Microevolution during Chronic Infection May Lead T. asahii to Coexist with the Host

Background Trichosporon asahii (T. asahii) is part of the cutaneous fungal microbiota in humans and can cause lethal opportunistic infection. During infection, microorganisms can adapt to their environment by adjusting gene expression and cellular activities. Objectives Investigation of the microevolutionary changes in T. asahii during chronic infection. Methods Two T. asahii strains were isolated from a chronic trichosporonosis patient between a 15-year interval, and the microevolutionary changes were compared by the immune response of dendritic cell (DC), mice survival model, and transcriptome sequencing analysis. Results Compared with the primary T. asahii strain, the microevolved strain induced much lower expression of TNF-α by mice bone marrow-derived DC and had a much superior survival rate, a total of 2212 significantly differentially expressed genes were identified in the microevolved strain, and functional analysis showed significance in the downregulated transcription and metabolic process, especially the valine, leucine, and isoleucine degradation pathways, which were associated with pathogenicity and virulence; hence, the results were highly consistent with the decreased immunogenicity and virulence of the microevolved strain. Conclusions These results demonstrated that the microevolution during chronic infection could induce changes in immunogenicity, virulence, and transcriptome, which might lead T. asahii to coexist with the host.


Background
Trichosporon asahii (T.asahii) is part of the cutaneous fungal microbiota in humans and can cause lethal opportunistic infection in immunocompromised patients [1]; however, there are increasing cases of T. asahii infection in immunocompetent hosts [2][3][4].During infection, microorganisms can adapt to changes in their environment by adjusting gene expression and cellular activities [5], such microevolution can be illustrated by bioflm development in Candida albican through evolved transcriptional network controls [6] and hyphal formation of Candida albican to escape from phagocytosis [7].
As the most powerful antigen-presenting cell (APC), dendritic cell (DC) can motivate and regulate immune responses against fungi: phagocyte fungi and present fungal antigen, to mediate innate and adaptive immunity, at the same time release proinfammatory cytokines such as TNF-α [8,9].
We previously obtained two T. asahii strains from facial skin lesions of a chronic trichosporonosis patient in 2000 (called AS2.2174) and 2015 (called BM1403), the 26S rDNA D1/D2 region of AS2.2174 was sequenced and confrmed to be Trichosporon asahii strain, and BM1403 was 100% mapped onto AS2.2174 genome and morphologically and biochemically same with AS2.2174.Tus, AS2.2174 was the ancestor of BM1403 [10,11].Te patient displayed no evidence of underlying disease during infection, which reminded us of microevolution of T. asahii during chronic infection.Moreover, few works have focused on the interactions between DC or mice model and T. asahii undergoing microevolution.Hence, in this study, we compared the microevolutionary changes in T. asahii by the immune response of DC, mice survival model, and transcriptome sequencing analysis, and our results showed that T. asahii may perform microevolution to coexists with the host through immunogenicity, virulence, and transcriptome adaption.

Strains and Growth
Condition.Te two T. asahii strains, AS2.2174 and BM1403 samples, were both stored in 20% glycerol at −80 °C until use, and then, they were transferred into fresh yeast extract peptone dextrose (YPD) liquid medium in a shaker incubator and cultured at 150 rpm at 35 °C for 18 h.After that, 20 ml of each T. asahii strain was centrifuged at 3000 rpm at 4 °C for 10 min, then washed with normal saline, and resuspended in normal saline at 1 × 10 4 CFU/ml density.5 μl of each suspension was spotted onto potato dextrose agar (PDA) plates at 35 °C for 7 days.

Bone Marrow-Derived Dendritic Cell (BMDC) Culture.
Bone marrow cells were collected from the femur and tibia bones of C57BL/6 mice, erythrocyte lysate was added to remove red blood cells, and the supernatant was discarded after centrifugation (1500 r/min, 5 min).Cells were resuspended by RPMI 1640 medium with 10% fetal bovine serum and plated at a concentration of 2 × 10 6 cells well in 6-well culture plates, supplemented with GM-CSF (20 ng/ml, PeproTech, USA) and IL-4 (10 ng/ml, PeproTech, USA) in a cell incubator (37 °C, 5% CO 2 ).Half of the medium was changed, and cytokines were supplemented every other day.On day 8, suspended and semisuspended cells were harvested in new 6-well plates at the same concentration for coincubation with T. asahii strains.

