Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

Summary

Two assays for microscopy-based high-throughput screening of host factors involved in Brucella infection are described. The entry assay detects host factors required for Brucella entry and the endpoint assay those required for intracellular replication. While applicable for alternative approaches, siRNA screening in HeLa cells is used to illustrate the protocols.

Abstract

Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by direct contact with infected animals or by ingestion of contaminated food and can lead to severe chronic infections.

Brucella can invade professional and non-professional phagocytic cells and replicates within endoplasmic reticulum (ER)-derived vacuoles. The host factors required for Brucella entry into host cells, avoidance of lysosomal degradation, and replication in the ER-like compartment remain largely unknown. Here we describe two assays to identify host factors involved in Brucella entry and replication in HeLa cells. The protocols describe the use of RNA interference, while alternative screening methods could be applied. The assays are based on the detection of fluorescently labeled bacteria in fluorescently labeled host cells using automated wide-field microscopy. The fluorescent images are analyzed using a standardized image analysis pipeline in CellProfiler which allows single cell-based infection scoring.

In the endpoint assay, intracellular replication is measured two days after infection. This allows bacteria to traffic to their replicative niche where proliferation is initiated around 12 hr after bacterial entry. Brucella which have successfully established an intracellular niche will thus have strongly proliferated inside host cells. Since intracellular bacteria will greatly outnumber individual extracellular or intracellular non-replicative bacteria, a strain constitutively expressing GFP can be used. The strong GFP signal is then used to identify infected cells.

In contrast, for the entry assay it is essential to differentiate between intracellular and extracellular bacteria. Here, a strain encoding for a tetracycline-inducible GFP is used. Induction of GFP with simultaneous inactivation of extracellular bacteria by gentamicin enables the differentiation between intracellular and extracellular bacteria based on the GFP signal, with only intracellular bacteria being able to express GFP. This allows the robust detection of single intracellular bacteria before intracellular proliferation is initiated.

Introduction

Brucella species are gram-negative, facultative intracellular pathogens belonging to the class of α-Proteobacteria. They cause abortions and infertility in their natural hosts such as cattle, goats, or sheep resulting in severe economic losses in endemic areas. Brucellosis is one of the most important zoonotic diseases worldwide causing over half a million new human infections annually¹. Transmission to human is most commonly caused by direct contact with infected animals or by ingestion of contaminated food such as unpasteurized milk. Symptoms of the febrile disease are unspecific, which causes difficulties in the diagnosis of brucellosis. If....

Protocol

Note: All work with live Brucella abortus strains must be performed inside a biosafety level 3 (BSL3) laboratory considering all required regulations and safety precautions.

1. Preparation of Screening Plates and Culturing of Bacteria and Cells

1. Preparation of Brucella abortus Starter Cultures
   1. Streak Brucella abortus 2308 (B. abortus) from -80 °C milk stock on a Tryptic Soy Agar (TSA) plate containing 50 µg/ml kanamycin (TSA/Km). Incubate plate for 3-4 days at 37 °C. Use strain Brucella abortus 2308 pJC43 (apHT::GFP)¹³ for the endpoint assay and strain <....

Results

Figure 1A shows an example of image analysis used to automatically identify infected cells in the endpoint assay. Nuclei of HeLa cells stained with DAPI were identified, a peri-nucleus of 8 pixels width surrounding the nucleus, and a Voronoi cell body by extension of the nucleus by 25 pixels were calculated. Since bacteria mainly proliferate in the peri-nuclear space, the GFP intensity in this area of the cell is the most robust measurement to discriminate between infecte.......

