Quantifying Yersinia pseudotuberculosis Type III Secretion System Activity Following Iron Starvation and Anaerobic Growth

Summary

Bacteria colonize host tissues that vary in oxygen and iron bioavailability, yet most approaches to studying bacteria use aerated, rich media. This protocol describes culturing the human pathogen Yersinia pseudotuberculosis under varying iron concentrations and oxygen tension, and quantifying activity of the Yersinia type III secretion system, which is an important virulence factor.

Abstract

A key virulence mechanism for many Gram-negative pathogens is the type III secretion system (T3SS), a needle-like appendage that translocates cytotoxic or immunomodulatory effector proteins into host cells. The T3SS is a target for antimicrobial discovery campaigns since it is accessible extracellularly and largely absent from non-pathogenic bacteria. Recent studies demonstrated that the T3SS of Yersinia and Salmonella are regulated by factors responsive to iron and oxygen, which are important niche-specific signals encountered during mammalian infection. Described here is a method for iron starvation of Yersinia pseudotuberculosis, with subsequent optional supplementation of inorganic iron. To assess the impact of oxygen availability, this iron starvation process is demonstrated under both aerobic and anaerobic conditions. Finally, incubating the cultures at the mammalian host temperature of 37 °C induces T3SS expression and allows quantification of Yersinia T3SS activity by visualizing effector proteins released into the supernatant. The steps detailed here offer an advantage over the use of iron chelators in the absence of iron starvation, which is insufficient for inducing robust iron starvation, presumably due to efficient Yersinia iron uptake and scavenging systems. Likewise, acid-washing laboratory glassware is detailed to ensure the removal of residual iron, which is essential for inducing robust iron starvation. Additionally, using a chelating agent is described to remove residual iron from media, and culturing the bacteria for several generations in the absence of iron to deplete bacterial iron stores. By incorporating standard protocols of trichloroacetic acid-induced protein precipitation, SDS-PAGE, and silver staining, this procedure demonstrates accessible ways to measure T3SS activity. While this procedure is optimized for Y. pseudotuberculosis, it offers a framework for studies in pathogens with similar robust iron uptake systems. In the age of antibiotic resistance, these methods can be expanded to assess the efficacy of antimicrobial compounds targeting the T3SS under host-relevant conditions.

Introduction

Many clinically relevant Gram-negative pathogens like Yersinia, Vibrio, Escherichia, Pseudomonas, and Shigella encode the type III secretion system (T3SS) to inject effector proteins into host cells¹. In many bacterial species, the T3SS is under strict regulatory control². For example, translocation of Yersinia T3SS effector proteins into target host cells is critical to subvert host defense mechanisms and enable bacterial colonization of host tissues. However, Yersinia T3SS activity is metabolically burdensome and can trigger recognition by host immune receptors³. Accordingly, regulators that sense specific environmental cues control the expression of T3SS genes in many bacterial species. As pathogens such as Yersinia experience environmental changes during their infection cycle that impact the expression of critical virulence factors, it is important to develop laboratory conditions that mimic salient features of host niches occupied by bacterial pathogens. Specifically, oxygen tension and iron availability differ among various tissue sites in a spatiotemporal manner and impact the expression of virulence genes such as the T3SS^4,5,6. Therefore, the goal of this method is to assess how oxygen and iron impact the expression of the Yersinia T3SS. This will provide insight into the dynamics of the host-pathogen interaction.

The method described here details how to culture Yersinia pseudotuberculosis aerobically and anaerobically, as well as how to deplete Yersinia iron stores during aerobic or anaerobic growth. There are a few important considerations highlighted here regarding successfully culturing bacteria under these variable conditions. First, anaerobic culturing requires additional glucose supplementation, a modification that is noted in the media recipe. Second, since Y. pseudotuberculosis employs siderophores and other iron uptake systems that can robustly scavenge iron from the environment, special attention is devoted to ensuring the culture media and laboratory glassware are as free of iron as possible⁷. Previous studies have used iron chelators such as dipyridyl to deplete iron from rich-media bacterial cultures to mimic iron starvation^8,9. However, depleting Yersinia iron stores to induce iron starvation requires the removal of residual iron in glassware and media as well as prolonged growth in the absence of iron. This protocol details how to acid wash glassware and chelate media to remove residual iron, in addition to culturing the bacteria for several generations to ensure thorough iron starvation. Iron starvation can be ensured by measuring relative transcript levels of well-characterized iron-responsive genes across conditions, as demonstrated here with yfeA, and bfd.

The culmination of this protocol demonstrates how to precipitate secreted T3SS effector proteins from each of these conditions by treating the culture supernatant with trichloroacetic acid (TCA) and visualizing secreted proteins through SDS-PAGE. Finally, relative T3SS activity is assessed by visualizing secreted proteins via silver staining and quantifying relative levels of T3SS effector proteins, referred to as Yersinia outer proteins (Yops)¹⁰.

