway and the dimorphic switching mechanism of T.marneffei.Methods: Using the homologous recombination to construct the gene deletion mutant,and analyze the function of genes in various aspects after compared with the Spm4 strain,such as the changes of phenotypes,the changes of tryptophan catabolic ability,the changes in drug,oxidative stress,osmotic stress and cell wall stress sensitivity,and the changes in expression levels of genes specifically expressed in mycelial phase and yeast phase.Results:(1)Tcr1,IDO1 and IDO2 gene deletion mutants were successfully constructed;(2)At 25°C,the Tcr1,IDO1 gene deletion mutants could not grown on tryptophan as the sole carbon or nitrogen source plate,but IDO2 gene deletion could.(3)The Tcr1 deletion mutant slightly increases the sensitivity to osmotic stress,amphotericin B and micafungin,and is particularly vulnerable to cell wall stress,while compared to Spm4;(4)The IDO1 gene deletion mutants show decreased sensitivity to Amphotericin B.(5)The IDO2 gene deletion mutants show decreased sensitivity to Amphotericin B and micafungin.(6)After the deletion of IDO1 and Tcr1,the expression level of the yeast-specific highly expressed genes were significantly lower than that of Spm4.Conclusions:(1)At 25°C,the IDO1 gene plays a major role in the tryptophan catabolism of T.marneffei.IDO1 gene may related to the structure of the amphotericin B binding site on the cell membrane.(2)At 25°C,the Tcr1 transcription factor gene is involved in the regulation of tryptophan catabolism.,and may also affect the composition and integrity of the cell wall.(3)The IDO2 gene isn't a key gene in the tryptophan catabolism of T.marneffei,but may be related to the sterol binding site of the amphotericin B membrane and the glucan binding site on the cell wall of micafungin.(4)The Tcr1 and IDO1 gene may involved in the formation and maintenance of yeast phase.