The effects of microbial priming with reactive oxygen species: an in vitro and an in vivo approach

Abstract

Organisms need efficient ways to store environmental information so that they can easily use it in the future and react to their environment appropriately. This ability has usually been associated with organisms having a central nervous system. Recently more and more evidence has piled up suggesting organisms like plants, fungi and bacteria also possess the ability to store the encounters from the past and inform their future decisions based on these past encounters. This thesis deals with one such instance of microbes storing past stresses and using this information for improved survival in case of future stresses. This phenomenon has been termed “priming”, which refers to organisms’ ability to show heightened immune responses on a second exposure to stress. The first exposure to stress is a sub-lethal dose followed by a lethal dose, referred to as the challenge dose. Priming helps organisms survive stresses that would have otherwise been lethal, providing a considerable survival advantage, but nothing comes without a cost. Priming also comes at a cost since organisms spend energy in heightening their immune responses. Still, this cost is usually negligible compared to the survival benefits it provides in a stressful environment. Microbes are constantly present in settings such as wastewater treatment plants and antibiotic production units, where they are always under exposure to low doses of antibiotics. This exposure to low doses of antibiotics could arm them against the lethal doses of antibiotics, adding to an already growing problem of resistance emergence. The priming phenomenon has been tested in several microbial species and stresses such as pH, temperature, osmotic pressure, salt stress, antimicrobial peptides, etc. All the existing literature on priming deals with priming and challenge stress where microbes are in the same environment. This gives us a robust idea of priming being a generalised phenomenon but does not tell us if priming confers an advantage in natural settings. In nature, most microbes do not stay in the same environment and are dynamically jumping across environments. Among other stresses, microbes are constantly exposed to oxidative stress in nature, either because of the presence of reactive oxygen species (ROS) in antiseptics or ROS immune defences inside the host. ROS levels are tightly regulated in organisms by enzymes such as peroxidases and catalases since excess ROS can be lethal. When a host, such as an insect, is infected with microbes, ROS is the first line of defence to fight the infection. In this thesis, ROS priming in an in vitro and in vivo setting was studied using a phytopathogen Erwinia carotovora carotovora 15 (Ecc15), a causal agent of soft rot in crop plants. It was tested if in vitro priming with ROS, specifically hydrogen peroxide, leads to improved survival upon receiving a challenge. It was found that priming leads to enhanced survival upon in vitro challenge. The phenotypic markers associated with peroxide priming were then investigated using LC-mass spectrometry. Testing the advantage of primed bacteria inside the host 2 Drosophila melanogaster highlighted the need to consider costs associated with in vitro priming, leading to differential bacterial numbers in primed and non-primed treatments. These costs were then reduced from 50% to 4% by testing a lower range of priming concentrations before testing the advantages of priming with lower concentrations of ROS inside the host. It was established that the effect of priming inside the host differs with time and sex of the host, possibly due to sexual dimorphism in ROS amounts inside male and female D. melanogaster.