Isolation of Chlamydia trachomatis and membrane vesicles derived from host and bacteria

Abstract

The study of intracellular bacteria and nanometer-size membrane vesicles within infected host cells poses an important challenge as it is difficult to identify each distinct population in the context of the complex populations generated from active host-pathogen interactions. Here, suspension cultures of L929 cells infected with the prevalent obligate intracellular bacterium Chlamydia trachomatis strain F/Cal-IC-13 are utilized for the large scale preparation and isolation of natural membrane vesicles and bacterial forms. Cell lysis with nitrogen cavitation in combination with differential centrifugation, OptiPrep™ density gradient separation, and immunoenrichment using anti-chlamydial lipopolysaccharide antibodies and MagnaBind beads allows for the isolation of both productive and persistent bacterial forms, as well as membrane vesicles derived from the host and pathogen. We have evaluated these populations by electron microscopy and Western blot analysis for identification of biomarkers. In addition, purified persistent forms of C. trachomatis induced by ampicillin display adenosine-5'-triphosphate (ATP) transport activity, suggesting that ampicillin-induced persistent C. trachomatis organisms, at least in part, rely upon host ATP as an energy source. Importantly, several chlamydial cytotoxic and/or secreted proteins are demonstrated to be associated with these vesicles, supporting the idea that membrane vesicles are generated by Chlamydia as a means of carrying and delivering virulence factors necessary for pathogenesis. The ability to produce large-scale infections and generate distinct bacteria and host-derived populations for biochemical analysis, while reducing the burdens of time and cost have implications in all areas of chlamydiology. These protocols can be applied to other strains of C. trachomatis or other intracellular bacteria.

Figure 1

Schematic of procedure for suspension cell infection with C. trachomatis serovar F/Cal-I-13, purification of chlamydial forms and vesicle isolation. Multiple concentrations of OptiPrep™ density gradient medium used in this study are as indicated. Abbreviations: EB, elementary body; RB, reticulate body; PF, persistent form; SPG, sucrose-phosphate-glutamate buffer; PBS, phosphate buffered saline.

Figure 2

Dose and volume optimization of suspension culture infection with C. trachomatis serovar F/Cal-I-13. (a) Suspension cultures were incubated with increasing amounts of bacteria. (b) Bacteria and host cells were incubated in varying volumes of infection buffer (SPG) to determine optimal infection conditions. Endpoint infection assays determining the IFU of progeny on monolayers of L929 cells resulting from each infection were used as an indication of quality of infection for both experiments. Each result is indicated as the mean value of at least three independent experiments ± the standard deviation (SD). Significant difference was analyzed by Student’s t-test. A p-value <0.05 was used as the cut-off for statistical significance.

Figure 3

The persistent form purification. Representative transmission electron micrographs of infected L929 cells (a) exposed or (b) unexposed to ampicillin and (c) the purified persistent form fraction containing aberrant RBs with a less electron dense appearance. Based on multiple cross-sections purified persistent form size is estimated at approximately 0.7–2.0 µm. Scale bars in (a) and (b) represent 2 µm and 0.5 µm in (c). (d) Endpoint infection assays determining IFUs on monolayers of L929 cells in each isolated density gradient fraction. Values represent mean IFU ± SD of three independent chlamydial preparations. (e) Representative images of the cell adherence assays demonstrating the inability of the persistent form to produce infection and inhibit cover slip adherence. Abbreviations: PF (+), persistent fraction purified from an infection exposed to ampicillin; PF (−) persistent fraction purified from an infection with no ampicillin exposure; RB (+), reticulate body fraction purified from an infection exposed to ampicillin; RB (−), reticulate body fraction purified from an infection with no ampicillin exposure; EB (+), elementary body fraction purified from an infection exposed to ampicillin; EB (−), elementary body fraction purified from an infection with no ampicillin exposure.

Figure 4

Characteristics of the persistent form fraction. (a) Western blot analysis of isolated chlamydial forms with monoclonal antibodies specific to chlamydial MOMP, OmcA, CPAF and RpoB. Sample loading normalized based on total protein. All samples prepared in Laemmli sample buffer (1:1, v:v) supplemented with 5% 2-Mercaptoethanol, 10 mM dithiothreitol and heated for 10 min at 100 °C. (b) ATP transport assays conducted with isolated persistent forms. ATP indicates control transport assays containing 20-times the determined K_m for C. trachomatis nucleoside phosphate transporter 1. All data normalized by adjusting maximum transport to 100%. Experiments were performed in triplicate on three independent chlamydial preparations. Abbreviations: EB, elementary body; RB, reticulate body; PF, persistent form; ATP, adenosine-5'-triphosphate.

Figure 5

Vesicle purification. (a–e) Representative transmission electron micrographs of various vesicle purifications from infected cultures that are (a) non-ampicillin-exposed, non-filtered; (b) ampicillin-exposed, non-filtered; (c) ampicillin-exposed, filtered; (d) mock infected; or (e) pellet collected from the vesicle isolation supernatant after 16 hr of centrifugation. Experiments performed with the initial fraction (15,300 ×g vesicle supernatant) confirmed that vesicles remain intact after 16 hr of centrifugation. (f) An endpoint assay determining the IFU present in each fraction was conducted to determine possible C. trachomatis elementary body contamination. Values represent mean IFU ± SD from three independent vesicle preparations. Scale bars represent 0.5 µm. Abbreviations: (+) Ap, vesicles isolated from an infection exposed to ampicillin; (−) Ap, vesicles isolated from an infection not exposed to ampicillin.

Figure 6

Analysis of vesicle populations by Western blot. (a) Relative enrichment of chlamydial LPS-containing vesicles results in a decrease of host derived vesicles. (b) Purity of the vesicle fraction and the association of putative virulence factors with the chlamydial vesicle population. Vesicle samples prepared in Laemmli sample buffer (1:1, v:v) supplemented with 5% 2-Mercaptoethanol, 10 mM dithiothreitol and heated for 10 min at 100 °C. Abbreviation: EB, elementary body.