An Automated Image-Based Method for Rapid Analysis of Chlamydia Infection as a Tool for Screening Antichlamydial Agents

Abstract

A major limitation in the identification of novel antichlamydial compounds is the paucity of effective methods for large-scale compound screening. The immunofluorescence assay is the preferred approach for accurate quantification of the intracellular growth of Chlamydia. In this study, an immunofluorescence image-based method (termed image-based automated chlamydial identification and enumeration [iBAChIE]) was customized for fully automated quantification of Chlamydia infection using the freely available open-source image analysis software program CellProfiler and the complementary data exploration software program CellProfiler Analyst. The method yielded enumeration of different species and strains of Chlamydia highly comparably to the conventional manual methods while drastically reducing the analysis time. The inhibitory capability of established antichlamydial activity was also evaluated. Overall, these data support that iBAChIE is a highly effective tool for automated quantification of Chlamydia infection and assessment of antichlamydial activities of molecules. Furthermore, iBAChIE is expected to be amenable to high-throughput screening studies for inhibitory compounds and fluorescently labeled molecules to study hostpathogen interactions.

Interrogating Genes That Mediate Chlamydia trachomatis Survival in Cell Culture Using Conditional Mutants and Recombination

“…Inclusions were imaged at ϫ10 magnification on an EVOS fluorescence microscope (Thermo Scientific). Inclusion cross-sectional areas were measured in CellProfiler (27) using a modified pipeline (28). Mutants were sorted into developmental cycle classes based on the first time interval during which their mean normalized inclusion area was Յ80% of that measured for L2-GFP.…”

Lipopolysaccharide-Binding Alkylpolyamine DS-96 Inhibits Chlamydia trachomatis Infection by Blocking Attachment and Entry

“…Images were captured using a QImaging QICAM digital camera, 12-bit Mono Fast 1394 cooled camera, and QCapture Suite software, version 3.1.3.5 (QImaging Corp., Surrey, BC, Canada). Image-based automated quantification using CellProfier and CellProfiler Analyst (iBAChIE) was performed as described previously (32).…”

“…When visualized by immunofluorescence mi- g for 1 h). Infected cultures were fixed with methanol, immunofluorescently stained, and analyzed for inhibition of inclusion formation 24 hpi using iBAChIE (32). Error bars indicate standard deviations from triplicate samples.…”

“…Chlamydia was stained with anti-MOMP antibody (green), and host cells were counterstained with DAPI (nuclei; blue) and Evan's blue (cytoplasm; red). (C) Inhibition of inclusion formation among the samples treated with various concentrations of DS-96 was quantified 24 hpi using the automated enumeration method iBAChIE (32). Percent inhibition was determined based on the number of inclusion-positive cells in DS-96-treated samples relative to DMSO-treated samples.…”

Simple Resazurin-Based Microplate Assay for Measuring Chlamydia Infections

“…This process is laborintensive and not suitable for large-scale screening required for antimicrobial compound development. Effective methods for largescale, image-based quantification have been reported for Chlamydia infection (2)(3)(4). However, automation of image acquisition typically requires specialized equipment and extensive computational resources.…”