T3SS activity assays generally utilize specific antibodies to detect T3SS effector protein levels in the culture supernatant. However, western blotting antibodies for T3SS effector proteins are often not commercially available. Therefore, special attention has been taken to ensure that the final visualization of T3SS activity in this method does not require specific antibodies, and instead can leverage silver staining, which allows for the visualization of all secreted proteins. While this method is specifically tailored and optimized for Y. pseudotuberculosis, it can be adapted to other bacterial species, though the exact media conditions and incubation times will vary.

Protocol

The details of the reagents, media composition, primer sequences, and equipment are listed in the Table of Materials. Figure 1 illustrates the overall experimental workflow.

1. Preparation of acid washed glassware and chelated M9 media

NOTE: Before starting, refer to the material section for the exact reagents and recipes that will be used. M9 media was first used for Yersinia T3SS assays in Cheng et al.¹¹.

1. Pour 100 mL of 6 N HCl into a 250 mL glass flask.
   CAUTION: HCl is extremely hazardous; ensure appropriate precautions are taken when handling this substance.
2. Seal the mouth of the glass container with a glass stopper and carefully swirl the flask for 1 min to distribute the HCl thoroughly, occasionally changing directions.
3. Dispose of the 6 N HCl appropriately.
4. Rinse the glass container by adding 100 mL of deionized H₂O, sealing the mouth, shaking for 20 s, then dumping the water.
5. Repeat the rinsing step for a total of 3 times.
6. Let air dry. Autoclave to sterilize.
7. To chelate media, mix M9 components together with the chelating reagent and stir with a magnetic stir bar at room temperature for ~18 h. Filter-sterilize and add MgSO₄ to a final concentration of 1 mM.

2. Culturing Y. pseudotuberculosis under varying iron levels and oxygen tension

1. Streak Y. pseudotuberculosis (strain IP2666pIB1 is used in this study) on Lysogeny Broth (LB) agar plates and incubate at room temperature.
   NOTE: Incubating Yersinia at 37 °C, particularly in low calcium medium, leads to T3SS activity, growth arrest, and ultimately selects for loss of the plasmid for Yersinia virulence (pYV) that encodes the T3SS¹². Therefore, this protocol uses 26 °C incubation of Yersinia until T3SS activity needs to be measured.
2. After 48 h, once visible colonies form, inoculate 4 mL of M9 media containing 0.2% glucose, 1 mg/L FeSO₄.7H₂O, and supplemented with casamino acids (referred to here as M9 media) from a single, isolated colony and culture overnight at 26 °C with aeration at 250 rpm for approximately 18 h.
   NOTE: Use acid-washed glassware starting with the following step and moving on.
3. Subculture into chelated M9 media following the steps below.
   NOTE: For all subsequent steps, M9 media with 0.9% glucose is used rather than the standard 0.2% glucose.
   1. Using a spectrophotometer, measure the optical density (OD₆₀₀) of the overnight culture.
   2. Dilute the overnight culture to an OD₆₀₀ value of 0.1 in a total of 14 mL of sterile chelated M9 media containing 0.9% glucose and no FeSO₄.7H₂O in a 250 mL acid-washed flask with no iron supplementation.
   3. Incubate for 8 h at 26 °C with aeration at 250 rpm.
4. Subculture into aerobic and anaerobic cultures in parallel.
   1. Measure the OD₆₀₀ of the growing cultures.
   2. For continuing aerobic incubation, subculture the growing cultures to an OD₆₀₀ value of 0.1 in 14 mL of sterile chelated M9 media containing 0.9% glucose and no FeSO₄.7H₂O in a 250 mL acid-washed flask and culture with aeration at 26 °C for 12 h with no iron supplementation.
   3. For anaerobic culturing, subculture to an OD₆₀₀ value of 0.1 in 14 mL of sterile chelated M9 media into two acid-washed glass tubes. Into the first tube, add filter-sterilized (using 0.22 µm polyethersulfone membrane) FeSO₄.7H₂O for a final concentration of 1 mg/L (referred to as "high iron"). In the second tube, add FeSO₄.7H₂O for a final concentration of 0.01 mg/L (referred to as "low iron"). This can be done by using a 10 mg/mL FeSO₄.7H₂O stock solution. Incubate both tubes anaerobically in an anaerobic chamber at room temperature for 12 h.
      NOTE: 1 mg/L FeSO₄.7H₂O provides iron-replete conditions for robust Yersinia growth. Conversely, adding only 0.01 mg/L FeSO₄.7H₂O to anaerobic cultures ensures sufficient growth under anaerobic conditions but still enables iron starvation responses, whereas aerobic cultures are grown for 12 h in the absence of any added iron to stimulate iron starvation responses prior to stimulation of T3SS activity.
5. Induce type III secretion system activity.
   1. Shift the anaerobic cultures to 37 °C and continue anaerobic incubation for 4 h to induce the T3SS.
   2. Measure the OD₆₀₀ of the aerobically growing culture.
   3. Into two acid-washed flasks, subculture the aerobically growing cultures to an OD₆₀₀ value of 0.2 in 14 mL of sterile chelated M9 media. Into the first flask, add filter-sterilized FeSO₄.7H₂O for a final concentration of 1 mg/L. Into the second flask, add FeSO₄.7H₂O for a final concentration of 0.01 mg/L. Incubate both flasks with aeration at 26 °C at 250 rpm for 2 h.
   4. After 2 h, shift the aerobic cultures to 37 °C with aeration at 250 rpm and incubate for 4 h to induce the T3SS.

