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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Here, we present a protocol to profile the interplay between host and pathogen during infection by mass spectrometry-based proteomics. This protocol uses label-free quantification to measure changes in protein abundance of both host (e.g., macrophages) and pathogen (e.g., Cryptococcus neoformans) in a single experiment.

Abstract

The technological achievements of mass spectrometry (MS)-based quantitative proteomics opens many undiscovered avenues for analyzing an organism’s global proteome under varying conditions. This powerful strategy applied to the interactions of microbial pathogens with the desired host comprehensively characterizes both perspectives towards infection. Herein, the workflow describes label-free quantification (LFQ) of the infectome of Cryptococcus neoformans, a fungal facultative intracellular pathogen that is the causative agent of the deadly disease cryptococcosis, in the presence of immortalized macrophage cells. The protocol details the proper protein preparation techniques for both pathogen and mammalian cells within a single experiment, resulting in appropriate peptide submission for liquid-chromatography (LC)-MS/MS analysis. The high throughput generic nature of LFQ allows a wide dynamic range of protein identification and quantification, as well as transferability to any host-pathogen infection setting, maintaining extreme sensitivity. The method is optimized to catalogue extensive, unbiased protein abundance profiles of a pathogen within infection-mimicking conditions. Specifically, the method demonstrated here provides essential information on C. neoformans pathogenesis, such as protein production necessary for virulence and identifies critical host proteins responding to microbial invasion.

Introduction

The prevalence of invasive fungal infections is vastly increasing and is correlated with unacceptably high mortality rates, most commonly reported in individuals with immunodeficient predispositions1. Cryptococcus neoformans is a notorious opportunistic fungal pathogen capable of intracellular survival within host macrophage cells. Inadequate antifungal intervention results in fungal dissemination and life-threatening manifestations of cryptococcal meningitis and meningoencephalitis2,3. The global increase in immunocompromised status has demanded a parallel increase in the use ....

Protocol

An immortalized line of macrophages derived from BALB/c mice were used for the following protocol approved by the University of Guelph Animal Utilization Protocol 4193. Notably, other strains of mice or other sources of immortalized cells can be applied to the outlined protocol with sufficient testing to optimize the detailed parameters. The following protocol will navigate the steps beginning with a frozen vial of macrophage cells. Cells are stored in 10% FBS (fetal bovine serum), 1% L-glutamine and 5% Pen/Strep (Penici.......

Representative Results

The protocol outlined above enables identification and quantification of proteins derived from both the fungal pathogen, C. neoformans, and the host, macrophage cells, in a single experiment. Following co-culture, cells are collected and processed together and bioinformatically separated based on peptide profiles specific to each species. This is a powerful approach for defining the interplay of the host-pathogen relationship during infection. The number of proteins identified from the experiment depends on the .......

Discussion

Critical steps in the protocol include preparation of macrophage cells and collection of co-culture samples for protein processing with minimal disruption to the cells. It is important to perform steps of washing, inoculating, and removing adherent macrophage cells gently and carefully to prevent unnecessary lysis of cells prior to collection. Establishing the correct MOI for the experiment is also critical as inoculating with an excessively high MOI can cause rapid macrophage cell death and difficulty in collecting and .......

Acknowledgements

The authors thank Dr. Jonathan Krieger of Bioinformatics Solutions Inc. for operating the mass spectrometer for representative experiments, as well as members of the Geddes-McAlister group for their assistance with experimental set-up and manuscript feedback. The authors acknowledge funding support, in part, from the Banting Research Foundation – The Jarislowsky Fellowship Discovery Award, New Frontiers Research Fund – Exploration (NFRFE-2019-00425), and the Canadian Foundation for Innovation (JELF 38798) for J.G.M., as well as NSERC Canada Graduate Scholarship – Masters and Ontario Graduate Scholarship for B.B., and Queen Elizabeth II Graduate Schol....

