Qualitative and Quantitative Analysis of Siderophore Production from Pseudomonas aeruginosa

Summary

This protocol provides both qualitative and quantitative analyses of total siderophores, pyoverdine, and pyochelin from Pseudomonas aeruginosa.

Abstract

Pseudomonas aeruginosa (P. aeruginosa) is known for its production of a diverse range of virulence factors to establish infections in the host. One such mechanism is the scavenging of iron through siderophore production. P. aeruginosa produces two different siderophores: pyochelin, which has lower iron-chelating affinity, and pyoverdine, which has higher iron-chelating affinity. This report demonstrates that pyoverdine can be directly quantified from bacterial supernatants, while pyochelin needs to be extracted from supernatants before quantification.

The primary method for qualitatively analyzing siderophore production is the Chrome Azurol Sulfonate (CAS) agar plate assay. In this assay, the release of CAS dye from the Fe³⁺-Dye complex leads to a color change from blue to orange, indicating siderophore production. For the quantification of total siderophores, bacterial supernatants were mixed in equal proportions with CAS dye in a microtiter plate, followed by spectrophotometric analysis at 630 nm. Pyoverdine was directly quantified from the bacterial supernatant by mixing it in equal proportions with 50 mM Tris-HCl, followed by spectrophotometric analysis. A peak at 380 nm confirmed the presence of pyoverdine. As for Pyochelin, direct quantification from the bacterial supernatant was not possible, so it had to be extracted first. Subsequent spectrophotometric analysis revealed the presence of pyochelin, with a peak at 313 nm.

Introduction

Organisms require iron to perform various vital functions, such as electron transport and DNA replication¹. Pseudomonas aeruginosa, a Gram-negative opportunistic pathogen, is known to possess a variety of virulence factors to establish infection in the host, among which one mechanism is siderophore formation². During iron-depleting conditions, P. aeruginosa releases specialized molecules called siderophores, which quench iron from the surrounding environment. Siderophores chelate iron extracellularly, and the resulting ferric-siderophore complex is actively transported back to the cell³.

P. aeruginosa is known to produce two siderophores, pyoverdine and pyochelin. Pyoverdine is known to have a higher iron chelating affinity (1:1), whereas pyochelin is known to have a lesser iron chelating affinity (2:1)⁴. Pyochelin is also called a secondary siderophore because it has a lower iron chelating affinity⁵. The production and regulation of siderophores are actively controlled by Quorum Sensing (QS) systems in P. aeruginosa⁶.

Besides iron quenching, siderophores are also involved in regulating virulence factors and play an active role in biofilm formation⁷. Siderophores serve additional crucial roles, including involvement in cell signaling, defense against oxidative stress, and facilitation of interactions between microbial communities⁸. Siderophores are typically categorized based on the specific functional groups through which they chelate iron. The three primary bidentate ligands in this classification are catecholate, hydroxamate, and α-hydroxycarboxylate³. Pyoverdines are hallmarks of fluorescent Pseudomonas species such as P. aeruginosa and P. fluorescens⁵. They consist of a mixed green fluorescent chromophore coupled to an oligopeptide containing 6-12 amino acids. Several non-ribosomal peptide synthetases (NRPs) are involved in their synthesis⁹. Four genes involved in pyoverdine production and regulation are pvdL, pvdI, pvdJ, and pvdD¹⁰. Pyoverdine is also responsible for infection and virulence in mammals¹¹. P. aeruginosa is noted to produce pyochelin in moderate iron-limiting conditions, while pyoverdine is produced during severe iron-limiting environments¹². Two operons involved in pyochelin production are pchDCBA and pchEFGHI¹³. It is noted that in the presence of pyocyanin, pyochelin (catecholate) induces oxidative damage and inflammation and generates hydroxyl radicals, which are harmful to host tissues¹¹.

