I plan to study the changes in metabolite content in chicken infected tissue with a single strain of Gallibacterium anatis and I would like to monitor in time the concentrations of the metabolites for the host and bacteria, separately. Is there a way to separate host cells from bacteria?
Hi Nicola,
one could think about techniques like FACS to separate host cells from bacteria but I don't think it makes sense for metabolomics. You would need to break up the tissue, get it into suspension, maybe do a staining step and then do the sorting, diluting the cells even more. By the time you have the cells separated the metabolome will be nothing like it was at the time of sampling. Some enzymatic reactions will continue to happen and soluble metabolites will be washed out.
There might be some metabolites for which that works, I could imagine membrane lipids, but for an accurate metabolome the sample would have to be quenched right away.
I would recommend you to look into spatial metabolomics instead. Mass spectrometry imaging can be combined with labeling techniques like FISH to find metabolites associated with bacteria in tissue sections. My colleagues published a beautiful study on the technique: https://www.nature.com/articles/s41564-019-0664-6
This approach could help you to identify marker molecules for the bacteria in different stages of infection which you could then analyze in tissue extracts with more traditional techniques like LC/MS.
It really all depends on what your research questions are though. Hope I could help.
Thank you for the detailed reply. It seems quite a complex technique. My initial idea was to see whether certain host metabolites can impose selective pressures on the pathogen and select specific mutants that are better adapted to the tissue micro-environment within the host. But this would require having the host and pathogen metabolomes separated. An alternative would be to just compare the metabolome of infected vs. uninfected host tissue to get a general picture of what happens during infection.
Hi Nicola, further to Patric's answer, I just wanted to say not to use FACS to do cell sorting prior to metabolomics, it puts a lot of stress on the cells - see the reference below. I also wonder if it's possible to do some sort of cell co-culture where you could at least compare the co-cultured cells with each type (host/bacteria) cultured separately. I realise this would produce a less optimal/realistic picture than the tissue, but might help get leads on the metabolites to investigate. Good luck!
Flow Cytometry Has a Significant Impact on the Cellular Metabolome, J. Proteome Res. 2019, 18, 1, 169–181 https://doi.org/10.1021/acs.jproteome.8b00472
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