Co-cultivation induces elicitation of unique Fungus-derived Natural products that leads to thiol stress mediated killing of Mycobacterium tuberculosis [F51]. Co-cultivation induces elicitation of unique Fungus-derived Natural products that leads to thiol stress mediated killing of Mycobacterium tuberculosis [F51]

Tuberculosis, caused by Mycobacterium tuberculosis has remained a leading cause of death worldwide even after decades of it being declared as global health emergency by the WHO. Newer drugs with novel modes of action are urgently needed to combat the threats imposed by the constantly emerging drug resistant strains. Natural products (NPs) derived anti-mycobacterials appear lucrative because of their complex structural features and unique cellular targets they bind to. Herein, we employed co-cultivation approach to identify cryptic biosynthetic gene clusters (BGCs) from fungal genomes eliciting the expression of genes that are silent or poorly transcribed in axenic cultures. Fungi were isolated from sphagnum peat bog samples collected from different regions of North-eastern USA because aspects of this ecological niche reflect the critical microenvironment of the human tuberculosis granuloma and are a natural habitat for slow growing mycobacterial species that compete for limited nutrients with other microbes. Bioactivity-guided assay led us to identify three unique fungal isolates that selectively produce growth inhibitory metabolites during co-cultivation with Mtb. Fungal mRNA sequencing from co-cultured isolates facilitated the identification of elicited Type I Polyketide Synthase BGCs that were silent/cryptic in monoculture conditions. Bioinformatic analyses followed by chemical validation identified these molecules to be patulin, citrinin and nidulalin A. Interestingly, these induced fungal metabolites led to a highly responsive redox-stress homeostasis within Mtb. Our study thus, illustrates a co-cultivation mediated elicitation of unique fungal NPs resulting in a thiol-burst oxidative stress mediated killing of Mtb. We believe that the identification of vulnerable drug targets may yield insights into further understanding of this essential thiol stress mediated killing of the mycobacteria Overall design: In an effort to identify the intrinsic ability of microorganisms to produce prolific, highly specific and active small molecules against Mtb, we focused on utilizing the co-cultivation approach to identify cryptic BGCs from fungal genomes eliciting the expression of genes that are silent or poorly transcribed in axenic cultures. Fungi were isolated from sphagnum peat bog samples collected from different regions of North-eastern USA and were screened using co-cultivation based approach for their specific anti-Mtb activity induced upon co-cultivation with Mtb as assessed by performing growth inhibition of reprter strain of Mtb using cell free media from mono and co-cultures. Unique fungi obtained in our study F2, F50, F51, C7 and F31 were taken forward for RNA seq to identify the differentially expressed gene clusters responsible for producing anti-Mtb metabolites in cocultivation conditions. Fungal spores were grown in PDB media for 24h following which they were either treated with phosphate buffer or Mtb. The mono- and co-cultures were incubated for different day points at 32'C. Mycelia was then harvested, ground by mortar pestle in Liquid N2 and the ground mycelia was used for Total RNA extraction. mRNA was then extracted from Total RNA following rRNA depletion method

结核分枝杆菌（Mycobacterium tuberculosis）引发的结核病，即便在世界卫生组织（World Health Organization, WHO）将其列为全球卫生紧急事件数十年后，仍是全球范围内的主要致死病因之一。当前亟需开发具有全新作用机制的新型抗结核药物，以应对不断涌现的耐药菌株所带来的公共卫生威胁。
天然产物（Natural Products, NPs）来源的抗分枝杆菌候选药物，因其复杂的结构特征以及独特的细胞结合靶点而展现出良好的开发前景。
本研究采用共培养策略，旨在从真菌基因组中挖掘在纯培养（axenic culture）条件下沉默或转录水平极低的隐蔽生物合成基因簇（biosynthetic gene clusters, BGCs）。
研究人员从美国东北部不同区域采集的泥炭藓泥炭沼样本中分离得到真菌菌株。该生态环境的诸多特征与人类结核病肉芽肿的关键微环境高度相似，同时也是慢生长分枝杆菌物种的天然栖息地——这类分枝杆菌可与其他微生物竞争有限的营养资源。
通过生物活性导向的筛选实验，我们获得了3株独特的真菌分离株，它们在与结核分枝杆菌（Mtb）共培养时可选择性合成生长抑制性代谢产物。
对共培养条件下的真菌进行mRNA测序，成功鉴定出在单培养条件下处于沉默或隐蔽状态的I型聚酮合酶（Type I Polyketide Synthase）生物合成基因簇。
经生物信息学分析与化学验证，这些诱导产生的真菌代谢产物被鉴定为棒曲霉素（patulin）、桔霉素（citrinin）与尼杜拉林A（nidulalin A）。值得注意的是，这些诱导产生的代谢产物可在结核分枝杆菌内引发强烈的氧化还原应激稳态反应。
综上，本研究证实了通过共培养策略可诱导真菌产生独特的天然产物，进而通过硫醇爆发型氧化应激介导的途径杀伤结核分枝杆菌。我们认为，本次鉴定出的易感药物靶点，可为进一步阐明这种依赖硫醇应激的分枝杆菌杀伤机制提供关键的研究思路。

### 整体实验设计
为挖掘微生物合成针对结核分枝杆菌的高效、高特异性活性小分子的内在能力，本研究聚焦于利用共培养策略，从真菌基因组中筛选在纯培养条件下沉默或转录水平低下的隐蔽生物合成基因簇。
研究人员从美国东北部不同区域采集的泥炭藓泥炭沼样本中分离真菌，并采用基于共培养的筛选方法：通过分别利用单培养与共培养体系的无细胞培养基，检测结核分枝杆菌报告菌株的生长抑制情况，以此筛选出经共培养诱导可产生特异性抗结核分枝杆菌活性的真菌菌株。
本研究中获得的F2、F50、F51、C7及F31共5株独特真菌菌株被用于RNA测序，以鉴定在共培养条件下负责产生抗结核分枝杆菌代谢产物的差异表达基因簇。
真菌孢子在马铃薯葡萄糖肉汤（Potato Dextrose Broth, PDB）培养基中培养24小时后，分别用磷酸盐缓冲液或结核分枝杆菌进行处理。单培养与共培养体系于32℃下分别培养不同时长。随后收集菌丝体，在液氮中用研钵和研杵进行研磨，以提取总RNA。最终通过核糖体RNA depletion法从总RNA中分离得到mRNA。