A Genome-Wide Screen in Macrophages Defines Host Genes Regulating the Uptake of Mycobacterium abscessus.
A Genome-Wide Screen in Macrophages Defines Host Genes Regulating the Uptake of Mycobacterium abscessus.
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宿主细胞和病原体之间的相互作用可以决定疾病的结果，并且是宿主定向治疗的重要靶点。结核分枝杆菌（Mycobacterium dumessus，Mab）是一种高度耐药、生长迅速的非结核分枝杆菌，可感染慢性肺部疾病患者。单克隆抗体可以感染宿主免疫细胞，如巨噬细胞，这有助于其发病机制。然而，我们对最初的宿主-单克隆抗体相互作用的理解仍然不清楚。在这里，我们开发了一种功能遗传学方法来定义这些主机单克隆抗体的相互作用，通过耦合单克隆抗体的荧光报告与全基因组敲除库在小鼠巨噬细胞。我们使用这种方法进行正向遗传筛选，以确定有助于巨噬细胞摄取Mab的宿主基因。我们鉴定了已知的吞噬作用调节因子，如整合素ITGB 2，并揭示了巨噬细胞有效摄取Mab的糖胺聚糖（sGAG）合成的关键要求。CRISPR-Cas9靶向三种关键sGAG生物合成调节剂Ugdh、B3 gat 3和B4 galt 7导致巨噬细胞对光滑和粗糙Mab变体的摄取减少。机制研究表明，sGAG的功能上游的病原体吞噬和所需的单克隆抗体的摄取，而不是大肠杆菌或乳胶珠。进一步的研究发现，sGAG的损失降低了关键整联蛋白的表面表达，但不降低mRNA表达，这表明sGAG在调节表面受体可用性中的重要作用。总之，这些研究在全球范围内定义和表征了巨噬细胞-单克隆抗体相互作用的重要调节因子，是了解导致单克隆抗体发病机制和疾病的宿主基因的第一步。重要性病原体与免疫细胞如巨噬细胞的相互作用有助于发病机制，但这些相互作用的机制在很大程度上仍不明确。对于新出现的呼吸道病原体，如结核分枝杆菌，了解这些宿主-病原体相互作用对于充分了解疾病进展至关重要。鉴于M。抗生素治疗广泛应用，需要新的治疗方法。在这里，我们利用小鼠巨噬细胞中的全基因组敲除文库来全面定义M。吸收。我们在M.包括整合素亚类和糖胺聚糖合成（sGAG）途径。虽然已知sGAG的离子特性驱动病原体-细胞相互作用，但我们发现了以前未认识到的sGAG维持关键摄取受体的稳健表面表达的要求。因此，我们开发了一个灵活的正向遗传管道来定义M期间的重要相互作用。Escherichia coli感染，并更广泛地确定了sGAG控制病原体摄取的新机制。
The interactions between a host cell and a pathogen can dictate disease outcomes and are important targets for host-directed therapies. Mycobacterium abscessus (Mab) is a highly antibiotic resistant, rapidly growing nontuberculous mycobacterium that infects patients with chronic lung diseases. Mab can infect host immune cells, such as macrophages, which contribute to its pathogenesis. However, our understanding of initial host-Mab interactions remains unclear. Here, we developed a functional genetic approach to define these host-Mab interactions by coupling a Mab fluorescent reporter with a genome-wide knockout library in murine macrophages. We used this approach to conduct a forward genetic screen to define host genes that contribute to the uptake of Mab by macrophages. We identified known regulators of phagocytosis, such as the integrin ITGB2, and uncovered a key requirement for glycosaminoglycan (sGAG) synthesis for macrophages to efficiently take up Mab. CRISPR-Cas9 targeting of three key sGAG biosynthesis regulators, Ugdh, B3gat3, and B4galt7 resulted in reduced uptake of both smooth and rough Mab variants by macrophages. Mechanistic studies suggest that sGAGs function upstream of pathogen engulfment and are required for the uptake of Mab, but not Escherichia coli or latex beads. Further investigation found that the loss of sGAGs reduced the surface expression, but not the mRNA expression, of key integrins, suggesting an important role for sGAGs in modulating surface receptor availability. Together, these studies globally define and characterize important regulators of macrophage-Mab interactions and are a first step to understanding host genes that contribute to Mab pathogenesis and disease. IMPORTANCE Pathogen interactions with immune cells like macrophages contribute to pathogenesis, yet the mechanisms underlying these interactions remain largely undefined. For emerging respiratory pathogens, like Mycobacterium abscessus, understanding these host-pathogen interactions is important to fully understand disease progression. Given that M. abscessus is broadly recalcitrant to antibiotic treatments, new therapeutic approaches are needed. Here, we leveraged a genome-wide knockout library in murine macrophages to globally define host genes required for M. abscessus uptake. We identified new macrophage uptake regulators during M. abscessus infection, including a subset of integrins and the glycosaminoglycan synthesis (sGAG) pathway. While ionic characteristics of sGAGs are known to drive pathogen-cell interactions, we discovered a previously unrecognized requirement for sGAGs to maintain robust surface expression of key uptake receptors. Thus, we developed a flexible forward-genetic pipeline to define important interactions during M. abscessus infection and more broadly identified a new mechanism by which sGAGs control pathogen uptake.
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影响因子：
7.7
作者：
Akusobi, Chidiebere;Benghomari, Bouchra S.;Zhu, Junhao;Wolf, Ian D.;Singhvi, Shreya;Dulberger, Charles L.;Ioerger, Thomas R.;Rubin, Eric J.;Kana, Bavesh D.
通讯作者：
Kana, Bavesh D.
影响因子：
4.5
作者：
Lee HJ;Woo Y;Hahn TW;Jung YM;Jung YJ
通讯作者：
Jung YJ
影响因子：
30.8
作者：
Haney MS;Bohlen CJ;Morgens DW;Ousey JA;Barkal AA;Tsui CK;Ego BK;Levin R;Kamber RA;Collins H;Tucker A;Li A;Vorselen D;Labitigan L;Crane E;Boyle E;Jiang L;Chan J;Rincón E;Greenleaf WJ;Li B;Snyder MP;Weissman IL;Theriot JA;Collins SR;Barres BA;Bassik MC
通讯作者：
Bassik MC
DOI：
10.3390/antibiotics9100711
发表时间：
2020-10-17
期刊：
Antibiotics (Basel, Switzerland)
影响因子：
--
作者：
Brzostek J;Fatin A;Chua WH;Tan HY;Dick T;Gascoigne NRJ
通讯作者：
Gascoigne NRJ
影响因子：
3.1
作者：
Bucior, Iwona;Mostov, Keith;Engel, Joanne N.
通讯作者：
Engel, Joanne N.