The Chrome Azurol Sulfonate (CAS) assay is widely adopted due to its comprehensiveness, high sensitivity, and greater convenience compared to microbiological assays, which, although sensitive, can be overly specific¹⁴. The CAS assay can be conducted on agar surfaces or in a solution. It relies on the color change that occurs when the ferric ion transitions from its intense blue complex to orange. The CAS colorimetric assay quantifies the depletion of iron from a Fe-CAS-surfactant ternary complex. This particular complex, consisting of metal, organic dye, and surfactant, has a blue color and exhibits an absorption peak at 630 nm.

This report presents a method for the qualitative detection of siderophore production, where one can detect the production of siderophores on an agar plate. A method for the quantitative estimation of total siderophore production in a microtiter plate and the detection and quantitative analysis of two siderophores, pyoverdine and pyochelin, from P. aeruginosa, is also provided.

Protocol

All bacterial isolates of P. aeruginosa were obtained from medical microbiology laboratories from Vadodara and Jaipur, India. All selected clinical isolates were handled in Biosafety Cabinet (BSL2) and utmost care was taken while handling bacterial isolates during the experiments. The commercial details of all the reagents/solutions are provided in the Table of Materials.

1. Preparation of Chrome Azurol Sulfonate (CAS) dye and agar media

1. Prepare CAS dye (100 mL) with the following composition:
   1. Dissolve 60 mg of CAS in 50 mL of distilled water (Solution 1).
   2. Dissolve 2.7 mg of FeCl₃·6H₂O in 10 mL of 10 mM HCl (Solution 2).
   3. Dissolve 73 mg of Cetrimonium bromide (HDTMA) in 40 mL of distilled water (Solution 3).
   4. Carefully mix Solution 1, Solution 2, and Solution 3. Store it in a glass bottle.
2. Prepare CAS agar following the steps below:
   1. Add 100 mL of MM9 salt solution to 750 mL of distilled water.
   2. Dissolve 32.24 g of piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES) free acid.
   3. Add 15 g of Agar agar. Autoclave and allow it to cool.
   4. Add 30 mL of sterile Casamino acid solution and 10 mL of sterile 20% glucose solution to the mixture.
   5. Add 100 mL of CAS dye, mix, and pour it in aseptic conditions.
      NOTE: Preparation of MM9 media, Casamino acid solution, and 8-Hydroxyquinoline are provided in Supplementary File 1. PIPES buffer is pH-sensitive. It will not dissolve until pH 5.6 is achieved. Ensure that the pH is constantly monitored because when PIPES starts to dissolve in water, it will further lower the pH. Extract Casamino acid with the same volume of 3% 8-hydroxyquinoline dissolved in chloroform. Leave the immiscible solution at 4 °C for around 20 min and carefully collect the upper phase of the solution without disturbing the lower phase.

2. Qualitative analysis of siderophores production

1. Set the OD₆₀₀ nm to 0.2 for 24 h grown cultures of P. aeruginosa.
2. Using a sterile wire loop, streak the bacterial culture on a CAS agar plate.
3. Incubate at 30 °C for 24 h.
   NOTE: Peptone water media or 0.8% normal saline can be used to dilute the bacterial culture. If no bacterial growth is observed at 24 h, incubate CAS plates for 48 to 72 h.

3. Quantitative estimation of total siderophores

1. Re-inoculate 24-h-grown cultures of P. aeruginosa in Peptone water media after adjusting OD₆₀₀ nm to 0.25, and incubate at 30 °C for 48 h.
2. After 48 h, centrifuge the bacterial culture at 4650 x g for 10 min at room temperature.
3. After centrifugation, add 100 µL of cell-free supernatant to a 96-well microtiter plate and add 100 µL of CAS dye to it.
4. Cover the plate with aluminum foil and incubate at room temperature for 20 min.
5. After incubation, take spectrophotometric readings at 630 nm.
6. Calculate the results obtained for the quantification of total siderophores as Percent Siderophore Unit (PSU).
   NOTE: PSU can be calculated as: [(A_r - A_s)/A_r] x 100
   where, A_r = absorbance of the reference at 630 nm, A_s = absorbance of the cell-free supernatant of the sample. For the reference, CAS dye should be added to uninoculated peptone water media. Fill all glassware, such as test tubes, flasks, etc., with 6 M HCl for 2 h and rinse them twice with distilled water to remove any trace of iron on them.

