A Tandem Liquid Chromatography–Mass Spectrometry-based Approach for Metabolite Analysis of Staphylococcus aureus

Summary

Here we describe a protocol for the extraction of metabolites from Staphylococcus aureus and their subsequent analysis via liquid chromatography and mass spectrometry.

Abstract

In an effort to thwart bacterial pathogens, hosts often limit the availability of nutrients at the site of infection. This limitation can alter the abundances of key metabolites to which regulatory factors respond, adjusting cellular metabolism. In recent years, a number of proteins and RNA have emerged as important regulators of virulence gene expression. For example, the CodY protein responds to levels of branched-chain amino acids and GTP and is widely conserved in low G+C Gram-positive bacteria. As a global regulator in Staphylococcus aureus, CodY controls the expression of dozens of virulence and metabolic genes. We hypothesize that S. aureus uses CodY, in part, to alter its metabolic state in an effort to adapt to nutrient-limiting conditions potentially encountered in the host environment. This manuscript describes a method for extracting and analyzing metabolites from S. aureus using liquid chromatography coupled with mass spectrometry, a protocol that was developed to test this hypothesis. The method also highlights best practices that will ensure rigor and reproducibility, such as maintaining biological steady state and constant aeration without the use of continuous chemostat cultures. Relative to the USA200 methicillin-susceptible S. aureus isolate UAMS-1 parental strain, the isogenic codY mutant exhibited significant increases in amino acids derived from aspartate (e.g., threonine and isoleucine) and decreases in their precursors (e.g., aspartate and O-acetylhomoserine). These findings correlate well with transcriptional data obtained with RNA-seq analysis: genes in these pathways were up-regulated between 10- and 800-fold in the codY null mutant. Coupling global analyses of the transcriptome and the metabolome can reveal how bacteria alter their metabolism when faced with environmental or nutritional stress, providing potential insight into the physiological changes associated with nutrient depletion experienced during infection. Such discoveries may pave the way for the development of novel anti-infectives and therapeutics.

Introduction

Bacterial pathogens must contend with many challenges within the host environment. In addition to direct attack by immune cells, the host also sequesters nutrients essential for bacterial survival and replication, generating nutritional immunity^1,2. To survive these hostile environments, bacterial pathogens deploy virulence factors. Some of these factors allow the bacteria to evade the immune response; other factors include secreted digestive enzymes, such as hyaluronidase, thermonuclease, and lipase, which may enable the bacteria to replenish missing nutrients by consuming tissue-derived constituents

Protocol

1. Preparation of Buffer Solutions

1. Prepare phosphate-buffered saline (PBS; pH 7.4) by diluting a stock solution of 10x PBS to a final concentration of 1x with ultrapure (distilled and deionized) water.
2. Prepare quenching solution by combining 2 mL of acetonitrile, 2 mL of methanol, 1 mL of ultrapure H₂O, and 19 µL (0.1 mM final concentration) of formic acid.
3. Prepare LC-MS solvent A by adding formic acid (0.2% [v/v] final concentration) to ultrapure water.
4. Prepare LC-MS solvent B by adding formic acid (0.2% [v/v] final concentration) to acetonitrile.
   NOTE: All solutions should be prepared using the highest....

Results

We have analyzed intracellular metabolite pools in S. aureus during in vitro growth in a rich, complex medium. As proof of principle, we compared metabolite profiles between the methicillin-susceptible S. aureus osteomyelitis isolate UAMS-1 (wild-type [WT]) and an isogenic strain lacking the global transcriptional regulator CodY (ΔcodY)²⁶. Steady-state, exponential cultures of the WT and codY strains were established in .......

Discussion

All small-molecule metabolites are connected to one another through their common origins in central metabolic pathways. During exponential growth, bacterial cells are at biological and metabolic steady state, providing a snapshot of the physiological state under specific conditions. CodY monitors nutrient sufficiency by responding to ILV and GTP. As ILV and GTP pools drop, CodY activity is likely progressively reduced, adjusting the expression of its target genes to adapt to increasing nutrient depletion

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Material/Equipmenta
DeLong Culture Flask (250 mL)	Belco	2510-00250
Sidearm Flask, 500 mL	Pyrex	5340
3-hole Rubber Stopper, #7	Fisher	14-131E
Stainless Steel Filter holder/frit	VWR	89428-936
Petri Dish, 35 mm	Corning	430588	Not tissue culture treated
Mixed cellulose ester membrane, 0.22 μm pore size	Millipore	GSWP02500
Impact-resistant tubes, 2 mL	USA Scientific	1420-9600
Silica Beads, 0.1 mm	Biospec Products Inc	11079101Z
Precellys 24 homogenizer	Bertin Instruments	EQ03119-200-RD000.0
Micro BCA Protein Assay Kit	Pierce (Thermo Scientific)	23235
Cogent Diamond hydride type C column	Agilent	70000-15P-2
Accurate-Mass Time-of-Flight (TOF) LC-MS, 6200 Series	Agilent	G6230B
Quat Pump, 1290 Series	Agilent	G4204A
Bin Pump, 1290 Series	Agilent	G4220A
Valve Drive, 1290 Series	Agilent	G1107A
Isocratic Pump, 1290 Series	Agilent	G1310B
TCC, 1290 Series	Agilent	G1316C
Sampler, 1290 Series	Agilent	G4226A
Thermostat, 1290 Series	Agilent	G1330B
Chemical
Tryptic Soy Broth	Becton Dickinson	211825
Difco Agar, Granulated	Becton Dickinson	214530	Solid media contains 1.5% [w/v] agar
Phosphate-buffered saline (pH 7.4) 10x	Ambion	AM9624	Dilute fresh to 1x with ultra-pure water
Acetonitrile	Fisher Scientific	A955-500	Optima LC-MS
Methanol	Fisher Scientific	A456-500	Optima LC-MS; toxic
Formic Acid	Sigma Aldrich	94318	For mass spectrometry, 98%
Software
MassHunter	Agilent	G3337AA
Bacterial Strain	Species	Strain	Genotype
SRB 337	Staphylococcus aureus	USA200 MSSA UAMS-1	wild type
SRB 372	Staphylococcus aureus	USA200 MSSA UAMS-1	ΔcodY::erm
aChemicals and materials listed are specific to the method described and do not include standard laboratory chemicals or supplies.