MADS-box transcriptional regulation of dimorphic transition in Penicillium marneffei

系统性二相性真菌每年总共造成100多万新感染。潜伏感染 在全球范围内增长到数千万，使它们成为当今研究的优先事项。马尔尼菲青霉菌(PM) 由于青霉病病例在过去20年中显著增加， 与此同时，由于艾滋病毒感染的全球传播，免疫抑制现象也在上升。下午 在环境中以菌丝的形式生长，在哺乳动物的宿主中以酵母的形式生长。它们之间的相变 这两种生长形式被认为是其致病性和青霉病传播所必需的。 菌丝和酵母菌之间的转变是可逆的，可以在 37°C下，PM的热敏和相变的精确机制尚不清楚。 因此，迫切需要确定与形态发生有关的关键基因和调控过程 PM中的控制申请者最近的发现为识别目标提供了一个很有希望的新机会。 使用一种创新的、基于连续培养的实验进化(EE)技术，申请者获得了突变 PM菌株在30°C而不是37°C经历菌丝到酵母的相变。DNA-和RNA- 对突变株和野生型菌株的测序揭示了MADS-box转录家族的重要作用 转录因子基因，特别是MADS B和MADS A在调节PM热二型性中的作用的表达方式 野生型的MadsB在菌丝到酵母的转变过程中上调了1500倍。MADSB功能丧失 突变株的基因组缺失引起的突变似乎是导致阈值较低的原因 相变温度。值得注意的是，EE衍生的突变体被发现在 小鼠模型，表明相变的准确阈值温度在37℃是必不可少的 以便PM感染和/或适应宿主条件。此外，MadsA的过度表达导致PM 在37℃下生长为菌丝而不是酵母。这些初步数据导致了一个中心假设，即PM 热敏系统通过MADS盒热敏元件的温度响应活动起作用。基于这些 根据初步结果，申请者提出了以下两个具体目标：(1)确定MADS B的作用 以及(2)确定MADS B和MADS A的下游靶标。 MADSA。在完成建议的研究后，申请人预期会严格测试 两个MADS-box转录因子在调节PM二态发育中的作用(目标1)。申请者将确认 MADS B和/或MADS A的下游目标，并审查选定的目标在项目管理中的作用(目标2)。 总而言之，这些结果将提供急需的切入点，以进一步调查原本神秘的 这一重要但未被充分研究的真菌病原体的热二型形成机制。

Systemic dimorphic fungi collectively cause over one million new infections every year. Latent infections worldwide grow to the tens of millions, making them a priority for today's research. Penicillium marneffei (Pm) is of particular concern due to a marked increase in the number of cases of penicilliosis in the last 20 years, which has been concurrent with the rise in immunosuppression due to the global spread of HIV infections. Pm grows as hypha in its environmental reservoir and as yeast in mammalian hosts. The phase transition between the two growth forms is considered to be essential for its pathogenicity and the transmission of penicilliosis. The transition between hypha and yeast is reversible and can be triggered at a sharp threshold temperature of 37°C. The precise mechanisms underlying the thermosensing and the phase transition in Pm remain unknown. Therefore, there is a critical need to identify key genes and regulatory processes involved in the morphogenetic control in Pm. The applicants' recent findings provide a promising, new opportunity for target identification. Using an innovative, serial culture-based experimental evolution (EE) technique, the applicants derived mutant Pm strains that undergo the hypha-to-yeast phase transition at 30°C rather than 37°C. DNA- and RNA- sequencing of the mutant and wild-type strains revealed important roles of a family of MADS-box transcription factor (TF) genes, especially madsB and madsA, in regulating thermal dimorphism in Pm. The expression of madsB in wild type is up-regulated 1,500-fold during the hypha-to-yeast transition. The madsB loss-of-function mutation caused by genomic deletion in mutant strains seems to be responsible for the lower threshold temperature of phase transition. Significantly, EE-derived mutants are found to be completely avirulent in the mouse model, suggesting that the precise threshold temperature at 37°C for the phase transition is essential for Pm to infect and/or adapt to the host condition. Furthermore, the overexpression of madsA causes Pm to grow as hypha instead of yeast at 37°C. These preliminary data led to the central hypothesis that the Pm thermosensing systems act through temperature-responsive activities of the MADS-box TFs. Based on these preliminary results, the applicants propose the following two Specific Aims: (1) determine the roles of madsB and madsA in regulating thermal dimorphism in Pm, and (2) identify the downstream targets of MadsB and MadsA. Upon completion of the proposed research, the applicants expect to critically test the functions of the two MADS-box TFs in regulating dimorphic development in Pm (Aim 1). The applicants will identify the downstream targets of MadsB and/or MadsA, and examine the functions of selected targets in Pm (Aim 2). Together, these results will provide much-needed entry points to further investigate the otherwise mysterious mechanisms underlying thermal dimorphism in this important but understudied fungal pathogen.