4. Quantitative estimation of pyoverdine

1. Re-inoculate 24-h-grown cultures of P. aeruginosa in Peptone water media after adjusting OD₆₀₀ nm to 0.25 and incubate at 30 °C for 48 h.
2. After 48 h, measure the OD₆₀₀ nm of the bacterial growth before proceeding further.
3. Centrifuge the bacterial culture at 4650 x g for 10 min at room temperature.
4. After centrifugation, add 100 µL of cell-free supernatant to a 96-well microtiter plate and add 100 µL of 50 mM Tris-HCl (pH 8.0) to it.
5. Take spectrophotometric readings at OD₄₀₅ nm.

5. Pyochelin extraction and spectrophotometry

1. Re-inoculate 24-h-grown cultures of P. aeruginosa in King's B media (Supplementary File 1) after adjusting OD₆₀₀ nm to 0.25 and incubate at 30 °C for 24 h.
2. After 24 h, take 100 mL of culture and centrifuge at 4650 x g for 10 min at room temperature.
3. After centrifugation, add 5 mL of 1 M citric acid to the supernatant. Extract twice with 50 mL of Ethyl acetate.
4. Filter the organic phase with magnesium sulfate through a syringe filter. Store the filtered organic phase at -20 °C.
5. Take spectrophotometric readings at 320 nm.
   NOTE: As pyochelin is a highly unstable compound at room temperature, perform the extraction process on ice. Use a 50 mL sterile syringe for filter separation. Place cotton at the tip of the syringe and add 1 gm of magnesium sulfate on it. Fix a sterile syringe filter at the tip of the syringe and collect the filtrate in a sterile tube.

Results

Before quantification of siderophores from clinical isolates, a qualitative screening for siderophore production was carried out to ensure siderophores production. Qualitative detection of siderophores from clinical isolates was observed by streaking bacteria on CAS agar plates. Three clinical isolates, namely MR1, TL7, J3, along with PAO1 (the reference strain), were selected for the study. All three clinical isolates and PAO1 showed positive results for siderophore production, where a c...

Discussion

This protocol enables researchers to quantitate total siderophores and two different siderophores of P. aeruginosa, namely pyoverdine and pyochelin, from the bacterial cell-free supernatant. In the CAS agar plates assay, CAS dye and Fe³⁺ ions form a complex. When bacteria produce siderophores, they quench Fe³⁺ ions from the CAS-Fe³⁺ complex, leading to a color change around the bacterial growth. This change results in a clear orange halo around the bacterial growth

Materials

Name	Company	Catalog Number	Comments
Agar Agar, Type I	HIMEDIA	GRM666
8-Hydroxyquinoline	Loba Chemie	4151
Casamino Acid	SRL Chemicals	68806
Cetyltrimethyl Ammonium Bromide (CTAB)	HIMEDIA	RM4867-100G
Chloroform	Merck	1070242521
Chrome azurol sulfonate	HIMEDIA	RM336-10G
Citric acid	Merck	100241
Dextrose monohydrate	Merck	108342
Dichloromethane	Merck	107020
Ferric chloride hexahydrate	HIMEDIA	GRM6353
Glass Flasks	Borosil	5100021
Glass Test-tubes	Borosil	9820U05
Hydrochloric acid	SDFCL	20125
King's medium B base	HIMEDIA	M1544-500G
M9 Minimal Medium Salts	HIMEDIA	G013-500G
Magnesium Sulphate	Qualigens	10034
MultiskanGO UV Spectrophotometer	Thermo Scientific	51119200
Peptone Type I, Bacteriological	HIMEDIA	RM667-500G
PIPES free acid	MP Biomedicals	190257
Potassium dihydrogen phosphate	Merck	1048731000
Proteose peptone	HIMEDIA	RM005-500G
Shimadzu UV-Vis Spectrophotometer	Shimadzu	2072310058
Sigma Laborzentrifuge	Sigma-Aldrich	3-18K
Sodium chloride	Qualigens	15915