Strains and BMDC Coincubation.
Te two strains of T. asahii cultured on YPD as described above were washed and resuspended with normal saline and took half for heat inactivate (65 °C, 3 h).Ten, the activated and inactivated T. asahii strains were coincubated with BMDC in each well (BMDC: T. asahii � 1: 5) in a cell incubator (37 °C, 5% CO 2 ) for 24 h.

Murine Survival Study.
C57BL/6 mice (male 6-8 week, 18 to 20 g) were obtained from the Tsinghua Laboratory Animal Resources Center (Beijing, China) and housed in standard cages with ad libitum access to water and food.Te two strains of T. asahii cultured in YPD as described above were washed and resuspended with normal saline at 1 × 10 7 CFU/ml density and were injected into the lateral tail vein at a dose of 0.1 mL per mouse, 9 received AS2.2174, and 10 received BM1403.All mice surviving to day 16 were humanely sacrifced.

RNA Extraction, Library Construction, and Sequencing.
Te two strains of T. asahii cultured on PDA as described above were used for RNA isolation.Te total RNA was extracted with TRIzol reagent (Invitrogen, USA), the purity was analysed by NanoDrop 2000 (Termo Fisher Scientifc, USA), and the integrity was evaluated by Agilent 2100 (Agilent Technologies, USA).Ten, mRNA was enriched by magnetic beads with Oligo (dT, Dynabeads, Norway) from the qualifed total RNA.cDNA libraries were constructed by mRNA templates and purifed by AMPure XP beads (Beckman, USA).

Transcriptome Statistics.
Te N-containing, low quality, adapter-related reads were removed after high-throughput sequencing, leaving the clean reads.Ten, the transcriptome reads of the two strains were mapped with the T. asahii genome (CBS 2479 strain) using the TopHat2 software.Gene abundance levels were evaluated by fragments per kilobase of exon model (FPKM) per million mapped reads.

Expression Level Analysis.
HTSeq software was used for expression level analysis, the threshold of FPKM was >1, and the gene expressions between two strains were compared by the log 10 (FPKM + 1) value.DEGseq software was used for diferentially expressed genes.

Statistical Analysis.
Statistical analyses were carried out using the GraphPad Prism software, and the results were expressed as means and standard deviations (x ± s).Murine survival study was assessed by log-rank chi square test.Multiple groups were compared by one-way analysis of variance (ANOVA) followed by the Student-Newman-Keuls (SNK-q) test.Statistical signifcance 2 Dermatology Research and Practice was set at P < 0.05, and GO analysis statistical signifcance "corrected P value" was set at P < 0.05.

Strains and BMDC Coincubation.
After 24 h of coincubation, most of the living T. asahii strains were adhered by BMDC because of flamentation response and heatinactivated T. asahii strains were mostly phagocytosed by BMDC (Figure 1).We selected TNF-α as the representative infammatory response of BMDC to T. asahii; real-time PCR showed living BM1403 induced much lower expression of TNF-α by BMDC than living AS2.2174, and there were no diferences when they were heat inactivated (Figure 2).

Murine Survival Study.
Experimental mice were inoculated intravenously with T. asahii strains for 16 consecutive days.Log-rank chi square test showed that mice infected with BM1403 performed signifcant higher survival rate than AS2.2174 (P < 0.05): the median survival time of mice infected with AS2.2174 was 9 days while all the mice infected with BM1403 survived till the end of the trial (Figure 3).

Expression Level Analysis.
We used HTSeq and DEGseq software to analyse the transcriptome profles of T. asahii strains AS2.2174 and BM1403.A total of 8149 shared DEGs between the two strains were identifed (Figure 4(a), Supplementary Table S1).Among these DEGs, 2212 were statistically signifcant according to the screening threshold previously described as |log 2 (FoldChange)| > 1 and q value<0.005, of which 1014 were downregulated and 1198 were upregulated in BM1403 compared to AS2.2174 (Figure 4(b), Supplementary Table S2, Supplementary Table S3).Heatmap and clustering analysis revealed signifcant changes in the mRNA expression profles of the two strains (Figure 4(c)).

