title: 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 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.
  
  
  
  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.
authors.affiliation: Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, California, USA.
authors.forename: Xander M
authors.initials: XM
authors.lastname: van Wijk
authors.affiliation: Department of Pediatrics, University of California, San Diego, La Jolla, California, USA.
authors.forename: Simon
authors.initials: S
authors.lastname: Döhrmann
authors.affiliation: Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark.|Royal Institute of Technology, Stockholm, Sweden.
authors.forename: Björn M
authors.initials: BM
authors.lastname: Hallström
authors.affiliation: Department of Pediatrics, University of California, San Diego, La Jolla, California, USA.|Department of Bioengineering, University of California, San Diego, La Jolla, California, USA.|Novo Nordisk Foundation Center for Biosustainability, University of California, San Diego, La Jolla, California, USA.
authors.forename: Shangzhong
authors.initials: S
authors.lastname: Li
authors.affiliation: Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark.
authors.forename: Bjørn G
authors.initials: BG
authors.lastname: Voldborg
authors.affiliation: Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, California, USA.
authors.forename: Brandon X
authors.initials: BX
authors.lastname: Meng
authors.affiliation: Department of Pathology and Laboratory Medicine, Robert Wood Johnson Medical School, Piscataway, New Jersey, USA.
authors.forename: Karen K
authors.initials: KK
authors.lastname: McKee
authors.affiliation: Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud University Medical Centre, Nijmegen, The Netherlands.
authors.forename: Toin H
authors.initials: TH
authors.lastname: van Kuppevelt
authors.affiliation: Department of Pathology and Laboratory Medicine, Robert Wood Johnson Medical School, Piscataway, New Jersey, USA.
authors.forename: Peter D
authors.initials: PD
authors.lastname: Yurchenco
authors.affiliation: Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark.|Department of Bioengineering, University of California, San Diego, La Jolla, California, USA.|Novo Nordisk Foundation Center for Biosustainability, University of California, San Diego, La Jolla, California, USA.
authors.forename: Bernhard O
authors.initials: BO
authors.lastname: Palsson
authors.affiliation: Department of Pediatrics, University of California, San Diego, La Jolla, California, USA.|Novo Nordisk Foundation Center for Biosustainability, University of California, San Diego, La Jolla, California, USA.
authors.forename: Nathan E
authors.initials: NE
authors.lastname: Lewis
authors.affiliation: Department of Pediatrics, University of California, San Diego, La Jolla, California, USA.
authors.forename: Victor
authors.initials: V
authors.lastname: Nizet
authors.affiliation: Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, California, USA jesko@ucsd.edu.
authors.forename: Jeffrey D
authors.initials: JD
authors.lastname: Esko
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004705:Endocytosis; D017353:Gene Deletion; D055786:Gene Knockout Techniques; D005816:Genetic Complementation Test; D054884:Host-Pathogen Interactions; D007797:Laminin / Q000235:genetics / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D013211:Staphylococcus aureus / Q000502:physiology*; D013292:Streptococcus agalactiae / Q000502:physiology*; D013297:Streptococcus pyogenes / Q000502:physiology*
chemical_list: D007797:Laminin; C102645:laminin alpha 2