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首页> 外文期刊>MBio >Whole-Genome Sequencing of Invasion-Resistant Cells Identifies Laminin α2 as a Host Factor for Bacterial Invasion
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Whole-Genome Sequencing of Invasion-Resistant Cells Identifies Laminin α2 as a Host Factor for Bacterial Invasion

机译：抗侵袭细胞的全基因组测序确定层粘连蛋白α2为细菌侵袭的宿主因子。

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To understand the role of glycosaminoglycans in bacterial cellular invasion, xylosyltransferase-deficient mutants of Chinese hamster ovary (CHO) cells were created using clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated gene 9 (CRISPR-cas9) gene targeting. When these mutants were compared to the pgsA745 cell line, a CHO xylosyltransferase mutant generated previously using chemical mutagenesis, an unexpected result was obtained. Bacterial invasion of pgsA745 cells by group B Streptococcus (GBS), group A Streptococcus, and Staphylococcus aureus was markedly reduced compared to the invasion of wild-type cells, but newly generated CRISPR-cas9 mutants were only resistant to GBS. Invasion of pgsA745 cells was not restored by transfection with xylosyltransferase, suggesting that an additional mutation conferring panresistance to multiple bacteria was present in pgsA745 cells. Whole-genome sequencing and transcriptome sequencing (RNA-Seq) uncovered a deletion in the gene encoding the laminin subunit α2 (Lama2) that eliminated much of domain L4a. Silencing of the long Lama2 isoform in wild-type cells strongly reduced bacterial invasion, whereas transfection with human LAMA2 cDNA significantly enhanced invasion in pgsA745 cells. The addition of exogenous laminin-α2β1γ1/laminin-α2β2γ1 strongly increased bacterial invasion in CHO cells, as well as in human alveolar basal epithelial and human brain microvascular endothelial cells. Thus, the L4a domain in laminin α2 is important for cellular invasion by a number of bacterial pathogens.
机译：为了解糖胺聚糖在细菌细胞侵袭中的作用，使用簇状规则间隔的短回文重复序列（CRISPR）和CRISPR相关基因9（CRISPR-cas9）基因定位创建了中国仓鼠卵巢（CHO）细胞的木糖基转移酶缺陷型突变体。将这些突变体与pgsA745细胞系（以前使用化学诱变产生的CHO木糖基转移酶突变体）进行比较时，获得了意外的结果。与野生型细胞相比，Bs链球菌（GBS），A组链球菌和金黄色葡萄球菌对pgsA745细胞的细菌入侵明显减少，但新产生的CRISPR-cas9突变体仅对GBS具有抗性。通过用木糖基转移酶转染不能恢复对pgsA745细胞的侵袭，这表明在pgsA745细胞中还存在一个赋予多种细菌全抗性的突变。全基因组测序和转录组测序（RNA-Seq）发现了编码层粘连蛋白亚基α2（Lama2）的基因中的缺失，从而消除了大部分结构域L4a。在野生型细胞中沉默长的Lama2同工型可大大减少细菌入侵，而用人LAMA2 cDNA转染可显着增强pgsA745细胞的入侵。外源层粘连蛋白-α2β1γ1/层粘连蛋白-α2β2γ1的添加大大增加了CHO细胞，人肺泡基底上皮细胞和人脑微血管内皮细胞中的细菌入侵。因此，层粘连蛋白α2中的L4a结构域对于多种细菌病原体的细胞侵袭是重要的。

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