Quantitative Monitoring of the Chlamydia trachomatis Developmental Cycle Using GFP-Expressing Bacteria, Microscopy and Flow Cytometry

Abstract

Chlamydiae are obligate intracellular bacteria. These pathogens develop inside host cells through a biphasic cycle alternating between two morphologically distinct forms, the infectious elementary body and the replicative reticulate body. Recently, C. trachomatis strains stably expressing fluorescent proteins were obtained. The fluorochromes are expressed during the intracellular growth of the microbe, allowing bacterial visualization by fluorescence microscopy. Whether they are also present in the infectious form, the elementary body, to a detectable level has not been studied. Here, we show that a C. trachomatis strain transformed with a plasmid expressing the green fluorescent protein (GFP) accumulates sufficient quantities of the probe in elementary bodies for detection by microscopy and flow cytometry. Adhesion of single bacteria was detected. The precise kinetics of bacterial entry were determined by microscopy using automated procedures. We show that during the intracellular replication phase, GFP is a convenient read-out for bacterial growth with several advantages over current methods. In particular, infection rates within a non-homogenous cell population are easily quantified. Finally, in spite of their small size, individual elementary bodies are detected by flow cytometers, allowing for direct enumeration of a bacterial preparation. In conclusion, GFP-expressing chlamydiae are suitable to monitor, in a quantitative manner, progression throughout the developmental cycle. This will facilitate the identification of the developmental steps targeted by anti-chlamydial drugs or host factors.

Novel Detection Strategy To Rapidly Evaluate the Efficacy of Antichlamydial Agents

“…The L2/P ompA -GFP system, which contains ompA promoter-driven GFP, may be best suited for this purpose, as it is constitutively expressed throughout the chlamydial developmental cycle (16). The traceability of the L2/P ompA -GFP system was compared with that of the L2/P incD -GFP system (24), which was previously used to track infection (15). Following infection of HeLa 229 cells with C. trachomatis, microscopy and flow cytometry were used to monitor the time course of GFP expression from 8 to 24 h postinfection (hpi) when RBs replicated and/or asynchronously began to transit back to EBs.…”

“…in situ. Among these important advances are the use of genetic labeling of C. trachomatis with fluorescent proteins for a variety of purposes, including the tracking of gene insertions made by allelic exchange, deletions, or complementation and quantifying C. trachomatis growth as well as the localization of proteins in live cells (12)(13)(14)(15).…”

A Coming of Age Story: Chlamydia in the Post-Genetic Era

“…As the Binet and Maurelli study selected for recombinants and not transformants, transformation frequency could not be directly assessed. Development of selection protocols allowing for first passage mutant isolation such as plaque assay or flow cytometry (43,44) …”

“…Repeated rounds of development lead to a localized clearing of the monolayer referred to as a plaque. Plaque assay is also a useful method for obtaining clonal strains, although fluorescence-activated cell sorting methods appear promising for recombinant strains expressing fluorescent proteins (44). Drugs such as blasticidin and zeocin, which are toxic to both host cells and Chlamydia, impede the use of the plaque assay (55).…”

“…Alternative approaches available for studying essential genes in Chlamydia include regulated overexpression of wild-type genes or dominant-negative mutant genes along with further development of antisense RNA approaches described by Mishra et al (79). In addition, validation of fluorescent protein expression in C. trachomatis coupled with detection of fluorescent EBs via flow cytometry (or enumeration in plaque assay) should allow for optimization of transformation procedures as new methods of mutagenesis are explored (44). Along this line, transformation via electroporation as utilized by Binet and Maurelli (and the method of choice for transformation in other obligate, intracellular bacteria) is worth revisiting to assess efficiency versus chemical transformation (43).…”