Materials

NameCompanyCatalog NumberComments
100 mM Tris-HCl, pH 8.5Fisher ScientificBP152-1Maintain at 4°C
60 x 15 mm Dish, Nunclon DeltaThermoFisher Scientific174888
6-well cell culture plateThermoFisher Scientific140675
Acetonitrile, MS gradePierceTS-51101
Acetic AcidSigma Aldrich1099510001
AcetoneSigma Aldrich34850-1L
Ammonium bicarbonate (ABC)ThermoFisher ScientificA643-500Prepare a stock 50 mM ABC solution, stable at room temperature for up to one month.
Bel-Artâ„¢ HiFlow Vacuum Aspirator Collection SystemFisher Scientific13-717-300Not essential, serological pipettes can be used to remove media.
C18 resin3M Empore3M2215
Cell ScrapersVWR10062-906Not essential, other methods to release macrophage cells can be used.
Centrifugal vaccuum concentratorEppendorf07-748-15
Complete Filtration UnitVWR10040-436
Conical falcon tubes (15 mL)Fisher Scientific05-539-12
Countess II Automated Cell CounterThermoFisher ScientificAMQAX1000Not essential, haemocytometer can be used as an alternative.
CytoTox 96 Non-Radioactive Cytotoxicity AssayPromegaG1780
Dithiothreitol (DTT)ThermoFisher ScientificR0861Prepare bulk stock solution of 1 M DTT, flash frozen and stored at -20 °C until use. Discard after each use (do not freeze-thaw repeatedly).
DMEM, high glucose, GlutaMAX SupplementThermoFisher Scientific10566016
Fetal Bovine Serum (FBS)ThermoFisher Scientific12483020Heat inactivate by incubating at 60°C for 30 minutes. Prepare 50 ml aliquots and flash freeze. Thaw prior to media preparation
HaemocytometerVWR15170-208
HEPESSigma AldrichH3375Prepare 40 mM HEPES/8 M Urea in bulk stock solution, flash frozen, store at -20°C until use. Discard after each use (do not freeze-thaw repeatedly).
High-performance liquid chromatography systemThermoFisher ScientificLC140Gradient length is based on sample complexity, recommended 120 min gradient for infectome samples.
High-resolution mass spectrometerThermoFisher Scientific726042
Iodoacetamide (IAA)Sigma AldrichI6125Prepare 0.55 M bulk stock solution, flash frozen, store at -20°C until use. Discard after each use (do not freeze-thaw repeatedly).
L-glutamineThermoFisher Scientific25030081Can be aliquot and frozen for storage. Thaw prior to media preparation.
LoBind Microcentrifuge tubesEppendorf13-698-794
MaxQuanthttps://maxquant.org/MaxQuant is a public platform that offers tutorials, such as the MaxQuant Summer School, outlining the computational analysis steps of large MS data sets
MicrocentrifugeEppendorf13864457
Penicillin : Streptomycin 10k/10kVWRCA12001-692Can be aliquot and frozen for storage. Thaw prior to media preparation.
Peptide separation columnsThermoFisher ScientificES803
Perseus Softwarehttp://maxquant.net/perseus/
Phosphate Buffered SalineVWRCA12001-676Puchase not required. PBS can also be prepared but sterile filteration must be performed before use.
Pierce BCA Protein AssayThermoFisher Scientific 23225
Pipette, Disposable Serological (10 mL)Fisher Scientific13-678-11E
Pipette, Disposable Serological (25 mL) BasixFisher Scientific14955235
Probe sonciatorThermoFisher Scientific100-132-894
Protease inhibitor cocktail tabletRoche4693159001
Sodium dodecyl sulfateThermoFisher Scientific2836420% (w/v)
Spectrophotometer (Nanodrop)ThermoFisher ScientificND-2000
STAGE tipping centrifugeSonationSTC-V2
Thermal ShakerVWRNO89232-908
Trifluoroacetic acidThermoFisher Scientific85183
Trypsin/Lys-C protease mix, MS gradePierceA40007Maintain at -20 °C.
Ultrasonic bathBransonicA89375-450Stored in cold room (4C)
UreaSigma AldrichU1250-1KGPrepare 40 mM HEPES/8 M Urea in bulk stock solution, flash frozen, store at -20 °C until use. Discard after each use (do not freeze-thaw repeatedly).
Yeast-extract peptone dextrose brothBD DifcoBM20

References

  1. Bongomin, F., Gago, S., Oladele, R., Denning, D. Global and Multi-National Prevalence of Fungal Diseases-Estimate Precision. Journal of Fungi. , (2017).
  2. Tugume, L., et al. HIV-Associated cryptococcal meni....

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