Generation of In-Frame Gene Deletion Mutants in Pseudomonas aeruginosa and Testing for Virulence Attenuation in a Simple Mouse Model of Infection

Summary

Here, we describe a simple and reproducible protocol of mouse model of infection to evaluate the attenuation of the genetically modified strains of Pseudomonas aeruginosa in comparison to the United States Food and Drug Administration (FDA)-approved Escherichia coli for commercial applications.

Abstract

Microorganisms are genetically versatile and diverse and have become a major source of many commercial products and biopharmaceuticals. Though some of these products are naturally produced by the organisms, other products require genetic engineering of the organism to increase the yields of production. Avirulent strains of Escherichia coli have traditionally been the preferred bacterial species for producing biopharmaceuticals; however, some products are difficult for E. coli to produce. Thus, avirulent strains of other bacterial species could provide useful alternatives for production of some commercial products. Pseudomonas aeruginosa is a common and well-studied Gram-negative bacterium that could provide a suitable alternative to E. coli. However, P. aeruginosa is an opportunistic human pathogen. Here, we detail a procedure that can be used to generate nonpathogenic strains of P. aeruginosa through sequential genomic deletions using the pEX100T-NotI plasmid. The main advantage of this method is to produce a marker-free strain. This method may be used to generate highly attenuated P. aeruginosa strains for the production of commercial products, or to design strains for other specific uses. We also describe a simple and reproducible mouse model of bacterial systemic infection via intraperitoneal injection of validated test strains to test the attenuation of the genetically engineered strain in comparison to the FDA-approved BL21 strain of E. coli.

Introduction

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that can cause life-threatening diseases in humans, especially in the immunocompromised. The pathogenicity of P. aeruginosa is due to the expression of many virulence factors, including proteases and lipopolysaccharide, as well as its ability to form a protective biofilm¹. Because of its ability to produce virulence factors and cause disease in humans, using P. aeruginosa to make commercial products presents safety concerns. Nonpathogenic strains of E. coli have traditionally been used to bioengineer medical and commercial products for human use. H....

Protocol

Before beginning animal experiments, the protocol to be used must be approved by the Institutional Animal Care and Use Committee (IACUC). Approval for the protocol described was obtained through the IACUC at Marshall University (Huntington, WV, USA).

1. Plasmid Design

1. To generate a genetic deletion using the pEX100T-NotI plasmid, clone the regions of DNA flanking the desired deletion sequence and insert into the NotI restriction site of the plasmid. The plasmid insert should contai.......

Discussion

The pEX100T-Not1 plasmid is an efficient mediator of sequential genomic deletions that are marker-free and in-frame. When engineering bacterial strains for attenuated virulence, deletion of entire gene sequences rather than generating point mutations decreases the likelihood of reversion to a virulent phenotype. Additionally, each pathogenicity gene deletion attenuates the pathogen further, reinforcing the stability of the attenuation.

This method can also be used to generate genomic modificat.......

Materials

Name	Company	Catalog Number	Comments
0.2 mL tubes with flat caps	ThermoScientific	AB-0620	via Fisher Scientific
1 mL Syringe	BD	22-253-260	via Fisher Scientific
1.5 mL disposable polystyrene cuvette	Fisher Scientific	14955127
1.5 mL Microcentrifuge Tubes	Fisher Scientific	05-408-129
2.0 mL Cryogenic Vials	Corning	430659	via Fisher Scientific
27G needle	BD	14-821-13B	via Fisher Scientific
50 mL tubes	Fisher Scientific	05-539-13	via Fisher Scientific
Accu block Digital Dry Bath	Labnet	NC0205808	via Fisher Scientific
Benchtop Centrifuge 5804R	Eppendorf	04-987-372	via Fisher Scientific
Benchtop Microcentrifuge	Sorvall	75-003-287	via Fisher Scientific
Cabinet Incubator	VWR	1540
Carbenicillin disodium salt	Fisher Scientific	BP2648250
Culture Test Tube, Polystyrene	Fisher Scientific	14-956-6D	via Fisher Scientific
Diposable Inoculation Loops	Fisher Scientific	22-363-597
Dneasy UltraClean Microbial Kit (50)	Qiagen	12224-50	or preferred method/vendor
E.Z.N.A. Cycle Pure Kit (50)	Omega bio-tek	D6493-01	or preferred method/vendor
EcoRI-HF, restriction endonuclease	New England BioLabs	R3101L
Electroporation Cuvettes	Bulldog Bio	NC0492929	via Fisher Scientific
FastLink II DNA Ligation Kit	Epicentre Technologies	LK6201H	via Fisher Scientific
Gentamycin Sulfate	Fisher Scientific	BP918-1
Glycerol	Fisher Scientific	BP229-4
GoTaq G2 Colorless Master Mix	Promega	M7833	via Fisher Scientific
Isothesia Isoflurane	Henry Schein Animal Health	29405
IVIS Lumina XRMS Series III In Vivo Imaging System	Perkins and Elmer	CLS136340
Kanamycin monosulfate	Fisher Scientific	BP906-5
LE agarose	Genemate	3120-500	via Fisher Scientific
Luria Broth	Difco	240230	via Fisher Scientific
MicroPulser Electroporator	BioRad	1652100
Noble agar, ultrapure	Affymetris/USB	AAJ10907A1	via Fisher Scientific
NotI-HF, restriction endonuclease	New England BioLabs	R3189
One Shot TOP10 Electrocomp E. coli	Invitrogen	C404052	via Fisher Scientific
Phosphate buffered saline powder	Sigma	P3813-10PAK	Sigma-Aldrich
Prism 7	GraphPad		https://www.graphpad.com/scientific-software/prism/
Pseudomonas isolation agar	Difco	292710	via Fisher Scientific
Pseudomonas isolation broth	Alpha Biosciences	P16-115	Custom made batch
QIAprep Spin Miniprep Kit (250)	Qiagen	27106	or preferred method/vendor
Shaking Incubator	New Brunswick Scientific	Innova 4080	shake at 200 rpm
SimpliAmp Thermal Cycler	Applied Biosystems	A24811
Skim Milk	Difco	DF0032-17-3	via Fisher Scientific
Small Plates (100 O.D. x 10 mm)	Fisher Scientific	FB0875713
SmartSpec Plus Spectrophotometer	Bio-Rad	170-2525	or preferred method/vendor
Sucrose	Fisher Scientific	S5-500
Toothpicks, round	Diamond		Any brand of toothpicks, autoclaved
TOPO TA Cloning Kit, for seqeuncing	Invitrogen	45-0030
XAF-8 Anesthesia System Filters	Perkins and Elmer	118999
XGI 8 Gas Anesthesia System	Caliper Life Sciences/Xenogen