Live-Cell Forward Genetic Approach to Identify and Isolate Developmental Mutants in Chlamydia trachomatis

Summary

This protocol utilizes fluorescent promoter-reporters, live-cell microscopy, and individual inclusion extraction in a directed forward genetic approach to identify and isolate developmental mutants of Chlamydia trachomatis.

Abstract

The intracellular bacterial pathogen Chlamydia trachomatis undergoes a developmental cycle consisting of two morphologically discrete developmental forms. The non-replicative elementary body (EB) initiates infection of the host. Once inside, the EB differentiates into the reticulate body (RB). The RB then undergoes multiple rounds of replication, before differentiating back to the infectious EB form. This cycle is essential for chlamydial survival as failure to switch between cell types prevents either host invasion or replication.

Limitations in genetic techniques due to the obligate intracellular nature of Chlamydia have hampered identification of the molecular mechanisms involved in the cell-type development. We designed a novel dual promoter-reporter plasmid system that, in conjunction with live-cell microscopy, allows for the visualization of cell type switching in real time. To identify genes involved in the regulation of cell-type development, the live-cell promoter-reporter system was leveraged for the development of a forward genetic approach by combining chemical mutagenesis of the dual reporter strain, imaging and tracking of Chlamydia with altered developmental kinetics, followed by clonal isolation of mutants. This forward genetic workflow is a flexible tool that can be modified for directed interrogation into a wide range of genetic pathways.

Introduction

Chlamydia trachomatis (Ctr) is an obligate intracellular pathogen that progresses through a biphasic developmental cycle that is essential for its survival and proliferation¹. This cycle consists of two developmental forms, the elementary body (EB) and the reticulate body (RB). The EB is replication incompetent but mediates cell invasion through effector induced endocytosis². Once in the host, the EB matures to the replicative RB. The RB carries out multiple rounds of replication prior to converting back to the EB in order to initiate subsequent rounds of infection.

The limited array ....

Protocol

All Python scripts used in this protocol are available on Github https://github.com/SGrasshopper/Live-cell-data-processing

1. Mutagenize Reporter  Chlamydia

NOTE: Ctr-L2-hctBprom-mKate2/euoprom-Clover EBs were directly mutagenized using ethyl methanesulfonate (EMS) in the axenic media CIP-1 as this media supports EB metabolism and maintenance of EB infectivity¹².

1. Thaw a chlamydial stock on ice .......

Discussion

Dissecting the mechanisms that control the chlamydial developmental cycle has been hindered by the limitations of the currently available genetic tools. Employing our promoter-reporter Chlamydia in conjunction with live-cell automated microscopy, a system was built which enables monitoring of cell-type development in individual inclusions over a 24 h period. This system, in combination with chemical mutagenesis and direct inclusion isolation has established a method to rapidly and clonally select Chlamydia

Materials

Name	Company	Catalog Number	Comments
24-well polystyrene plates	Corning	3524	Cell culture growth for reinfection of isolates
6-well glass bottom plates	Cellvis	P06-1.5H-N	Cell culture growth for imaging
96-well glass bottom plates	Nunc	165305	Cell culture growth for imaging
Bold line CO2 Unit	OKO Labs	CO2 UNIT BL	Stage incubator CO2 control
Bold line T Unit	OKO Labs	H301-T-UNIT-BL-PLUS	Stage incubator temperature control
Borosilicate glass capillary tubes	Sutter Instrument	B1005010	Capillary tubes
BrightLine bandpass emissions filter (514/30nm)	Semrock	FF01-514/30-25	Fluoescent filter cube
BrightLine bandpass emissions filter (641/75nm)	Semrock	FF02-641/75-25	Fluoescent filter cube
CellTram Vario	Eppendorf	5196000030	Microinjector
Chlamydia trachmatis serovar L2	ATCC	VR-577	Chlamydia trachomatis
CIP-1 media	In house	NA	Axenic media. IPB supplemented with 1% FBS, 25 μM amino acids, 0.5 mM G6P, 1.0 mM ATP, 0.5 mM DTT, and 50 μM GTP, UTP, and CTP. (Omsland, A. 2012) made in-house.
Cos-7 cells (ATCC)	ATCC	CRL-1651	African green monkey kidney cell (host cells)
Cycloheximide	MP Biomedicals	194527	Host cell growth inhibitor
Ethyl methanesulfonate, 99%	Acros Organics	AC205260100	Mutagen
Fetal Plex	Gemini Bio-Products	100-602	Supplement for base growth media
Fiji/ImageJ	https://imagej.net/Fiji	NA	Open sourse Image analysis software. https://imagej.net/Fiji
Galaxy 170 S CO2 incubator	Eppendorf	CO1700100X	Cell culture incubation
gblocks (Fluorescent FP variants: Clover and mKate2)	Integrated DNA Technologies	NA	gblock ORFs of Ctr optimized FP varients for cloning into p2TK2SW2
Gentamycin 10mg/ml	Gibco	15710-064	Antibiotic for growth media
HBSS (Hank's Balanced Salt Solution)	Corning	21-020-CM	Host cells rinse
Heparin sodium	Amersham Life Science	16920	inhibits and reverses the early electrostatic interactions between the host cell and EBs
HEPES 1M	GE Life Sciences	SH30237.01	pH buffer for growth media
InjectMan	Eppendorf	5179 000.018	Micromanipulator
Jupyter Notebook	https://jupyter.org/	NA	Visualization of inclusion traces. https://jupyter.org/
Lambda 10-3	Sutter Instrument	LB10-3	Filter wheel controler
Oko Touch	OKO Labs	Oko Touch	Interface to control the Bold line T and CO2 Unit
Prior XY stage	Prior	H107	Motorized XY microscope stage
PrismR Centrifuge	Labnet	C2500-R	Temperature controlled microcentrifuge
Problot Hybridization oven	Labnet	H1200A	Rocking Incubator for infection with Chlamydia
Proscan II	Prior	H30V4	XYZ microscope stage controler
Purifier Class 2 Biosafety Cabinet	Labconco	362804	Cell culture work
RPMI-1640 (no phenol red)	Gibco	11835-030	Base growth media for imaging
RPMI-1640 (phenol red)	GE Life Sciences	SH30027.01	Base growth media
scopeLED excitation LEDs (470nm,595nm)	scopeLED	F140	Excitation light
Sonic Dismembrator Model 500	Fisher Scientific	15-338-550	Sonicator, resuspending chlamydial pellet
Stage incubator	OKO Labs	H301-K-FRAME	Cluster well plate incubation chamber
sucrose-phosphate-glutamate buffer 1X (SPG)	In house	NA	Chlamydial storage buffer. (10 mM sodium phosphate [8 mM K 2HPO 4, 2 mM KH 2PO 4], 220 mM sucrose, 0.50 mM L-glutamic acid; pH 7.4)
T-75 Flasks	Thermo Scientific	156499	Cell culture growth
TE 300 inverted microscope	Nikon	16724	microscope
THOR LED	Thor Labs	LEDD1B	White light
Trypsin	Corning	25-052-CI	Dislodges host cells from flask for seeding into plates
Zyla sCMOS	Andor	ZYLA-5.5-USB3	imaging camera
µManager 2.0gamma	https://github.com/micro-manager/micro-manager	NA	Open sourse automated microscope control software package