AbstractColonization of mammal hosts by bacterial pathogens requires various adaptation mechanisms. One of these, is the fine-tuning of bacterial metabolism according to nutrients availability and physical conditions (O2, pH, …) that are encountered during the infection. Each colonized niche is likely to be characterized by specific physico-chemical properties imposing a metabolic plasticity to bacterial pathogens.
Brucella spp. are facultative intracellular pathogens, which manage to adapt, feed and multiply in various mammal hosts, in different organs and tissues, as well as in diverse cell-types either professional or non-professional phagocytes. Despite this impressive adaptability, little is known on the metabolism of these bacteria, especially in vivo. Current understanding of Brucella metabolism can be summarized as: (i) hexose metabolism is based on the pentose phosphate pathway with the TCA cycle as the glycolysis is interrupted and the Entner-Doudoroff pathway does not appear to be active; (ii) erythritol is a preferential carbon source and is catabolized in 5 steps to produce dihydroxyacetone phosphate. Interestingly, this polyol might represent a relevant carbon source in vivo as it is found in substantial amounts in the reproductive tracts of some hosts for which these bacteria have a particular tropism. During our investigation on Brucella spp. metabolism, data inconsistent with these models were quickly gathered, however, leading us to re-examine what was thought to be known.
First, we have revisited the erythritol catabolic pathway in B. abortus 2308, which was described nearly 40 years ago. We identified and purified five proteins that were required for bacterial growth on this polyol as sole carbon source. We have demonstrated that these five enzymes work sequentially for the conversion of erythritol to erythrose-4-P, a metabolite of the pentose phosphate pathway and not to dihydroxyacetone phosphate unlike previously considered. By doing so, four reactions out of the five initially described were revised and include three new enzymatic reactions. In parallel, we investigated the relevancy of erythritol as carbon source during the infection. Our results demonstrate that erythritol catabolism is not required for the infection of RAW 264.7 murine macrophages, human THP-1 macrophage-like cells or BeWo and JEG-3 human trophoblasts. In the latter cells, we nonetheless demonstrate that the polyol is present in substantial amount and is in all likelihood used by intracellular bacteria.
Second, we provide isotopic and genetic evidences that the catabolism of glucose in B. suis bv.5 is not based on the pentose phosphate pathway but rather on the Entner-Doudoroff pathway in chemically defined medium. Our data demonstrate that almost 70% of the assimilated glucose is indeed catabolized by this pathway while only the 30 remaining % are metabolized by the pentose phosphate pathway. We also provide first evidences that the TCA cycle operates in an oxidative manner and is supplied by both acetyl-CoA and oxaloacetate. Such EDP-dependent glucose catabolism appears to be a feature common to several phylogenetically close α-proteobacteria. Finally, it appears that glucose and hexose metabolisms do not play a major role for bacterial physiology during the infection of RAW 264.7 macrophages.
|Date of Award||14 Nov 2015|
|Supervisor||JEAN-JACQUES LETESSON (Supervisor), MICHEL JADOT (President), Emile Van Scaftingen (Jury), Ignacio Moriyón (Jury) & Olivier Neyrolle (Jury)|