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Demonstrate

Effect of Nor-Pyochelin, Pyochelin and Desferrithiocin complexed with gallium on P. aeruginosa and Cystic Fibrosis Bronchial Epithelial cells

Determination of the Minimal Inhibitory Concentration
In the following the three substances Nor-Pyochelin, Pyochelin and Desferrithiocin will be named siderophores in order to enhance readability.

To analyse the effect of our siderophores and siderophore-analogs we conducted several Minimal Inhibitory Concentration (MIC) assays.

For this purpose, we prepared dilution series of the siderophores complexed with gallium overnight (Ga-complex), siderophores with gallium added right before (+Ga(NO3)3) and siderophores without gallium. For the first MIC assay the siderophores and gallium were mixed in a ratio of 2:1. The dilution series ranged from 50 µM to 0.195 µM.
The siderophores were solvated in absolute ethanol. In order to be able to differentiate between the toxic effect of the ethanol and the toxic effect of the siderophores, a dilution series of ethanol was prepared that contained the same amount of ethanol as the siderophore solutions.
In parallel we used a gallium dilution series to show that gallium alone has not the same effect compared to a complex with the siderophore.
As positive control we used two wells with bacteria only and no additional substances and as a negative control two wells with pure medium.
These controls were carried out in all following MIC assays.

To keep the assay as standardized as possible we used Müller Hinton medium (Roth) provided by the Heisig research group, which is the standard medium for MIC assays. In the following the pipetting scheme and a picture of the first MIC assay are shown.

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Figure 1: Pipetting scheme MIC of P. aeruginosa with siderophore-gallium-complexes (2:1) and siderophores in Müller Hinton medium. Pipetting errors (cells were either accidentally added or forgotten) are marked in blue.
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Figure 2: MIC of P. aeruginosa with siderophore-gallium-complexes (2:1) and siderophores in Müller Hinton medium after 16 hours of incubation at 37 °C.

The MIC is indicated with 12.5 µM in the literature. Therefore we expected our MIC at the same range.
The results from literature could not be replicated, although a slightly inhibitory effect is visible with the gallium complexed siderophores. After a whole day of trouble shooting it was found that the missing effect is probably based on the fact that the Müller Hinton medium contains beef infusion which also contains bovine serum. The serum contains a protein called lipocalin 2 which binds siderophores (like yersiniabactin) and is also a part of human serum to fend off bacterial infections. [1]

The next MIC was conducted with the same dilution series but in LB medium. The pipetting scheme and the picture are shown below.

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Figure 3: Pipetting scheme MIC of P.aeruginosa with siderophore-gallium-complexes (2:1) and siderophores in L.B. medium.
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Figure 4: MIC of P.aeruginosa with siderophore-gallium-complexes (2:1) and siderophores in L.B. medium after 16 hours incubation at 37 °C.

The MIC conducted in L.B. medium did not show the expected effect either. After several discussions it was found that a miscommunication had occurred referring to the ratio of siderophores and gallium ions during the binding process. The used complexes were formed with a ratio of 2:1 (siderophores: gallium). This ratio is wrong and in the following MIC - assays the correct ratio of 1:1 siderophores to gallium was used. With free siderophores in iron-containing L.B. medium the iron supply was warranted for P.aeruginosa.

After eliminating this mistake, the MIC was repeated with new, correctly built gallium-siderophore-complexes and a different dilution series ranging from 400 µM to 1.563 µM. The dilution series of the gallium complexes were parallel duplicated on a second 96-well plate and two new concentrations between 12 µM and 6.25 µM were added. In the following the new pipetting schemes and the associated pictures are shown.

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Figure 5: Pipetting scheme MIC of P.aeruginosa with siderophore-gallium-complexes (1:1) and siderophores in L.B. medium.
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Figure 6: MIC of P.aeruginosa with siderophore-gallium-complexes (1:1) and siderophores in L.B. medium after 16 hours incubation at 37 °C.
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Figure 7: Pipetting scheme MIC of P.aeruginosa with siderophore-gallium-complexes (1:1) and siderophores in L.B. medium.
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Figure 8: MIC of P.aeruginosa with siderophore-gallium-complexes (1:1) and siderophores in L.B. medium after 16 hours incubation at 37 °C.

In this assays the siderophore-gallium-complexes show no significant effect on the cell growth. One explanation could be that the L.B. medium contains enough iron for P.aeruginosa to maintain their iron uptake so that the bacteria do not depend on gallium loaded siderophores.

