On the link between Brucella abortus cell cycle and adaptation to its intracellular lifestyle

Promoter

Prof. Xavier DE BOLLE, UNamur, Research Unit in Biology of Microorganisms (URBM)

Jury

Xavier De Bolle (Supervisor), Patricia Renard (President), Michel Jadot (Jury), Jean-Yves Matroule (Jury), Suzana P Salcedo (Jury) & Jean-François Collet (Jury)

Summary

Brucellae are the agent responsible for a worldwide zoonosis called « brucellosis ». This intracellular bacterium is able to infect professional and non-professional phagocytes. Therein, the infection occurs in two successive stages, composed of an early phase characterized by a stable number of viable bacteria, followed by a proliferative phase. Inside the cell, the bacterium resides in a vacuole, which interacts transiently with the endocytic pathway (carrying the LAMP-1 marker) before reaching its replicative niche, an endoplasmic reticulum-like compartment. All cells, either prokaryotic or eukaryotic, are characterized by a cell cycle. There are two major aspects of the bacterial cell cycle, the replication of the genome and the growth of the bacteria. The bacterial cell cycle occurs in three main stages, first the "newborn" (Nb) stage between cell division and the initiation of genome replication, then the "intermediate" (Int) stage corresponding to growing bacteria without detectable constriction site, and finally the predivisional (Pd) stage. Given the biphasic infectious mode of Brucella abortus, we hypothesized that the progression of its cell cycle could be modulated during infection. However, the Brucella cell cycle in bacterial culture and a fortiori during infection is poorly investigated, as well as its regulation. Such as other Rhizobiales of the a-proteobacteria group, Brucella abortus displays a unipolar growth and we took advantage of the TRSE labeling to characterize the "growth" aspect of the cell cycle during infection. Indeeed, this TRSE labelling allows the monitoring of growth for individual bacteria. During this thesis, we showed that during the first 6 h PI, constricting bacteria were almost inexistent and bacterial growth was undetectable. This suggests a non-proliferation and an arrest of the unipolar growth of B. abortus during the first stage of the infection. In addition, the newborn bacteria (originating from a division event) were predominantly found during the early phase of the infection. Consistently, the majority of bacteria were blocked in a G1 stage of the cell cycle during this phase (M. Deghelt). All together those data define newborn bacteria as the specific infectious subpopulation. Surprisingly, we also observed growing bacteria in both LAMP-1 positive or LAMP-1 negative compartments. Moreover, after proliferation, a fraction of the mother bacteria are in LAMP-1 positive compartments while daughter bacteria are almost all in LAMP-1 negative compartments. We also observed that the growth of the virB mutant resumed in LAMP-1 positive compartments with the same timing as a wild type strain, but this mutant remains in LAMP-1 positive compartments and does not proliferate. At the early stage of a cellular infection, the Brucella containing vacuole encounters harsh conditions, such as starvation, reactive oxygen species-related damages and an acidic pH. Therefore, we propose that the regulation and modulation of Brucella abortus cell cycle along intracellular trafficking during host cells infection is probably linked to the adaptation of Brucella for survival in the presence of those detrimental stresses.