Using Fluorescent Proteins to Visualize and Quantitate <em>Chlamydia </em>Vacuole Growth Dynamics in Living Cells

Abstract

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C. trachomatis is the leading bacterial cause of sexually transmitted infection and also the primary cause of preventable blindness in the world. An essential determinant for successful infection of host cells by Chlamydia is the bacterium's ability to manipulate host cell signaling from within a novel, vacuolar compartment called the inclusion. From within the inclusion, Chlamydia acquire nutrients required for their 2-3 day developmental growth, and they additionally secrete a panel of effector proteins onto the cytosolic face of the vacuole membrane and into the host cytosol. Gaps in our understanding of Chlamydia biology, however, present significant challenges for visualizing and analyzing this intracellular compartment. Recently, a reverse-imaging strategy for visualizing the inclusion using GFP expressing host cells was described. This approach rationally exploits the intrinsic impermeability of the inclusion membrane to large molecules such as GFP. In this work, we describe how GFP-or mCherry-expressing host cells are generated for subsequent visualization of chlamydial inclusions. Furthermore, this method is shown to effectively substitute for costly antibody-based enumeration methods, can be used in tandem with other fluorescent labels, such as GFP-expressing Chlamydia, and can be exploited to derive key quantitative data about inclusion membrane growth from a range of Chlamydia species and strains. Video LinkThe video component of this article can be found at

The Chlamydia trachomatis Extrusion Exit Mechanism Is Regulated by Host Abscission Proteins

“…To determine whether the depletion of abscission proteins by RNAi had any adverse effects on early stages of Chlamydia infection, we measured the diameters of primary C. trachomatis inclusions in cells treated with siRNA compared to cells treated with a nontargeting siRNA control. mCherry-HeLa cells [37] were treated with siRNA, infected with GFP-expressing C. trachomatis L2, and inclusion diameters were measured by live cell fluorescence microscopy. Inclusions were determined by the areas containing GFP-expressing bacteria, and diameters of inclusions were calculated at 48 hpi on a per cell basis by imaging software analysis.…”

In-cell western assay as a high-throughput approach for Chlamydia trachomatis quantification and susceptibility testing to antimicrobials

“…Recently, other alternative approaches have been suggested for improving or automating C. trachomatis quantification or susceptibility testing. In particular, genetically modified C. trachomatis strains expressing Green Fluorescent Protein (GFP), in-silico analysis of Immuno-Spot data or modified plaque-forming assays [27][28][29][30][31] have been proposed. However, these approaches have not gained a lot of traction in the scientific community since, for example, the engineering of GFP-producing C. trachomatis is challenging and time-consuming [27][28][29][30][31], the ImmunoSpot imaging system shows a low resolving power, underestimating chlamydial quantification [30], and the plaque assay lack accuracy and reproducibility [31].…”

Establishment and evaluation of a rat model of extracorporeal membrane oxygenation (ECMO) thrombosis using a 3D-printed mock-oxygenator

“…The carotid artery was cannulated with a 24-gauge drainage catheter (Insyte™ 0.7 × 19 mm, BD), which was advanced into the aortic arch and connected to the ECMO circuit inlet (see Fig. 1 a below) [ 15 ]. The right external jugular vein was cannulated with a 20-gauge infusion catheter (Insyte™ 1.1 × 30 mm, BD), which was advanced to the bifurcation at the right external jugular vein and the right subclavian vein and attached to the circuit outlet [ 16 ].…”