3. Trichloroacetic acid (TCA) precipitation of T3SS effector proteins

1. Once the anaerobic incubation is complete, measure the OD₆₀₀ of the cultures.
2. Normalize all anaerobic samples to achieve equal cell mass (refer to the example provided in Table 1). For anaerobic culturing, generally expect an OD₆₀₀ value of ~0.5 in iron-starved cultures and ~1 for iron-replete cultures. In this case, use 6 mL of the iron-starved cultures and 3 mL of the iron-replete cultures.
   NOTE: The remaining culture can be used for other purposes, such as harvesting total RNA and using quantitative PCR to measure steady-state levels of target mRNA (not described here).
3. Transfer the normalized volumes of each culture into 15 mL tubes.
4. To control for the efficiency of secreted protein precipitation, add 4 µL of 0.5 mg/mL bovine serum albumin (BSA) into each tube.
5. Pellet cultures at 3200 x g for 15 min at 4 °C.
6. Attach a 0.22 µm PVDF filter to a 10 mL syringe, and filter the supernatant of each pelleted culture into a fresh 15 mL tube.
   NOTE: This filtration step minimizes the chances of transferring whole bacterial cells, which would result in unwanted cytoplasmic proteins in the sample.
7. Add 10% of the supernatant volume of 6.1 N TCA to each sample.
8. Vortex vigorously for 1 min. Incubate tubes on ice in a 4 ˚C cold room overnight.
   NOTE: The duration of incubation can be optimized based on need. As little as a 1 h incubation can work for conditions or strains where secretion is robust.
9. Repeat the pellet collection and TCA precipitation steps with the aerobic cultures once the 4 h incubation is complete.
   NOTE: Normalize the aerobic cultures by pelleting volumes with equivalent cell mass.
10. Add 2 mL of each sample to a fresh 2 mL tube and centrifuge at 21,000 x g for 15 min at 4 °C.
11. Aspirate supernatant using a vacuum attachment, and be careful not to touch the bottom or sides of the tube.
    NOTE: The pellet can be difficult to visualize and may appear as a haze along the length of the tube.
12. Repeat steps 3.10 and 3.11 until all contents from each precipitation reaction are precipitated into a single 2 mL tube. This may take three or four sequential centrifugation steps but allows the concentration of all secreted proteins from each sample into a 2 mL tube.
13. To wash the pellets, gently add 1 mL of ice-cold 100% acetone into each tube. To avoid sample loss, do not resuspend and do not touch the sides of the tube.
14. Centrifuge the samples at 21,000 x g for 15 min at 4 °C.
15. Aspirate the supernatant and be careful not to touch the bottom or sides of the tube.
16. Repeat ice-cold acetone washing steps 3.13 and 3.14.
17. After aspirating the supernatant for the final time, open the tubes and allow the pellet to dry completely on the bench for approximately 1 h.
18. Add 50 µL of the FSB: DTT solution to each dried sample. To avoid protein loss, do not resuspend.
19. Thoroughly vortex each sample for 1 min, ensuring the FSB: DTT solution coats the walls of the entire tube. This can be optimized by using a vortex attachment capable of holding multiple tubes at once.
20. Boil the samples at 95 °C for 15 min.
21. Centrifuge the samples briefly for 30 s at the maximum speed at room temperature.
22. Store at -80 °C until future use.

4. SDS-PAGE and silver staining to visualize T3SS effector proteins

1. Load 15 µL of each anaerobic sample and 10 µL of each aerobic sample into a 12.5% SDS-PAGE gel, along with 3 µL of a commercially available unstained standard as the ladder.
2. Run the gel for about 90 min at 100 V. These settings can vary depending on the apparatus used.
3. Follow the silver staining protocol following the manufacturer's instructions, resulting in a gel, as shown in Figure 2.

