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...
| Main Authors: | , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
American Society for Microbiology
2017-03-01
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| Series: | mBio |
| Online Access: | https://journals.asm.org/doi/10.1128/mBio.02128-16 |
| _version_ | 1818402091951980544 |
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| 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 |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-12-14T08:02:51Z |
| format | Article |
| id | doaj.art-b977856af4b74bdaa8a1ed1c0e80562c |
| institution | Directory Open Access Journal |
| issn | 2150-7511 |
| language | English |
| last_indexed | 2024-12-14T08:02:51Z |
| publishDate | 2017-03-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | mBio |
| spelling | doaj.art-b977856af4b74bdaa8a1ed1c0e80562c2022-12-21T23:10:19ZengAmerican Society for MicrobiologymBio2150-75112017-03-018110.1128/mBio.02128-16Whole-Genome Sequencing of Invasion-Resistant Cells Identifies Laminin α2 as a Host Factor for Bacterial InvasionXander M. van Wijk0Simon Döhrmann1Björn M. Hallström2Shangzhong Li3Bjørn G. Voldborg4Brandon X. Meng5Karen K. McKee6Toin H. van Kuppevelt7Peter D. Yurchenco8Bernhard O. Palsson9Nathan E. Lewis10Victor Nizet11Jeffrey D. Esko12Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, California, USADepartment of Pediatrics, University of California, San Diego, La Jolla, California, USANovo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, DenmarkDepartment of Pediatrics, University of California, San Diego, La Jolla, California, USANovo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, DenmarkDepartment of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, California, USADepartment of Pathology and Laboratory Medicine, Robert Wood Johnson Medical School, Piscataway, New Jersey, USADepartment of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud University Medical Centre, Nijmegen, The NetherlandsDepartment of Pathology and Laboratory Medicine, Robert Wood Johnson Medical School, Piscataway, New Jersey, USANovo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, DenmarkDepartment of Pediatrics, University of California, San Diego, La Jolla, California, USADepartment of Pediatrics, University of California, San Diego, La Jolla, California, USADepartment of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, California, USAABSTRACT 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.https://journals.asm.org/doi/10.1128/mBio.02128-16 |
| spellingShingle | 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 mBio |
| title | 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_short | 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 |
| url | https://journals.asm.org/doi/10.1128/mBio.02128-16 |
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