Identification of small molecule inhibitors of Pseudomonas aeruginosa exoenzyme S using a yeast phenotypic screen.
Identification of small molecule inhibitors of Pseudomonas aeruginosa exoenzyme S using a yeast phenotypic screen.
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DOI：
10.1371/journal.pgen.1000005
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发表时间：
2008-02-29
期刊：
影响因子：
4.5
通讯作者：
Stagljar I
中科院分区：
文献类型：
--
作者：
Arnoldo A;Curak J;Kittanakom S;Chevelev I;Lee VT;Sahebol-Amri M;Koscik B;Ljuma L;Roy PJ;Bedalov A;Giaever G;Nislow C;Merrill AR;Lory S;Stagljar I
铜绿假单胞菌是一种机会性的人类病原体，是囊性纤维化患者死亡的关键因素，感染在全球范围内对人类健康构成越来越大的威胁。由于铜绿假单胞菌对抗生素的耐药性正在增加，因此需要针对这种细菌的药理学有效靶点的新的抑制剂。在这里，我们证明了一种基于细胞的酵母表型试验，结合大规模的抑制剂筛选，确定了可以抑制选定的铜绿假单胞菌ORF异源表达所引起的毒性的小分子抑制剂。我们发现了第一个外酶S(ExOS)的小分子抑制物，它是一种与III型分泌有关的毒素。我们发现，这种抑制剂，外切蛋白，在体外调节EXOS ADP-核糖基转移酶的活性，表明这种抑制是直接的。此外，Exosin及其两个类似物在体内对假单胞菌感染具有显著的保护作用。此外，由于检测是在酵母中进行的，我们能够证明已知的人类EXOS靶标的几个酵母同源物很可能被毒素ADP-核糖化。例如，使用体外酶试验，我们证明了酵母Ras2p直接被EXOS修饰。最后，通过对酵母缺失突变体的研究，我们发现Bmh1p是一种与人Fas同源的酵母辅因子，揭示了细菌毒素的部分作用模式在酵母与人类之间是保守的。综上所述，我们集成的基于细胞的化学遗传学方法表明，这种筛选可以加强传统的药物筛选方法，并促进发现针对广泛的人类病原体的新化合物。微生物对抗生素的耐药性是对人类健康的一个严重且日益严重的威胁。在这里，我们使用了一种新的方法，将化学和遗传扰动结合在一起，以寻找新的靶点，这些靶点可能有效地控制由人类条件致病菌铜绿假单胞菌引起的感染。铜绿假单胞菌是囊性纤维化患者死亡的主要原因，并已显示出惊人的抗药性。在这项研究中，我们确定了EXOS的第一个小分子抑制剂，EXOS是一种在铜绿假单胞菌感染过程中发挥关键作用的毒素。其中一种化合物Exosin可能是通过调节毒素的酶活性来发挥作用的。我们进一步表明，这种抑制物保护哺乳动物细胞免受铜绿假单胞菌的感染。最后，我们使用酵母功能基因组学工具确定了几个已知的人类EXOS靶标的酵母同源物作为毒素的可能靶标。总之，这些观察结果验证了我们以酵母为基础的方法来发现新的抗生素。这些化合物可以作为新的治疗方法的起点，类似的策略也可以应用于广泛的人类病原体，如病毒或寄生虫。
Pseudomonas aeruginosa is an opportunistic human pathogen that is a key factor in the mortality of cystic fibrosis patients, and infection represents an increased threat for human health worldwide. Because resistance of Pseudomonas aeruginosa to antibiotics is increasing, new inhibitors of pharmacologically validated targets of this bacterium are needed. Here we demonstrate that a cell-based yeast phenotypic assay, combined with a large-scale inhibitor screen, identified small molecule inhibitors that can suppress the toxicity caused by heterologous expression of selected Pseudomonas aeruginosa ORFs. We identified the first small molecule inhibitor of Exoenzyme S (ExoS), a toxin involved in Type III secretion. We show that this inhibitor, exosin, modulates ExoS ADP-ribosyltransferase activity in vitro, suggesting the inhibition is direct. Moreover, exosin and two of its analogues display a significant protective effect against Pseudomonas infection in vivo. Furthermore, because the assay was performed in yeast, we were able to demonstrate that several yeast homologues of the known human ExoS targets are likely ADP-ribosylated by the toxin. For example, using an in vitro enzymatic assay, we demonstrate that yeast Ras2p is directly modified by ExoS. Lastly, by surveying a collection of yeast deletion mutants, we identified Bmh1p, a yeast homologue of the human FAS, as an ExoS cofactor, revealing that portions of the bacterial toxin mode of action are conserved from yeast to human. Taken together, our integrated cell-based, chemical-genetic approach demonstrates that such screens can augment traditional drug screening approaches and facilitate the discovery of new compounds against a broad range of human pathogens. Microbial resistance to antibiotics is a serious and growing threat to human health. Here, we used a novel approach that combines chemical and genetic perturbation of bakers yeast to find new targets that might be effective in controlling infections caused by the opportunistic human pathogen Pseudomonas aeruginosa. P. aeruginosa is the primary cause of mortality with cystic fibrosis patients and has demonstrated an alarming ability to resist antibiotics. In this study, we identified the first small molecule inhibitors of ExoS, a toxin playing a pivotal role during P. aeruginosa infections. One of these compounds, exosin, likely works by modulating the toxin's enzymatic activity. We further show that this inhibitor protects mammalian cells against P. aeruginosa infection. Finally, we used yeast functional genomics tools to identify several yeast homologues of the known human ExoS targets as possible targets for the toxin. Together, these observations validate our yeast-based approach for uncovering novel antibiotics. These compounds can be used as starting point for new therapeutic treatments, and a similar strategy could be applied to a broad range of human pathogens like viruses or parasites.
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DOI：
10.1073/pnas.90.6.2320
发表时间：
1993-03-15
影响因子：
11.1
作者：
FU, H;COBURN, J;COLLIER, RJ
通讯作者：
COLLIER, RJ
影响因子：
56.9
作者：
Hung, DT;Shakhnovich, EA;Mekalanos, JJ
通讯作者：
Mekalanos, JJ
影响因子：
3.2
作者：
Finck-Barbançon, V;Frank, DW
通讯作者：
Frank, DW
DOI：
10.1073/pnas.2036282100
发表时间：
2003-11-25
影响因子：
11.1
作者：
Jacobs, MA;Alwood, A;Manoil, C
通讯作者：
Manoil, C
DOI：
10.1053/srin.2002.34689
发表时间：
2002-09-01
期刊：
Seminars in respiratory infections
影响因子：
--
作者：
Cunha, Burke A
通讯作者：
Cunha, Burke A