5. Quantifying relative T3SS activity

1. Position the gel into the imaging system, ensuring all the bands intended for quantification are in the image frame.
   NOTE: The molecular weight of BSA is ~66 kDa, while the molecular weight of the T3SS effector protein YopE is ~23 kDa.
2. In the software, select all the YopE bands across all wells.
3. Set the reference YopE band to the appropriate sample.
4. Export the relative quantification values calculated by the software for all the YopE bands.
5. Going back into the software, select all the BSA bands across all wells.
6. Set the reference BSA band to ensure it is from the same sample as the reference YopE band.
7. Export the relative quantification values calculated by the software for all the BSA bands.
8. To calculate relative YopE expression levels, divide the YopE relative quantity value by the BSA relative quantity value for each sample.

Results

This method allows for the relative comparison of secreted Yops across various conditions relative to a reference condition of interest. The overall experimental workflow is depicted in Figure 1. Table 1 depicts a representation of how cell culture normalization would typically occur in the instance of each culture condition and the volume of TCA that would be added to each supernatant. Here, representative results are shown using wildtype (WT) Y. pseudotuberculosis...

Discussion

The T3SS is an important virulence factor in many pathogenic bacteria; therefore, developing laboratory techniques to study its regulation is important for understanding pathogenesis and developing potential therapeutics¹. Iron and oxygen are known to be important host cues sensed by bacterial pathogens to regulate T3SS expression⁵; therefore, this method presents a strategy for culturing Y. pseudotuberculosis under either anaerobic or aerobic conditions, with iron...

Materials

Name	Company	Catalog Number	Comments
10 mL Luer-Lok Tip syringe	BD	301029
10x SDS Running Buffer	Home made		0.25 M Tris base, 1.92 M Glycine, 1% SDS in 1 L volume
12.5% SDS-Page Gel	Home made
15 mL culture tubes	Falcon	352059	For initial overnight
15 mL Falcon tubes	Falcom	352196	For supernatant collection
250 mL culture flask	Belco	251000250
500 mL Filter System	Corning	431097
6 N Hydrochloric acid solution	Fisher Scientific	7732185
Acetone	Fisher Chemical	A949-4	4 L
Bio Rad ChemiDoc MP Imaging System	Bio Rad	Model Number: Universal Hood III
Borosilicate glass culture tubes	Fisherbrand	14-961-34	For anaerobic culturing
Chelex 100 Resin	Bio Rad	142-1253
Chelex M9 +0.9% Glucose media	Home made		6 g/L Na2HPO4, 3 g/L KH2PO4, 0.5 g/L NaCl, 1 g/L NH4Cl, 1% casamino acids, 0.9% dextrose, 0.0005% thiamine, 5 g/L Chelex 100 Resin. Stir media for 18 h at room temp, filter using 500 mL Corning filtration unit, then add MgSO4 for 1 mM MgSO4 final solution
Final Sample Buffer (FSB)	Home made		0.1 M Tris-HCl, 4% SDS, 20% glycerol, 0.2% of Bromophenol Blue
FSB:DTT solution	Home made		FSB+0.2M DTT
Image Lab Software	Bio Rad	https://www.bio-rad.com/en-us/product/image-lab-software?ID=KRE6P5E8Z	Software
Isotemp Heat Block	Fisher Scientific	88860021
LB Agar Plates	Home made		10 g Tryptone, 5 g Yeast extract, 10 g NaCl, 15 g Agar in 1 L total volume. Autoclaved
M9+0.2% Glucose Media	Home made		6 g/L Na2HPO4, 3 g/L KH2PO4, 0.5 g/L NaCl, 1 g/L NH4Cl, 1 mM MgSO4, 1 mg/L FeSO47H2O, 1% casamino acids, 0.2% dextrose, 0.0005% thiamine
Millex-GP PES 0.22um filter attachment for syringe	Millipore	SLGPR33RS	For FeSO47H2O filtration
Millex-GV PVDF 0.22um filter attachment for syringe	Millipore	SLGVR33RS	For supernatant filtration
Precision Plus Protein Unstained Standard	Bio Rad	1610363
SDS-PAGE Gel Apparatus	Bio Rad	Model Number: Mini PROTEAN Tetra Cell
SilverXpress Silver Staining Kit	Invitrogen	LC6100
The BellyDancer Shaker	IBI Scientific	BDRAA1155
Trichloroacetic acid solution 6.1N	Sigma Aldrich	T0699
Vinyl Anaerobic Chamber	Coy Lab Products		https://coylab.com/products/anaerobic-chambers/vinyl-anaerobic-chambers/#details
qPCR Primer sequences
yfeA forward - CAC AGT CAG CAG ACC TTA TCT T
yfeA reverse - GGC AGA CGG GAC ATC TTT AAT A
bfd forward - ccagcatcagccccatacag
bfd reverse - tggcttgtcggatgcacttc
yopE forward - CCATAAACCGGTGGTGAC
yopE reverse - CTTGGCATTGAGTGATACTG