Whole-Genome Sequencing of Invasion-Resistant Cells Identifies Laminin α2 as a Host Factor for Bacterial Invasion

ABSTRACT 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 targeti...

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Autores principales:	Xander M. van Wijk, Simon Döhrmann, Björn M. Hallström, Shangzhong Li, Bjørn G. Voldborg, Brandon X. Meng, Karen K. McKee, Toin H. van Kuppevelt, Peter D. Yurchenco, Bernhard O. Palsson, Nathan E. Lewis, Victor Nizet, Jeffrey D. Esko
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2017
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/b977856af4b74bdaa8a1ed1c0e80562c
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spelling	oai:doaj.org-article:b977856af4b74bdaa8a1ed1c0e80562c2021-11-15T15:51:07ZWhole-Genome Sequencing of Invasion-Resistant Cells Identifies Laminin α2 as a Host Factor for Bacterial Invasion10.1128/mBio.02128-162150-7511https://doaj.org/article/b977856af4b74bdaa8a1ed1c0e80562c2017-03-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.02128-16https://doaj.org/toc/2150-7511ABSTRACT 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. IMPORTANCE Pathogenic bacteria penetrate host cellular barriers by attachment to extracellular matrix molecules, such as proteoglycans, laminins, and collagens, leading to invasion of epithelial and endothelial cells. Here, we show that cellular invasion by the human pathogens group B Streptococcus, group A Streptococcus, and Staphylococcus aureus depends on a specific domain of the laminin α2 subunit. This finding may provide new leads for the molecular pathogenesis of these bacteria and the development of novel antimicrobial drugs.Xander M. van WijkSimon DöhrmannBjörn M. HallströmShangzhong LiBjørn G. VoldborgBrandon X. MengKaren K. McKeeToin H. van KuppeveltPeter D. YurchencoBernhard O. PalssonNathan E. LewisVictor NizetJeffrey D. EskoAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 8, Iss 1 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Xander M. van Wijk Simon Döhrmann Björn M. Hallström Shangzhong Li Bjørn G. Voldborg Brandon X. Meng Karen K. McKee Toin H. van Kuppevelt Peter D. Yurchenco Bernhard O. Palsson Nathan E. Lewis Victor Nizet Jeffrey D. Esko Whole-Genome Sequencing of Invasion-Resistant Cells Identifies Laminin α2 as a Host Factor for Bacterial Invasion
description	ABSTRACT 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. IMPORTANCE Pathogenic bacteria penetrate host cellular barriers by attachment to extracellular matrix molecules, such as proteoglycans, laminins, and collagens, leading to invasion of epithelial and endothelial cells. Here, we show that cellular invasion by the human pathogens group B Streptococcus, group A Streptococcus, and Staphylococcus aureus depends on a specific domain of the laminin α2 subunit. This finding may provide new leads for the molecular pathogenesis of these bacteria and the development of novel antimicrobial drugs.
format	article
author	Xander M. van Wijk Simon Döhrmann Björn M. Hallström Shangzhong Li Bjørn G. Voldborg Brandon X. Meng Karen K. McKee Toin H. van Kuppevelt Peter D. Yurchenco Bernhard O. Palsson Nathan E. Lewis Victor Nizet Jeffrey D. Esko
author_facet	Xander M. van Wijk Simon Döhrmann Björn M. Hallström Shangzhong Li Bjørn G. Voldborg Brandon X. Meng Karen K. McKee Toin H. van Kuppevelt Peter D. Yurchenco Bernhard O. Palsson Nathan E. Lewis Victor Nizet Jeffrey D. Esko
author_sort	Xander M. van Wijk
title	Whole-Genome Sequencing of Invasion-Resistant Cells Identifies Laminin α2 as a Host Factor for Bacterial Invasion
title_short	Whole-Genome Sequencing of Invasion-Resistant Cells Identifies Laminin α2 as a Host Factor for Bacterial Invasion
title_full	Whole-Genome Sequencing of Invasion-Resistant Cells Identifies Laminin α2 as a Host Factor for Bacterial Invasion
title_fullStr	Whole-Genome Sequencing of Invasion-Resistant Cells Identifies Laminin α2 as a Host Factor for Bacterial Invasion
title_full_unstemmed	Whole-Genome Sequencing of Invasion-Resistant Cells Identifies Laminin α2 as a Host Factor for Bacterial Invasion
title_sort	whole-genome sequencing of invasion-resistant cells identifies laminin α2 as a host factor for bacterial invasion
publisher	American Society for Microbiology
publishDate	2017
url	https://doaj.org/article/b977856af4b74bdaa8a1ed1c0e80562c
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Whole-Genome Sequencing of Invasion-Resistant Cells Identifies Laminin α2 as a Host Factor for Bacterial Invasion

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