Deciphering the Molecular Mechanism and Function of Pore-Forming Toxins Using Leishmania major

Summary

Presented here is a protocol using Leishmania major promastigotes to determine the binding, cytotoxicity, and signaling induced by pore-forming toxins. A proof-of-concept with streptolysin O is provided. Other toxins can also be used to leverage the genetic mutants available in L. major to define new mechanisms of toxin resistance.

Abstract

Understanding the function and mechanism of pore-forming toxins (PFTs) is challenging because cells resist the membrane damage caused by PFTs. While biophysical approaches help understand pore formation, they often rely on reductionist approaches lacking the full complement of membrane lipids and proteins. Cultured human cells provide an alternative system, but their complexity and redundancies in repair mechanisms make identifying specific mechanisms difficult. In contrast, the human protozoan pathogen responsible for cutaneous leishmaniasis, Leishmania major, offers an optimal balance between complexity and physiologic relevance. L. major is genetically tractable and can be cultured to high density in vitro, and any impact of perturbations on infection can be measured in established murine models. In addition, L. major synthesizes lipids distinct from their mammalian counterparts, which could alter membrane dynamics. These alterations in membrane dynamics can be probed with PFTs from the best-characterized toxin family, cholesterol-dependent cytolysins (CDCs). CDCs bind to ergosterol in the Leishmania membrane and can kill L. major promastigotes, indicating that L. major is a suitable model system for determining the cellular and molecular mechanisms of PFT function. This work describes methods for testing PFT function in L. major promastigotes, including parasite culture, genetic tools for assessing lipid susceptibility, membrane binding assays, and cell death assays. These assays will enable the rapid use of L. major as a powerful model system for understanding PFT function across a range of evolutionarily diverse organisms and commonalities in lipid organization.

Introduction

Pore-forming toxins (PFTs) are the largest family of bacterial toxins¹, but the mechanisms by which they perforate and destroy cells are poorly understood. The best-studied family of pore-forming toxins is that of cholesterol-dependent cytolysins (CDCs). CDCs are primarily synthesized by gram-positive bacteria, including the causative agent of necrotizing fasciitis, Streptococcus pyogenes². S. pyogenes secretes the CDC streptolysin O (SLO), which binds to sterols in the plasma membrane of host cells as monomers, oligomerizes, and inserts ~20-30 nm pores into the membrane¹. The ro....

Protocol

All appropriate guidelines and standard microbiological, safety, and cell culture practices were employed for the use and handling of the RG2 pathogen Leishmania major and recombinant DNA. All experiments with live L. major were performed in a biosafety cabinet in a BSL-2 certified laboratory. The work was overseen by the Texas Tech University Institutional Biosafety Committee.

NOTE: From a safety perspective, live L. major promastigotes are Risk Group 2 pathogens. Handle using appropriate containment, precautions, and oversight from the Institutional Biosafety Committee (IBC). Handle toxic substances and chemical....

Results

Increased promastigote sensitivity to SLO in Tyrode's buffer compared to M199
The SLO sensitivity of L. major promastigotes was compared between different assay buffers. Wild-type, spt2^-, and spt2^-/+SPT2 promastigotes were challenged with SLO in serum-free M199 or Tyrode's buffer supplemented with 2 mM CaCl₂ for 30 min prior to analysis on a flow cytometer. Suitable parasites for analysis were single cells identified by forwar.......

Discussion

In this study, methods to study the molecular mechanisms and functions of PFTs were described, using the human pathogen Leishmania major as a model system. A medium-throughput flow cytometry-based cytotoxicity assay to measure single-cell viability was developed. Viability is quantitative at the population level because LC₅₀ values can be calculated from the dose-response curve using logistic modeling. As a proof-of-principle, a flow cytometric assay was used to illustrate that the choice of media can.......

