High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

Summary

A high-throughput assay to in vitro phenotype Salmonella or other bacterial association, invasion, and replication in phagocytic cells with high-throughput capacity was developed. The method was employed to evaluate Salmonella gene knockout mutant strains for their involvements in host-pathogen interactions.

Abstract

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock¹. Understanding the mechanisms underlying Salmonella-host interactions are important to elucidate the molecular pathogenesis of Salmonella infection. The Gentamicin protection assay to phenotype Salmonella association, invasion and replication in phagocytic cells was adapted to allow high-throughput screening to define the roles of deletion mutants of Salmonella enterica serotype Typhimurium in host interactions using RAW 264.7 murine macrophages. Under this protocol, the variance in measurements is significantly reduced compared to the standard protocol, because wild-type and multiple mutant strains can be tested in the same culture dish and at the same time. The use of multichannel pipettes increases the throughput and enhances precision. Furthermore, concerns related to using less host cells per well in 96-well culture dish were addressed. Here, the protocol of the modified in vitro Salmonella invasion assay using phagocytic cells was successfully employed to phenotype 38 individual Salmonella deletion mutants for association, invasion and intracellular replication. The in vitro phenotypes are presented, some of which were subsequently confirmed to have in vivo phenotypes in an animal model. Thus, the modified, standardized assay to phenotype Salmonella association, invasion and replication in macrophages with high-throughput capacity could be utilized more broadly to study bacterial-host interactions.

Introduction

Nontyphoidal Salmonella are important causes of enteric diseases in all vertebrates. Salmonellosis in humans is among the top bacterial food-borne diseases¹. Characterization of the molecular mechanisms that underpin the interactions of Salmonella with their animal hosts is mainly achieved through the study of Salmonella enterica serotype Typhimurium (STM) in tissue culture and animal models of infection. Gaining insights in STM-host interactions will help us understand how Salmonella survive and grow inside host cells. The first challenge in studying these interactions is to identify as many participating factors as poss....

Protocol

1. Murine Macrophage RAW264.7 Cell Culture

1. Grow low passage number murine macrophage cells, RAW264.7 (The ATCC^® Number, TIB-71) in a T-75 cell culture flask vented filter cap in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 0.5% NaHCO₃, and 1% 100x Nonessential amino acids (NEAA) at 37°C, in a 5% CO₂ incubator.
2. Once cells reach a 60-80% confluence in the flask, use a cell scraper to harvest cells, and count the cells in a hemacytometer and calculate the cell concentration.
3. Resuspend the cells and dilute the cell concentration into 2.5 x 10⁵

Results

See representative results (Figure 2) after the data are plotted based on the modified phagocytic cell invasion assay. The data include five different strains, WT, ΔinvA, ΔphoP, mutant A, and mutant B. ΔinvA, known to be defective for invasion, and ΔphoP known to be defective for replication⁸, are used as positive controls to assess the experimental validity. Indeed, in the modified invasion assay, a ΔinvA mutant is internalized poorly by RAW264.7 c.......

Discussion

The Gentamicin protection assay is widely used to study the invasion and replication of intracellular bacterial pathogens inside host cell, and it is especially an important biological tool for studying pathogens, like Salmonella, whose invasion is the prerequisite step for establishing infection¹. The standard Gentamicin protection assay in Salmonella research community is implemented in 24-well culture dish⁵. Though the use of 48 or even 96-well plates were discussed before for h.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dulbecco's Modified Eagle Medium (DMEM)	Life Technologies	11965
Fetal bovine serum	HyClone	SH30910.03
T-75 Cell culture flask vented filter cap	Nest Biotechnology	708003
100x Non-Essential Amino Acids	Life Technologies	11140
Cell scraper	BD Falcon	353086
96-well Cell culture plate	Corning Incorporated	3595
Luria-Bertani (LB) broth	MP Biomedicals	3002-075
14 ml Polypropylene Round-Bottom Tube	BD Falcon	352059
PBS pH 7.4 (1x)	Life Technologies	10010
Triton X-100	Sigma	T-8787
Kanamycin solution	Sigma	K0254
Gentamicin solution	Sigma	G1272
0.25% Trypsin-EDTA	Life Technologies	25200
Trypan blue	Sigma	T8154