title: Quantification of bacterial internalization by host cells using a beta-lactamase reporter strain: Neisseria gonorrhoeae invasion into cervical epithelial cells requires bacterial viability.
abstract: Neisseria gonorrhoeae can invade into cervical epithelial cells to overcome this host defense barrier. We developed a beta-lactamase reporter system that allowed us to quantify at the single cell level if a host cell internalized a viable or nonviable microorganism. We autodisplayed beta-lactamase on the surface of FA1090 [FA1090Phi(bla-iga')] and demonstrated by confocal fluorescence microscopy and flow cytometry that FA1090Phi(bla-iga') cleaved the beta-lactamase substrate CCF2-AM loaded into host cells only when gonococci were internalized by these host cells. While FA1090Phi(bla-iga') adhered to almost all ME180 cells, viable N. gonorrhoeae were internalized by only a subset of cells during infection. Nonviable gonococci adhered to, but were not internalized by ME180 cells, and failed to recruit F-actin to sites of adherent bacteria. Overall, we show that epithelial cell invasion is a dynamic process that requires viable N. gonorrhoeae. We demonstrate the advantages of the beta-lactamase reporter system over the gentamicin protection assay in quantifying bacterial invasion. The reporter system that we have developed can be adapted to studying the internalization of any bacterial species into any host cell.
authors.affiliation: Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742, USA.
authors.forename: Samuel E
authors.initials: SE
authors.lastname: Bish
authors.forename: Wenxia
authors.initials: W
authors.lastname: Song
authors.forename: Daniel C
authors.initials: DC
authors.lastname: Stein
mesh_terms: D000199:Actins / Q000378:metabolism; D001422:Bacterial Adhesion; D045744:Cell Line, Tumor; D002584:Cervix Uteri / Q000166:cytology / Q000382:microbiology*; D003001:Cloning, Molecular; D004847:Epithelial Cells / Q000378:metabolism / Q000382:microbiology*; D005260:Female; D005434:Flow Cytometry; D005452:Fluoresceins / Q000378:metabolism; D054884:Host-Pathogen Interactions; D006801:Humans; D007769:Lactams / Q000378:metabolism; D050576:Membrane Fusion Proteins / Q000378:metabolism; D050296:Microbial Viability; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009344:Neisseria gonorrhoeae / Q000472:pathogenicity* / Q000502:physiology; D001618:beta-Lactamases / Q000378:metabolism*
chemical_list: D000199:Actins; C109764:CCF 2; D005452:Fluoresceins; D007769:Lactams; D050576:Membrane Fusion Proteins; D001618:beta-Lactamases