Discussion

Bacterial pathogens have evolved numerous strategies to manipulate eukaryotic host cells to their benefit. Pathogens causing acute infections often show rapid proliferation which is accompanied by significant alarming of the immune system and loss of viability of infected cells. In contrast, Brucella and other pathogens that cause chronic infections manage to establish long-lasting interactions within host cells. Therefore, bacteria need to fine tune host cell functions to their benefit without disrupting cellul.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Tryptic Soy Agar (TSA)	BD	236950
Tryptic Soy Broth (TSB)	Fluka	22092
Kanamycin sulfate	Sigma-Aldrich	60615
Skim milk
250 ml screw cap bottle	Corning	8396
DMEM	Sigma-Aldrich	D5796
Fetal Calf Serum (FCS)	Gibco	10270	Heat-inactivated
Trypsin-EDTA (0.5%)	Gibco	15400-054	10x stock solution, dilute 1:10 in PBS
Scepte 2.0 Cell Counter	Merck Milipore	PHCC20060	Alternative cell counting devices can be used
Greiner CELLSTAR 384-well plate	Sigma-Aldrich	M2062
Peelable aluminum foil	Costar	6570
Reagent dispenser: "Multidrop 384 Reagent Dispenser"	Thermo Scientific	5840150	Alternative reagent dispenser can be used.
Transfection reagent: "Lipofectamine RNAiMAX	Invitrogen	13778-150
Automated plate washer: "Plate washer ELx50-16"	BioTek	ELX5016	This plate washer contains a 16-channel manifold suitable for 384-well plates. It fits into a biosafety cabinet and has a lid covering the plate during washing which reduces the risk of aerosol production. Alternative plate washers with similar features could be used.
Gentamicin	Sigma-Aldrich	G1397
Anhydrotetracycline hydrochloride	Sigma-Aldrich	37919	100 μg/ml solution in 100% ethanol is kept at -20 °C protected from light in aluminum-foil
PBS	Gibco	20012
Paraformaldehyde	Sigma-Aldrich	P6148	Dissolve in 0.2 M HEPES buffer, pH 7.4. Store at -20 °C and thaw freshly the day before use. Caution, PFA is toxic by inhalation, in contact with skin and if swallowed.
HEPES	Sigma-Aldrich	H3375
Triton X-100	Sigma-Aldrich	T9284
DAPI	Roche	10236276001
Scrambled siRNA	Dharmacon	D-001810-10
Kif11 siRNA	Dharmacon	L-003317-00
ARPC3 siRNA	Dharmacon	L-005284-00
Brucella abortus 2308
pJC43 (apHT::GFP)			Celli et al.12
pAC042.08 (apht::dsRed, tetO::tetR-GFP)			Construction: pJC44 4 was digested with EcoRI followed by generation of blunt ends with Klenov enzyme and subsequent digestion with SalI. TetR-GFP was amplified from pNF106 using primer prAC090 and prAC092. Following digestion with SalI, the TetR-GFP product was ligated to the digested pJC44 vector.
Primer prAC090	Sigma-Aldrich		TTT TTG AAT TCT GGC AAT TCC GAC GTC TAA GAA ACC
Primer prAC092	Sigma-Aldrich		TTT TTG TCG ACT TTG TCC TAC TCA GGA GAG CGT TC
HeLa CCL-2	ATCC	CCL-2
ImageXpress Micro	Molecular Devices	IXM IMAGING MSCOPE	Automated cellular imaging microscope equipped with a precision motorized Z-stage. Alternative systems for automated microscopy and alternative components for hard- and software specified below can be employed.
High-Speed Laser Auto-Focus	Molecular Devices	1-2300-1037
CFI Super Fluor 10X objective	Nikon	MRF00100	N.A 0.50, W.D 1.20 mm, DIC Prism: 10X, Spring loaded
Photometrics CoolSNAP HQ Monochrome CCD Camera	Molecular Devices	1-2300-1060	1,392 x 1,040 imaging pixels, 6.45 x 6.45 µm pixels, 12 bits digitization
MetaXpress software	Molecular Devices	9500-0100
LUI-Spectra-X-7	Lumencor	SPECTRA X V-XXX-YZ	Light engine. The following light sources are used: violet (DAPI), cyan (GFP), green/yellow (RFP)
Single Band Exciter for DAPI	Semrock	FF01-377/50-25
Single Band Emitter for DAPI	Semrock	FF02-447/60-25
Single Band Dichroic for DAPI	Semrock	FF409-Di03-25x36
Single Band Exciter for GFP	Semrock	FF02-472/30-25
Single Band Emitter for GFP	Semrock	FF01-520/35-25
Single Band Dichroic for GFP	Semrock	FF495-Di03-25x36
Single Band Exciter for RFP	Semrock	FF01-562/40-25
Single Band Emitter for RFP	Semrock	FF01-624/40-25
Single Band Dichroic for RFP	Semrock	FF593-Di03-25x36