Development of a Larval Zebrafish Infection Model for Clostridioides difficile

Summary

Presented here is a safe and effective method to infect zebrafish larvae with fluorescently labeled anaerobic C. difficile by microinjection and noninvasive microgavage.

Abstract

Clostridioides difficile infection (CDI) is considered to be one of the most common healthcare-associated gastrointestinal infections in the United States. The innate immune response against C. difficile has been described, but the exact roles of neutrophils and macrophages in CDI are less understood. In the current study, Danio rerio (zebrafish) larvae are used to establish a C. difficile infection model for imaging the behavior and cooperation of these innate immune cells in vivo. To monitor C. difficile, a labeling protocol using a fluorescent dye has been established. A localized infection is achieved by microinjecting labeled C. difficile, which actively grows in the zebrafish intestinal tract and mimics the intestinal epithelial damage in CDI. However, this direct infection protocol is invasive and causes microscopic wounds, which can affect experimental results. Hence, a more noninvasive microgavage protocol is described here. The method involves delivery of C. difficile cells directly into the intestine of zebrafish larvae by intubation through the open mouth. This infection method closely mimics the natural infection route of C. difficile.

Introduction

C. difficile is a gram-positive, spore-forming, anaerobic, and toxin-producing bacillus that is the leading cause of severe infections in the gastrointestinal tract¹. Typical symptoms of CDI include diarrhea, abdominal pain, and fatal pseudomembranous colitis, and it can sometimes lead to death^1,2. Evidence has shown that host immune responses play a critical role in both the progression and outcome of this disease³. In addition to the immune response, the indigenous gut microbiota is crucial for the onset and pathogenesis of CDI⁴....

Protocol

All animal work described here was performed in accordance with legal regulations (EU-Directive 2010/63, license AZ 325.1.53/56.1-TUBS and license AZ 33.9-42502-04-14/1418).

1. Preparation of Low Melting Agarose, Gel Plate, and Microinjection/Microgavage Needles

1. Dissolve 0.08 g of low melting agarose (Table of Materials, agarose A2576) in 10 mL of 30% Danieau's medium (0.12 mM MgSO₄, 0.18 mM Ca [NO₃]₂), 0.21 mM KCl, 1.5 mM HEPES (pH = 7.2), and 17.4 mM NaCl, stored at room temperature (RT) to obtain a 0.8% solution.
   NOTE: Higher or lower concentrations of agarose can b....

Results

C. difficile is strictly anaerobic, but the chromophore of fluorescent proteins usually requires oxygen to mature. To overcome this problem, a fluorescent dye was used to stain live C. difficile cells that were actively growing (R20291, a ribotype 027 strain; Figure 1A). Using the Gal4/UAS system, stable transgenic zebrafish lines were generated for live imaging, in which the mpeg1.1 or lyZ promoters drove the expression of EGFP fluorescen.......

Discussion

The presented methods modify and extend an existing approach to infect zebrafish larvae by performing both injection and microgavage^10,14. It also demonstrates an approach to study anaerobic pathogens with zebrafish larvae²². In addition, the protocol facilitates the analysis of innate immune cell responses in vivo upon CDI and upon colonization of C. difficile in zebrafish. The method is reproducible and easy to conduct in a rout.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Agarose	Sigma-Aldrich	A2576	Ultra-low gelling agarose
Agarose low-melting (LM)	Pronadisa	8050	It is used in agarose plates
BacLight Red Bacterial Stain	Thermo Fisher Scientific	B35001	Fluorescent dye
Brain-Heart-Infusion Broth	Carl Roth GmbH	X916.1
Brass (wild-type)			deficient in melanin synthesis, used to generate stable transgenic lines
Calcium nitrate (Ca(NO3)2)	Sigma-Aldrich	C1396
Capillary Glass	Harvard Apparatus	30-0019	Injection needles
Clostridioides difficile			R20291,, a ribotype 027 strain, TcdA+/TcdB+/CDT+ production
DMSO	Carl Roth GmbH	A994
FIJI	open-source platform		Image processing
HEPES	Carl Roth GmbH	6763
Horizontal needle puller	Sutter instrument Inc	P-87
L-cysteine	Sigma-Aldrich	168149
Leica Application Suite X (LAS X)	Leica		Image processing
Magnesium sulfate (MgSO4)	Carl Roth GmbH	P026
Micro injector	eppendorf	5253000017
Microinjection molds	Adaptive Science Tools	TU1
Leica SP8 confocal microscope	Leica
Phenol Red	Sigma-Aldrich	P0290
Potassium chloride (KCl)	Carl Roth GmbH	5346
Sodium chloride (NaCl)	Carl Roth GmbH	9265
Taurocholate	Carl Roth GmbH	8149
Tg(lyZ: KalTA4)bz17/Tg(4xUAS-E1b:EGFP)hzm3			stable transgenic line in which in which the lyZ promoters drive the expression of EGFP fluorescent protein in neutrophils
Tg(mpeg1.1: KalTA4)bz16/Tg(4xUAS-E1b:EGFP)hzm3			stable transgenic line in which in which the mpeg1.1 drive the expression of EGFP fluorescent protein in macrophages
Tricaine	Sigma-Aldrich	E10521
Yeast extract	BD Bacto	212750