Reactive oxygen species are produced during early plant-pathogen interactions as a plant defence response. Sometimes, necrotrophic pathogens also produce ROS to cause necrosis or plant cell death. Now the question arises, how can we distinguish between both the ROS, specially during infection? Is there any approach available to do so?
Typically, superoxide (O2*-) is the first ROS being produced in most cells under aerobic conditions.
For bacterial cells, while it is true that a fraction of molecular oxygen consumed during respiration and ATP production is converted to superoxide, this amount of superoxide cannot be compared to the level of the host cells (which contains multiple mitochondria as a source of superoxide at resting state and during inflammation).
In modeling infection, typically host cell densities used are 1x10^4 to 1x10^5 cells /mL. In realistic terms, clinical infections involve infecting pathogens at densities at or below 10^7 microbes / mL at most (because the nutrient requirements including aeration in vivo won't allow them to grow to supraphysiological levels like 10^8 / mL) .
Also, typically, infection experiments use MOI (multiplicity of infection) ranging from 1 to 10. MOI like 100 (equivalent to 10^7/mL or more) again seems unrealistic for the reasons stated above.
Most bacteria possess multiple enzymatic and non-enzymatic antioxidant defenses to deal with superxide -- first by dismutation by SOD, then catalase or peroxiredoxins to terminate the ROS reactions. So, effectively, what you can expect to detect or see in resting bacteria for endogenous superoxide / ROS is very very low (we have some unpublished imaging data). Certainly not to be compared with a resting host cell.
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Having said that, it means endogenous bacterial superoxide (or ROS) in host-pathogen interactions may not be a key concern in measuring the overall ROS turnover. It is expected to be a small contribution.
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If you have an appropriately selective chemical probe for ROS (say superoxide), then what you do is to use enzyme inhibitors (e.g. NOX inhbitors) or antioxidants (say mito-TEMOL for mitochondrial superoxide) to measure the effects of the intervention, compared to un-intervened controls.
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Also, if you are absolutely interested in finding about ROS in the bacteria/microbe, fluorescent protein based H2O2 reporting has been established (the HyPer series). Those can be integrated into your model strains by transgenesis. Then, you can image or quantify H2O2 changes during infection.
Article Using Hyper as a Molecular Probe to Visualize Hydrogen Perox...
Currently with the known tools, precise investigation on ROS kinetics and dynamics remain quite challenging.
Good luck on your experiments!
@Nai-Kei Wang
Thank you for providing the very nicely explained and useful information.
I think it is difficult to distinguish the host generated and infection driven ROS quantities in cells, however one can always compare ROS levels in actively infected cells and non infected cells as control. The difference between the two is actual pathogen infection induced ROS in cells.
@Vimal Kumar Semwal
I agree with the comparative approach, But the question still remains who produced ROS?
Pathogen in response to host or Host in response to pathogen?
Hi Yeshveer,
The ROS are produced by host in response to pathogen attack.
Botrytis cinerea, a necrotroph and other pathogenic fungi also produce ROS through Nox-complex during their interaction with host plants.
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