AbstractAll living organisms must ensure that their DNA will remain protected against stresses. Intracellular bacteria are no exception, as they have developed strategies to face DNA damaging stresses inside their host cells. One well-known example is oxidative stress, which can result from the oxidative burst of macrophages. Another DNA damaging stress that is predicted to be encountered by intracellular bacteria is alkylating stress, resulting from the peroxidation of lipids, the N-nitrosation of metabolites or the presence of week endogenous alkylating agents, such as S-adenosyl-methionine. Despite previous attempts, to our knowledge, the occurrence of alkylating stress inside host cells still needs to be experimentally demonstrated.
Here, we studied the response of the class III pathogen Brucella abortus inside host cells, and we showed, for the first time, that an intracellular bacterium is meeting alkylating stress inside its host. Indeed, the construction of a fluorescence-based reporter system detecting alkylation events on DNA allowed us to find out that B. abortus is mainly subjected to alkylation stress during the first stage of its infection, while it is still residing inside its endosomal Brucella-containing vacuole (eBCV) and before to reach its replicative niche, the endoplasmic reticulum (rBCV). To go further, and in order to better assess the environment in which Brucella is residing inside its host cell, a probe was designed so it could be covalently attached at the bacterial surface and report, based on fluorescence emission, the level of N- nitrosation events occurring inside the eBCV. This technique revealed that N-nitrosation events do take place inside this compartment in host cells. However, exogenous N-nitrosation was innocuous to Brucella. Instead, we found that the alkylating stress felt by the bacterium was mainly due to the endogenous formation of N-nitroso compounds, which were produced by bacterial metabolism.
Without surprise, the deletion of DNA repair genes was inconclusive in showing an attenuation of the bacterium inside host cells, as it was the case for previous reports on other intracellular pathogens, such as Mycobacterium tuberculosis. It is thus likely that DNA is protected by numerous and redundant DNA repair pathways that ensure that it is protected at all times. Most bacteria possess a DNA repair pathway that is specialized in coping with alkylated DNA. It is the so-called adaptive system. Interestingly, B. abortus does not possess a functional adaptive system. Instead, we found that this bacterium is relying on its SOS system, as well as on the essential and well-conserved transcription factor GcrA to regulate a series of genes involved in DNA repair.
|Date of Award||11 Dec 2018|
|Sponsors||Fund for Research Training in Industry and Agriculture (FRIA)|
|Supervisor||Xavier De Bolle (Supervisor), Regis Hallez (President), Patrick VIOLLIER (Jury), Ivan Matić (Jury) & Bernard Hallet (Jury)|
- DNA repair