GO Enrichment Analysis.
Te GO enrichment analysis of DEGs was categorized as biological process (BP), molecular function (MF), and cellular component (CC).In the downregulated DEGs of BM1403 compared to AS2.2174, terms of cellular molecule metabolic process and nucleic acid metabolism were signifcantly enriched in BP (P < 0.05), such as regulation of cellular metabolic process (GO: 0031323), regulation of RNA metabolic process (GO: 0051252), and regulation of gene expression (GO: 0010468), and nucleic acid-binding transcription factor activity (GO: 0001071) and sequence-specifc DNA-binding transcription factor activity (GO: 0003700) were signifcantly enriched in MF (P < 0.05, Table 1, Supplementary Table S4).In the upregulated DEGs, terms of DNA repair were enriched in BP, such as response to DNA damage stimulus (GO: 0006974), and terms of host cell were enriched in CC, such as host intracellular part (GO: 0033646), but were not significant (P > 0.05, Table 2, Supplementary Table S5).

Discussion
To survive inside the host during chronic infection, fungus must be able to adapt to adverse environment and microevolution could be the driving force of immune response and virulence change, such as the experiment of continuous coincubation of Candida glabrata with a murine macrophage cell line for over six months [12], which resulted in alteration of morphology with increased immunogenicity (TNF-α) and increased virulence (mouse infection model).
Our results could also see signifcant changes in immunogenicity and virulence, but on the contrary, after a 15-yearperiod infection, the microevolved T. asahii strain BM1403 induced much lower expression of TNF-α by BMDC and had a much superior survival rate than the AS2.2174 strain, tending to "peacefully" coexist with the host.Tis diference might be because of the two strains of our study were isolated from a systemic disseminated trichosporonosis patient, which ofered a much more complicated environment that thrived most vitro studies.Ormerod et al. [13] analysed two serial isolates obtained from a immunodefciency patient sufered an initial and relapse episode of cryptococcal meningoencephalitis; consistent with our results, the microevolved isolate (F2) also exhibited reduced virulence such as capsule size and melanization at 37 °C than the initial strains (F0) and was amplifed by mice survival model, and whole-genome sequencing uncovered transcriptional regulatory gene mutants.We performed transcriptome sequencing analysis of the two serial isolates and found adaptions by DEGs of T. asahii in systemic infected patient.Our data identifed 8149 shared DEGs, and 2212 DEGs showed statistical signifcance in BM1403 compared to AS2.2174.Among these, the GO terms signifcantly enriched in downregulated transcription and metabolic process, and the KEGG pathway signifcantly enriched in downregulated valine, leucine, and isoleucine degradation.Valine, leucine, and isoleucine, also known as branched-chain amino acids (BCAAs), are synthesized by fungi for important components of proteins and secondary metabolites, especially ribosomal protein [14]; hence, the KEGG pathway was highly consistent with the GO enrichment.By reviewing the literature [15][16][17][18], we found that BCAA auxotrophs in Dermatology Research and Practice    Highly coincidence with these results, our team previously performed proteome analysis of the two T. asahii strains and demonstrated that the most common homologs in the more abundant DEPs (diferentially expressed proteins) were in reduced virulence category, followed by unafected pathogenicity and loss of pathogenicity [19].Furthermore, the enzyme in BCAA biosynthesis pathway is a promising new target for antifungal drug discovery, as humans do not synthesize BCAAs [15,20].
Tere are several limitations in our study.Firstly, genes involved in branched-chain amino acids (BCAAs) metabolism for T. asahii remained unclear in our study and previous work.Secondly, pathway-specifc transcriptional regulation of BCAAs biosynthesis needs to be confrmed in our future work.Tirdly, we also need to provide much more specifc evidences for the relationship between BCAAs auxotrophs and decreased pathogenicity and virulence in T. asahii.
In summary, our study demonstrated that microevolution induced by chronic infection could induce changes in immunogenicity, virulence, and transcriptome, which might lead T. asahii to coexist with the host.Future studies could amplify our deduction, and a better understanding of the BCAA pathway could provide new ways to deal with chronic T. asahii systemic infection.