Genome-scale CRISPR/Cas9 screening reveals the role of PSMD4 in colibactin-mediated cell cycle arrest.

IF 3.7 2区 生物学 Q2 MICROBIOLOGY
mSphere Pub Date : 2025-03-25 Epub Date: 2025-02-07 DOI:10.1128/msphere.00692-24
Michael W Dougherty, Ryan M Hoffmann, Maria C Hernandez, Yougant Airan, Raad Z Gharaibeh, Seth B Herzon, Ye Yang, Christian Jobin
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引用次数: 0

Abstract

Colibactin is a genotoxic secondary metabolite produced by certain Enterobacteriaceae strains that populate the intestine and produces a specific mutational signature in human colonocytes. However, the host pathways involved in colibactin response remain unclear. To address this gap, we performed genome-wide CRISPR/Cas9 knockout screens and RNA sequencing utilizing live pks+ bacteria and a synthetic colibactin analog. We identified 20 enriched genes with a MAGeCK score of >2.0 in both screens, including proteasomal subunits (e.g., PSMG4 and PSMD4), RNA processing factors (e.g., SF1 and PRPF8), and RNA polymerase III (e.g., CRCP), and validated the role of PSMD4 in colibactin sensitization. PSMD4 knockout in HEK293T and HT-29 cells promoted cell viability and ameliorated G2-M cell cycle arrest but did not affect the amount of phosphorylated H2AX foci after exposure to synthetic colibactin 742. Consistent with these observations, PSMD4-/- cells had a significantly higher colony formation rate and bigger colony size than control cells after 742 exposure. These findings suggest that PSMD4 regulates cell cycle arrest following colibactin-induced DNA damage and that cells with PSMD4 deficiency may continue to replicate despite DNA damage, potentially increasing the risk of malignant transformation.

Importance: Colibactin has been implicated as a causative agent of colorectal cancer. However, colibactin-producing bacteria are also present in many healthy individuals, leading to the hypothesis that some aspects of colibactin regulation or host response dictate the molecule's carcinogenic potential. Elucidating the host-response pathways involved in dictating cell fate after colibactin intoxication has been difficult, partially due to an inability to isolate the molecule. This study provides the first high-throughput CRISPR/Cas9 screening to identify genes conferring colibactin sensitivity. Here, we utilize both bacterial infection and a synthetic colibactin analog to identify genes directly involved in colibactin response. These findings provide insight into how differences in gene expression may render certain individuals more vulnerable to colibactin-initiated tumor formation after DNA damage.

基因组级CRISPR/Cas9筛选揭示了PSMD4在大肠杆菌素介导的细胞周期阻滞中的作用。
大肠杆菌素是由某些肠杆菌科菌株产生的遗传毒性次级代谢物,这些菌株在肠道中繁殖并在人类结肠细胞中产生特定的突变特征。然而,参与大肠杆菌素反应的宿主途径尚不清楚。为了解决这一空白,我们利用活的pks+细菌和合成的大肠杆菌蛋白类似物进行了全基因组CRISPR/Cas9敲除筛选和RNA测序。我们在两个筛选中鉴定了20个MAGeCK评分为b> 2.0的富集基因,包括蛋白酶体亚基(如PSMG4和PSMD4), RNA加工因子(如SF1和PRPF8)和RNA聚合酶III(如CRCP),并验证了PSMD4在大肠杆菌素致敏中的作用。在HEK293T和HT-29细胞中敲除PSMD4可促进细胞活力,改善G2-M细胞周期阻滞,但不影响暴露于合成大肠杆菌蛋白742后磷酸化的H2AX灶的数量。与这些观察结果一致,在742暴露后,PSMD4-/-细胞的集落形成率和集落大小明显高于对照细胞。这些发现表明,PSMD4调节大肠杆菌素诱导的DNA损伤后的细胞周期阻滞,PSMD4缺陷的细胞可能在DNA损伤后继续复制,潜在地增加了恶性转化的风险。重要性:大肠杆菌蛋白已被认为是结直肠癌的一种病原体。然而,产生大肠杆菌素的细菌也存在于许多健康个体中,这导致了大肠杆菌素调节或宿主反应的某些方面决定了该分子的致癌潜力的假设。阐明大肠杆菌蛋白中毒后决定细胞命运的宿主反应途径一直很困难,部分原因是无法分离该分子。这项研究提供了第一个高通量CRISPR/Cas9筛选来鉴定赋予大肠杆菌素敏感性的基因。在这里,我们利用细菌感染和合成的大肠杆菌蛋白类似物来鉴定直接参与大肠杆菌蛋白反应的基因。这些发现为基因表达差异如何使某些个体在DNA损伤后更容易受到大肠杆菌素引发的肿瘤形成提供了见解。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
mSphere
mSphere Immunology and Microbiology-Microbiology
CiteScore
8.50
自引率
2.10%
发文量
192
审稿时长
11 weeks
期刊介绍: mSphere™ is a multi-disciplinary open-access journal that will focus on rapid publication of fundamental contributions to our understanding of microbiology. Its scope will reflect the immense range of fields within the microbial sciences, creating new opportunities for researchers to share findings that are transforming our understanding of human health and disease, ecosystems, neuroscience, agriculture, energy production, climate change, evolution, biogeochemical cycling, and food and drug production. Submissions will be encouraged of all high-quality work that makes fundamental contributions to our understanding of microbiology. mSphere™ will provide streamlined decisions, while carrying on ASM''s tradition for rigorous peer review.
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