As you mentioned, to me it appears that Zn ions are binding to the -ve charged peptides, thereby increasing its antimycobacterial potency. One of the mechanisms could be that binding of Zn ions indicuces conformational change which makes the peptide more accessible to cross the membrane. If the peptide is working at the membrane then the its possible that Zn bound peptide my have better interaction with membrane lipids/proteins thereby causing greater destablization.

There could be several ways to test this hypothesis

1. Do a dose dependent Zn study and include Zn alone in control wells. In other words you will have wells with peptide alone, Zn alone and peptide+zn

2. Are there Cysteine or Histidine in your peptides. These amino acids are known to cor-ordinate Zn.

3. I would also test other divalent cations such as Mg++, Ca++, Cu++, etc to know if the phenomenon is Zn dependent or any divalet cation would do the job.

4. If you have Cys/His in you peptide try replacing that with other aminoacid (may be Ala). Don't replace all of them in one go (if you have more than one Cys/His)

5. Test if Zn ions bind to peptide. There are several ways to do this. Let me know if you are interested doing this. I can suggest several approach to do this.

Yes, cells have a kind of Zinc uptake mechanism which increased your peptide uptake in H37Ra cells. I can go for Zn as antibacterial agent but in case that it was Zn nanoparticles, they have unknown mechanism of antibacterial effect.

It may be due to synergetic effect of both caused increased inhibition...

After posting the question, synergy also crossed my mind. I guess a synergistic study will be in order? Using the concentrations in various combinations?

Although the mechanism is not known, we found that zinc is needed for some anionic antimicrobial peptides to work ( Brogden KA, De Lucca AJ, Bland J, Elliott S. Isolation of an ovine pulmonary surfactant-associated anionic peptide bactericidal for Pasteurella haemolytica. Proc Natl Acad Sci U S A 1996;93(1):412-6. PubMed PMID: 8552650).

As you mentioned, to me it appears that Zn ions are binding to the -ve charged peptides, thereby increasing its antimycobacterial potency. One of the mechanisms could be that binding of Zn ions indicuces conformational change which makes the peptide more accessible to cross the membrane. If the peptide is working at the membrane then the its possible that Zn bound peptide my have better interaction with membrane lipids/proteins thereby causing greater destablization.

There could be several ways to test this hypothesis

1. Do a dose dependent Zn study and include Zn alone in control wells. In other words you will have wells with peptide alone, Zn alone and peptide+zn

2. Are there Cysteine or Histidine in your peptides. These amino acids are known to cor-ordinate Zn.

3. I would also test other divalent cations such as Mg++, Ca++, Cu++, etc to know if the phenomenon is Zn dependent or any divalet cation would do the job.

4. If you have Cys/His in you peptide try replacing that with other aminoacid (may be Ala). Don't replace all of them in one go (if you have more than one Cys/His)

5. Test if Zn ions bind to peptide. There are several ways to do this. Let me know if you are interested doing this. I can suggest several approach to do this.

I think this question is very difficult to answer because the system is very complex. I assist Kim Brogden in his answer - in a recent study we published a zinc-dependent antimicrobial ion channel (peptide was anionic) -

http://www.pnas.org/content/110/12/4586.full

However I must admit that in another study of the same peptide, zinc did not seem to activate the peptide in vivo against Ecoli. However, from all the data we collected in vitro zinc was very important for activation.

Thanks for the usual info and literature. Suhail Alam, there is no cys or his amino acids present. Interestingly enough, Mg++ and Ca++ antagonize the peptide. Could please tell me how to test the binding of zinc ions?

Generally you can do this by ITC if the binding constant is significant. Or there are more sophisticated techniques such as X-ray absorption. How big is your peptide? But as I mentioned, there might another effect on zinc on the bacterium which is possibly more difficult to trace.

The peptide is around 1500 g/mol. I think the best starting point would be to do a synergy study using varying concentrations of the peptide and zinc. After the best combination is determined, determine how zinc at that concentration changes the viability of the cells compared to normal concentrations used. What do you think?

Dear Shane

To rule in or out the direct influence of zinc on your peptide you can also try circular dichroism to see of the zinc has an influence on the secondary structure of your peptide in for example extracellular buffer, intracellular buffer, liposomes and maybe TFE. If there is an influence, you can titrate you peptide with zinc to determine constants in the different environments for comparison. If you have a Trp in the peptide you can do a similar study with fluorescence to determine the influence of zinc on the Trp(s) in the peptide structure. You can do these experiments at Stellenbosch University - if you are interested contact me ([email protected]) .

Just a note on your use of Alamar Blue: I think 24 hours is a bit long for this dye as the colour change is from blue (resazurin) to pink (resofurin) to colourless (hydroresofurin) over such a long time. Also, you can look if the zinc has an influence on the dye reaction itself. You can do this by adding the zink and NADH to your dye in media.

Good luck

Marina

Thanks Marina, luckily Stellenbosch isn't to too far away. How much time do you think I will require for the investigation? I will be in contact.

About the Alamar blue, for MTB MIC investigations, a 24 hour incubation period is recommended. I also do the incubation in the dark. I have noticed the colorless form, but that has been due to longer incubation periods in the presence of light.

Hi Shane

If you have prepared you peptide analytically (weighed analytically, aliquoted and dried - 200ug per sample, preferably in glass), I recon it would take is about 8-10 hours over 2 days to get good repeatable results. We can do the analytical preparation on our side too if you do not have a 6 digit scale. We also have the facilities to do the liposome preparation (add a day or two for that type of work).

Regards

Marina

I wouldn't be surprised by your observation. This is because Zinc is a respiratory poison at the concentration you have added by mistake and M. tuberculosis is a strict aerobe. Therefore, what you observed was right. Now just ignore this mistake and proceed with your work.

Well, many antimicrobial peptides are known to be more/less potent in higher ionic strength media (salt dependence). It depends on the peptide and the system. I agree with Jai, in that you should probably continue to answer your primary research aims before moving on to investigations of salt/ion dependence. Most journal/grant reviewers have become pretty bored with the question of salt dependence in antimicrobial peptides.