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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.
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.
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....
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.......
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.......
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.......
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.
....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) |
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