title: Isolation of single Chlamydia-infected cells using laser microdissection.
abstract: Chlamydia are obligate intracellular parasites of humans and animals that cause a wide range of acute and chronic infections. To elucidate the genetic basis of chlamydial parasitism, several approaches for making genetic modifications to Chlamydia have recently been reported. However, the lack of the available methods for the fast and effective selection of genetically modified bacteria restricts the application of genetic tools. We suggest the use of laser microdissection to isolate of single live Chlamydia-infected cells for the re-cultivation and whole-genome sequencing of single inclusion-derived Chlamydia. To visualise individual infected cells, we made use of the vital labelling of inclusions with the fluorescent Golgi-specific dye BODIPY® FL C5-ceramide. We demonstrated that single Chlamydia-infected cells isolated by laser microdissection and placed onto a host cell monolayer resulted in new cycles of infection. We also demonstrated the successful use of whole-genome sequencing to study the genomic variability of Chlamydia derived from a single inclusion. Our work provides the first evidence of the successful use of laser microdissection for the isolation of single live Chlamydia-infected cells, thus demonstrating that this method can help overcome the barriers to the fast and effective selection of Chlamydia.
authors.affiliation: Scientific Research Institute of Physico-Chemical Medicine, Malaya Pirogovskaya Str. 1a, Moscow 119435, Russia; Koltzov Institute of Developmental Biology of the Russian Academy of Sciences, Vavilov Str. 26, Moscow 119334, Russia. Electronic address: olegpodgorny@inbox.ru.
authors.forename: Oleg V
authors.initials: OV
authors.lastname: Podgorny
authors.affiliation: Scientific Research Institute of Physico-Chemical Medicine, Malaya Pirogovskaya Str. 1a, Moscow 119435, Russia.
authors.forename: Nadezhda F
authors.initials: NF
authors.lastname: Polina
authors.affiliation: Scientific Research Institute of Physico-Chemical Medicine, Malaya Pirogovskaya Str. 1a, Moscow 119435, Russia.
authors.forename: Vladislav V
authors.initials: VV
authors.lastname: Babenko
authors.affiliation: Scientific Research Institute of Physico-Chemical Medicine, Malaya Pirogovskaya Str. 1a, Moscow 119435, Russia.
authors.forename: Irina Y
authors.initials: IY
authors.lastname: Karpova
authors.affiliation: Scientific Research Institute of Physico-Chemical Medicine, Malaya Pirogovskaya Str. 1a, Moscow 119435, Russia.
authors.forename: Elena S
authors.initials: ES
authors.lastname: Kostryukova
authors.affiliation: Scientific Research Institute of Physico-Chemical Medicine, Malaya Pirogovskaya Str. 1a, Moscow 119435, Russia; Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, Miklukho-Maklaya Str., 16/10, Moscow 117997, Russia; Moscow Institute of Physics and Technology (State University), Institutskiy per. 9, Dolgoprudny, Moscow Region 141700, Russia.
authors.forename: Vadim M
authors.initials: VM
authors.lastname: Govorun
authors.affiliation: Scientific Research Institute of Physico-Chemical Medicine, Malaya Pirogovskaya Str. 1a, Moscow 119435, Russia; Moscow Institute of Physics and Technology (State University), Institutskiy per. 9, Dolgoprudny, Moscow Region 141700, Russia.
authors.forename: Vassili N
authors.initials: VN
authors.lastname: Lazarev
mesh_terms: D001431:Bacteriological Techniques / Q000379:methods*; D002689:Chlamydia / Q000254:growth & development*; D003584:Cytological Techniques / Q000379:methods*; D005456:Fluorescent Dyes / Q000378:metabolism; D014644:Genetic Variation; D016680:Genome, Bacterial; D006801:Humans; D007834:Lasers; D042282:Microdissection / Q000379:methods*; D059010:Single-Cell Analysis / Q000379:methods*; D013194:Staining and Labeling
chemical_list: D005456:Fluorescent Dyes
keywords: Chlamydia; Laser microdissection; Whole genome sequencing