Mechanisms of host cell exit by the intracellular bacterium Chlamydia

Abstract

The mechanisms that mediate the release of intracellular bacteria from cells are poorly understood, particularly for those that live within a cellular vacuole. The release pathway of the obligate intracellular bacterium Chlamydia from cells is unknown. Using a GFP-based approach to visualize chlamydial inclusions within cells by live fluorescence videomicroscopy, we identified that Chlamydia release occurred by two mutually exclusive pathways. The first, lysis, consisted of an ordered sequence of membrane permeabilizations: inclusion, nucleus and plasma membrane rupture. Treatment with protease inhibitors abolished inclusion lysis. Intracellular calcium signaling was shown to be important for plasma membrane breakdown. The second release pathway was a packaged release mechanism, called extrusion. This slow process resulted in a pinching of the inclusion, protrusion out of the cell within a cell membrane compartment, and ultimately detachment from the cell. Treatment of Chlamydia-infected cells with specific pharmacological inhibitors of cellular factors demonstrated that extrusion required actin polymerization, neuronal Wiskott-Aldrich syndrome protein, myosin II and Rho GTPase. The participation of Rho was unique in that it functioned late in extrusion. The dual nature of release characterized for Chlamydia has not been observed as a strategy for intracellular bacteria.

Fig. 1.

Detailed visualization of the Chlamydia developmental cycle in GFP-HeLa cells. Panels are representative of GFP-HeLa cells infected with C. trachomatis L2, and imaged live by confocal microscopy at times indicated. Arrows denote inclusions. (Scale bar: 10 μm.)

Fig. 2.

Lysis mechanism of Chlamydia release. GFP-HeLa cells infected with C. trachomatis L2 (A), C. trachomatis D (B), and C. caviae GPIC (C) for 72 h were imaged by live time-lapse videomicroscopy. Representative still frames illustrating the sequence of lysis are shown. Arrows denote inclusions. Arrowheads denote nuclei. (Scale bars: 10 μm.) (D) Quantification of lysis by the release of cytosolic GFP into culture supernatants. Fluorometric values are expressed relative to the maximal levels obtained at 72 h. Error bars denote the standard error of the mean (n = 9).

Fig. 3.

Lysis is mediated by proteases and intracellular calcium. (A) Chlamydia-infected GFP-HeLa cells treated with protease inhibitors. (B) Quantification of lysis by release of cytosolic GFP in untreated cells (DMSO, white bars) or treated with protease inhibitors (gray bars). Fluorometric values are expressed relative the maximal levels obtained at 72 h. PFO+PI's is expressed relative to untreated (PFO+DMSO) cells. (C) Chlamydia-infected cell in mid-lysis during Ca²⁺-free conditions. (D) Duration of lysis under normal (white bars) or Ca²⁺-free (gray bars) conditions. (Scale bars: 10 μm.) Error bars denote the standard error of the mean (n = 9).

Fig. 4.

Extrusion mechanism of Chlamydia release. (A) Time-lapse videomicroscopy images of extrusion formation. (B) Extrusion in cells expressing cytosol-red fluorescent protein and membrane-GFP. Confocal immunofluorescence of extrusions in red fluorescent protein-HeLa cells, using antibodies to MOMP (C) and IncA (D). (E) Scanning EM of a field of cells with extrusions. (F) Scanning EM of a single extruded cell. (G) Scanning EM of a detached extrusion. Arrows denote extrusions. Minor image enhancements were performed in A, C, and D to improve visualization of extrusions. Enhancements consisted of adjusting brightness and contrast slightly to ensure that the thin cell outlines could be reliably seen by the reader. (Scale bars: 10 μm.)

Fig. 5.

Extrusion requires actin polymerization by N-WASP, myosin II, and Rho GTPase. Images of Chlamydia-infected GFP-HeLa cells untreated (A) or treated with Latrunculin B (B), Wiskostatin (C), Nocodazole (D), Jasplakinolide at 72 h (E), and Jasplakinolide at 40 h (F), Blebbistatin (G), and C3 transferase (H). Arrows in A and D–F denote extrusions. Arrows in H denote site of pinching. Minor image enhancements were performed in D–F to facilitate visualization of extrusions. Enhancements consisted of adjusting brightness and contrast slightly to ensure that the thin cell outlines could be reliably seen by the reader. (Scale bar: 10 μm.)

Fig. 6.

Dual mechanisms of cellular exit by Chlamydia. (A) Lysis. (B) Extrusion.