title: Raman microspectroscopy reveals long-term extracellular activity of Chlamydiae.
abstract: The phylum Chlamydiae consists exclusively of obligate intracellular bacteria. Some of them are formidable pathogens of humans, while others occur as symbionts of amoebae. These genetically intractable bacteria possess a developmental cycle consisting of replicative reticulate bodies and infectious elementary bodies, which are believed to be physiologically inactive. Confocal Raman microspectroscopy was applied to differentiate between reticulate bodies and elementary bodies of Protochlamydia amoebophila and to demonstrate in situ the labelling of this amoeba symbiont after addition of isotope-labelled phenylalanine. Unexpectedly, uptake of this amino acid was also observed for both developmental stages for up to 3 weeks, if incubated extracellularly with labelled phenylalanine, and P. amoebophila remained infective during this period. Furthermore, P. amoebophila energizes its membrane and performs protein synthesis outside of its host. Importantly, amino acid uptake and protein synthesis after extended extracellular incubation could also be demonstrated for the human pathogen Chlamydia trachomatis, which synthesizes stress-related proteins under these conditions as shown by 2-D gel electrophoresis and MALDI-TOF/TOF mass spectrometry. These findings change our perception of chlamydial biology and reveal that host-free analyses possess a previously not recognized potential for direct experimental access to these elusive microorganisms.
authors.affiliation: Department of Microbial Ecology, University of Vienna, Althanstrasse 14, 1090 Vienna, Austria.
authors.forename: Susanne
authors.initials: S
authors.lastname: Haider
authors.forename: Michael
authors.initials: M
authors.lastname: Wagner
authors.forename: Markus C
authors.initials: MC
authors.lastname: Schmid
authors.forename: Barbara S
authors.initials: BS
authors.lastname: Sixt
authors.forename: Jan G
authors.initials: JG
authors.lastname: Christian
authors.forename: Georg
authors.initials: G
authors.lastname: Häcker
authors.forename: Peter
authors.initials: P
authors.lastname: Pichler
authors.forename: Karl
authors.initials: K
authors.lastname: Mechtler
authors.forename: Albert
authors.initials: A
authors.lastname: Müller
authors.forename: Christian
authors.initials: C
authors.lastname: Baranyi
authors.forename: Elena R
authors.initials: ER
authors.lastname: Toenshoff
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Montanaro
authors.forename: Matthias
authors.initials: M
authors.lastname: Horn
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D002460:Cell Line; D002689:Chlamydia / Q000737:chemistry / Q000166:cytology* / Q000254:growth & development* / Q000378:metabolism; D002690:Chlamydia Infections / Q000175:diagnosis / Q000382:microbiology*; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D010649:Phenylalanine / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013059:Spectrum Analysis, Raman / Q000379:methods*
chemical_list: D001426:Bacterial Proteins; D010649:Phenylalanine