Sign In

A subscription to JoVE is required to view this content. Sign in or start your free trial.

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. H.......

Representative 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.......

Acknowledgements

The authors would like to thank members of the Keyel and Zhang labs for their critical review of the manuscript. The authors thank the College of Arts and Sciences Microscopy for the use of facilities.

....

Materials

NameCompanyCatalog NumberComments
1.2 mL microtiter (Marsh) tubesFisher02-681-376Cytotoxicity assay
1.5 mL microcentrifuge tubeFisher05-408-129Toxin dilutions
15 mL centrifuge tube Avantor VWR (Radnor, PA)89039-666To hold cells and media
1x Phosphate buffered saline (PBS)FisherBP399For cell processing
3% H2O2 Walmart  (Fayetteville, AR)N/AFor ECL
5x M199Cell-gro11150067Basal growth media for L. major promastigotes
Biosafety cabinetBakerTo culture cells in sterile conditions
Bovine serum albumin (BSA)FisherBP1605-100Fraction V acceptable purity
CaCl2FisherBP510-100Stock concentration 100 mM
CentrifugeThermo Fisher Heraeus Megafuge 40RTo pellet the cells from culture
Cy5 Mono-reactive dye packCytiva (Marlborough, MA)PA25031Fluorophore label for toxins
Digital dry bathBenchmarkBSH1002To denature protein samples
EGTAAmresco0732-100GStock concentration 0.5 M
ExcelMicrosoft (Redmond, VA)Data analysis software
Flow cytometer (4-laser Attune NxT)FisherCytometer for data acquisition
FlowJoBD (Ashland, OR)Software
FormaldehydeFisherBP531-500Fixative for counting cells
G418FisherBP673-1Selection agent for cells
Hellmanex IIISigmaZ805939Dilute 1:4 for cleaning cytometer
HemacytometerFisher0267151BFor counting cells
Human red blood cellsZen-bio (Durham, NC)SER-10MLRBCTo validate toxin activity
Ice bucket
Light microscopeNikonEclipse 55iTo visualize cells
NitrocelluloseFisher88018For probing proteins via antibodies
Pipettors and tipsAvantor VWRTo dispense reagents
Power supplyBio-RadTo run SDS-PAGE and transfers
Propidium iodideBiotium40016Stock concentration 2 mg/mL in water
Protein ladderBio-Rad161-0373To determine molecular weight of proteins
SDS-PAGE Running Apparatus (Mini Protean III)Bio-Rad165-3302To separate proteins based on their size
Sealing tapeR&DDY992To seal plates with cells
Streptolysin O C530A plasmid insertCloned into pBAD-gIII vector (Reference: 7)
Streptolysin O C530A toxinLab purifiedSpecific activity  4.34 x 105 HU/mg
Swinging bucket rotorThermo Fisher 75003607To centrifuge cells
V-bottom plateGreiner Bio-one651206For cytotoxicity assay
VortexBenchmarkBV1000To mix cells
Western blot imaging system (Chemi-doc)Bio-RadTo visualize proteins by western blot
Western Blot Transfer Apparatus (Mini Protean III)Bio-Rad170-3930Transfer proteins to nitrocellulose
Whatman Filter paperGE Healthcare Life Sciences3030-700Used in transfer of proteins to nitrocellulose
Antibody
Anti-ERK antibodyCell Signaling Technologies Cat# 9102SRabbit (1:1000 dilution)
Anti-lipophosphoglycan (LPG) antibodyCreativeBioLabs Cat# WIC79.3Mouse (1: 1000)
Anti-MEK antibodyCell Signaling Technologies Cat# 9122LRabbit (1:1000)
Anti-mouse IgG, HRP conjugateJackson Immunoresearch Cat#715-035-151Donkey (1:10000)
Anti-phosphoERK antibodyCell Signaling Technologies Cat# 9101SRabbit (1:1000)
Anti-pMEK antibodyCell Signaling Technologies Cat# 9121SRabbit (1:1000)
Anti-rabbit IgG, HRP conjugateJackson Immunoresearch Cat#711-035-152Donkey (1:10000)
Anti-tubulin antibodySigmaCat# T5168Mouse (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)

References

  1. Thapa, R., Ray, S., Keyel, P. A. Interaction of macrophages and cholesterol-dependent cytolysins: The impact on immune response and cellular survival. Toxins. 12 (9), 531 (2020).
  2. Limbago, B., Penumalli, V., Weinrick, B., Scott, J. R.

This article has been published

Video Coming Soon

We use cookies to enhance your experience on our website.

By continuing to use our website or clicking “Continue”, you are agreeing to accept our cookies.

Learn More