To eliminate that source of trouble it would be necessary to conduct the MIC-assays in an iron free medium or a medium that contains iron concentrations comparable to these in physiological, infectious conditions. For lack of such media and the lack of time to order those, the MIC was conducted again in M9 (minimal medium) and for completeness replicated in L.B. medium.

The pipetting schemes and pictures of the assays are shown in the following.

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Figure 9: Pipetting scheme MIC of P.aeruginosa with siderophore-gallium-complexes (1:1) and siderophores in L.B. medium.
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Figure 10: MIC of P.aeruginosa with siderophore-gallium-complexes (1:1) and siderophores in L.B. medium after 16 hours incubation at 37 °C.
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Figure 11: Pipetting scheme MIC of P.aeruginosa with siderophore-gallium-complexes (1:1) and siderophores in L.B. medium.
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12: MIC of P.aeruginosa with siderophore-gallium-complexes (1:1) and siderophores in L.B. medium after 16 hours incubation at 37 °C.

As it was observed before, the assays conducted in L.B. medium did not show any visible effect on the bacterial growth.

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Figure 13: Pipetting scheme MIC of P.aeruginosa with siderophore-gallium-complexes (1:1) and siderophores in M9 medium. Pipetting errors (no cells were added or accidentally added) are marked in blue).
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Figure 14: MIC of P.aeruginosa with siderophore-gallium-complexes (1:1) and siderophores in M9 medium after 16 hours incubation at 37 °C.
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Figure 15: Pipetting scheme MIC of P.aeruginosa with siderophore-gallium-complexes (1:1) and siderophores in M9 medium. Pipetting errors (no cells were added or accidentally added) are marked in blue).
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Figure 16: MIC of P.aeruginosa with siderophore-gallium-complexes (1:1) and siderophores in M9 medium after 16 hours incubation at 37 °C.

In the minimal M9 medium an inhibiting effect is visible, but strongly relativized by the ethanol control dilution. In figure 14 and 16 can be observed that nor-pyochelin-gallium-complexes have no significant inhibitory effect on cell growth. In contrast the pyochelin-gallium complex inhibits the cell growth at a concentration of 50 µM (figure 14) but this concentration cannot be determined as MIC that easily. Therefore the assay has to be replicated several times.

The main reason for the low effect is probably the iron-containing medium the assays were conducted in. Another reason could be that the chemical synthesis also produced stereoisomeres which probably are not as effective.

MTT-Assay to determine the toxicity of siderophore-Gallium-complexes on human lung epithelial cells

The MTT-assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) is a standard method to quantify the viability of cells. The yellow MTT is uptaken by the cells and metabolized in the mitochondria. The metabolized product forms blue crystals which are no longer able the cross the cell membrane. The production of blue crystals is directly proportional for the cell vitality.

To test the toxicology of the siderophores Cystic Fibrosis Bronchial Epithelial (CFBE) cells were used because in case of treating aP.aeruginosa infection, the application site of siderophores would be the respiratory tract.

The cells were incubated 24 hours with the siderophores following pipette schemes below.

The cells were incubated 24 hours with the siderophores.

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Figure 17: MIC of P.aeruginosa with siderophore-gallium-complexes (1:1) and siderophores in M9 medium after 16 hours incubation at 37 °C.
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Figure 18: MIC of P.aeruginosa with siderophore-gallium-complexes (1:1) and siderophores in M9 medium after 16 hours incubation at 37 °C.

After adding the MTT solution and incubation for three hours the cells were lysed and the blue crystals were dissolved. Then the absorption was measured in an ELISA plate reader at a measuring wavelength of 𝜆 = 550 nm and a reference wavelength of 𝜆 = 690 nm.

The ratio between the intensity at both wavelengths was calculated and is directly proportional to the cell vitality. The double determination was averaged and the resulting data is shown in the following figure.

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Figure 19: Cell vitality of the CFBE cells after 24 h incubation with the siderophore-Ga-complexes and the corresponding EtOH concentration as control.

The MTT-assay shows that compared to the EtOH control the siderophores have a much lower toxic effect on the CFBE cells. The assay should be replicated several times, also with higher concentrations. Regarding this results it can be said that the toxic effect is due to the alcohol the siderophores are dissolved in and not to the siderophores themselves.

Sources:

  • [1]Ahmed, M., Brode, E., Brown, T., Eltoweissy, S., Gross, S., Markowitz, S., … Woodard, B. (n.d.). Effects of Gallium-Desferrioxamine Compounds on Bacteria.


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