Conservation of extrusion as an exit mechanism for Chlamydia

Abstract

Chlamydiae exit via membrane-encased extrusion or through lysis of the host cell. Extrusions are novel, pathogen-containing structures that confer infectious advantages to Chlamydia, and are hypothesized to promote cell-to-cell spread, dissemination to distant tissues and facilitate immune evasion. The extrusion phenomenon has been characterized for several Chlamydia trachomatis serovars, but a thorough investigation of extrusion for additional clinically relevant C. trachomatis strains and Chlamydia species has yet to be performed. The key parameters investigated in this study were: (i) the conservation of extrusion across the Chlamydia genus, (ii) the functional requirement for candidate Chlamydia genes in extrusion formation i.e. IncA and CT228 and (iii) extrusion-mediated uptake, and consequent survival of Chlamydia inside macrophages. Inclusion morphology was characterized by live fluorescence microscopy, using an inverted GFP strategy, at early and mid-stages of infection. Enriched extrusions were used to infect bone marrow-derived macrophages, and bacterial viability was measured following macrophage engulfment. Our results demonstrate that extrusion is highly conserved across chlamydiae, including ocular, STD and LGV biovars and divergent Chlamydia species. Consequently, this exit mechanism for Chlamydia may fulfill common advantages important for pathogenesis.

Keywords: Chlamydia; dissemination; exit; extrusion; macrophage.

Figure 1.

Conservation of extrusion production, size and survival in macrophages across Chlamydiae. (A) Inclusion morphology of Chlamydia spp.; HeLa cell (GFP), inclusion is shown by absence of GFP, at 60× magnification. Top row: CT L2 (left), CT D^incA− (middle) and C. psittaci (right). Bottom row: single, isolated extrusions matching top row Chlamydia species shown in brightfield at 60× magnification. Scale bar = 5 μm. (B) Normalized extrusion production, calculated by counting number of extrusions relative to % of cells infected in original infection. Columns show mean ± SEM. n = 3 independent experiments. Statistics were performed by one-way ANOVA analysis using Tukey's multiple comparisons test, and no significance was observed between species or C. trachomatis strains. (C) Extrusion size graph displaying each extrusion as a single point on the graph. Extrusions counted by Volocity microscope software parameters to measure diameter (μm), n = 3 independent experiments. Statistics were performed using one-way ANOVA analysis using Tukey's multiple comparisons test. No significance was observed between species or C. trachomatis strain extrusion diameter or spread. (D) Extrusion uptake by bone marrow macrophages. CT L2 extrusion (GFP), host cell actin (purple) and nucleus (DAPI) at 60× magnification with z stacking. Scale bar = 5 μm. (E) Summary table displaying normalized extrusion production and survival within macrophages up to 6 h. +++ denotes >100 IFU/field, ++ denotes 50–100 IFU/field, + denotes < 50 IFU/field and – denotes no IFU seen. ND = no data.