Materials

Name	Company	Catalog Number	Comments
1.2 mL microtiter (Marsh) tubes	Fisher	02-681-376	Cytotoxicity assay
1.5 mL microcentrifuge tube	Fisher	05-408-129	Toxin dilutions
15 mL centrifuge tube	Avantor VWR (Radnor, PA)	89039-666	To hold cells and media
1x Phosphate buffered saline (PBS)	Fisher	BP399	For cell processing
3% H2O2	Walmart  (Fayetteville, AR)	N/A	For ECL
5x M199	Cell-gro	11150067	Basal growth media for L. major promastigotes
Biosafety cabinet	Baker		To culture cells in sterile conditions
Bovine serum albumin (BSA)	Fisher	BP1605-100	Fraction V acceptable purity
CaCl2	Fisher	BP510-100	Stock concentration 100 mM
Centrifuge	Thermo Fisher	Heraeus Megafuge 40R	To pellet the cells from culture
Cy5 Mono-reactive dye pack	Cytiva (Marlborough, MA)	PA25031	Fluorophore label for toxins
Digital dry bath	Benchmark	BSH1002	To denature protein samples
EGTA	Amresco	0732-100G	Stock concentration 0.5 M
Excel	Microsoft (Redmond, VA)		Data analysis software
Flow cytometer (4-laser Attune NxT)	Fisher		Cytometer for data acquisition
FlowJo	BD (Ashland, OR)		Software
Formaldehyde	Fisher	BP531-500	Fixative for counting cells
G418	Fisher	BP673-1	Selection agent for cells
Hellmanex III	Sigma	Z805939	Dilute 1:4 for cleaning cytometer
Hemacytometer	Fisher	0267151B	For counting cells
Human red blood cells	Zen-bio (Durham, NC)	SER-10MLRBC	To validate toxin activity
Ice bucket
Light microscope	Nikon	Eclipse 55i	To visualize cells
Nitrocellulose	Fisher	88018	For probing proteins via antibodies
Pipettors and tips	Avantor VWR		To dispense reagents
Power supply	Bio-Rad		To run SDS-PAGE and transfers
Propidium iodide	Biotium	40016	Stock concentration 2 mg/mL in water
Protein ladder	Bio-Rad	161-0373	To determine molecular weight of proteins
SDS-PAGE Running Apparatus (Mini Protean III)	Bio-Rad	165-3302	To separate proteins based on their size
Sealing tape	R&D	DY992	To seal plates with cells
Streptolysin O C530A plasmid insert			Cloned into pBAD-gIII vector (Reference: 7)
Streptolysin O C530A toxin	Lab purified		Specific activity  4.34 x 105 HU/mg
Swinging bucket rotor	Thermo Fisher	75003607	To centrifuge cells
V-bottom plate	Greiner Bio-one	651206	For cytotoxicity assay
Vortex	Benchmark	BV1000	To mix cells
Western blot imaging system (Chemi-doc)	Bio-Rad		To visualize proteins by western blot
Western Blot Transfer Apparatus (Mini Protean III)	Bio-Rad	170-3930	Transfer proteins to nitrocellulose
Whatman Filter paper	GE Healthcare Life Sciences	3030-700	Used in transfer of proteins to nitrocellulose
Antibody
Anti-ERK antibody	Cell Signaling Technologies	Cat# 9102S	Rabbit (1:1000 dilution)
Anti-lipophosphoglycan (LPG) antibody	CreativeBioLabs	Cat# WIC79.3	Mouse (1: 1000)
Anti-MEK antibody	Cell Signaling Technologies	Cat# 9122L	Rabbit (1:1000)
Anti-mouse IgG, HRP conjugate	Jackson Immunoresearch	Cat#715-035-151	Donkey (1:10000)
Anti-phosphoERK antibody	Cell Signaling Technologies	Cat# 9101S	Rabbit (1:1000)
Anti-pMEK antibody	Cell Signaling Technologies	Cat# 9121S	Rabbit (1:1000)
Anti-rabbit IgG, HRP conjugate	Jackson Immunoresearch	Cat#711-035-152	Donkey (1:10000)
Anti-tubulin antibody	Sigma	Cat# T5168	Mouse (1: 2000)
Leishmania major Genotypes			Reference: 13
Episomal addback (spt2-/+SPT2)			Δspt2::HYG/Δspt2:PAC/+pXG-SPT2
Serine palmitoyltransferase subunit 2 knockout (spt2-)			Δspt2::HYG/Δspt2::PAC
Wild type (WT)			LV39 clone 5 (Rho/SU/59/P)