Downstream analysis of Tn-seq data and characterization of iaIB and oppB genes from Brucella abortus

Résumé

The genus Brucella, part of the alpha-proteobacteria, is composed of facultative intracellular pathogens. One of its member, Brucella abortus is the causative agent of the bovine brucellosis. A large library of B. abortus transpositional mutants was previously used to perform a timeresolved Tn-seq in macrophage infection, with the aim to unravel genes required for the infection fitness. Based on those results, this work focused on two genes reported as important at 2 h post infection in RA W 264.7 macrophages. One (BAB1_0491) is homologous to ialB, and the other (BAB2_0701) is part of an operon encoding an oligopeptide ABC transporter (homologous to Opp ). While IalB had been reported to play a role for human erythrocyte parasitism in Bartonella bacilliformis, Opp seems to have a broad range of possible fonctions. The aim of this master thesis was to characterize their fonctions. First, mutant strains by disruption of ialB or oppB were constructed and used to perform macrophages infection where the ialB mutant showed a decreased survival. If the oppB mutant survival was similar to the wild type (WT), it displayed smaller colonies that could be due to the infection process. Endosomal conditions (i .e. nutrient depletion and acidic pH) were then tested for the formation of this small colony phenotype. The acidic pH did not seem to have an impact, but the nutrient depletion appeared to have the same outcome than the infection. Also, phase contrast microscopy of the ialB mutant revealed a morphology impairment. Based on those results, deletion strains were designed. Because the ialB deletion was not possible, the second part of this work is solely based on Opp for which a deletion of the complete opp operon was generated. The !':iopp mutant displayed a growth delay in rich medium, compared to the WT strain. The opp mutant behaved similarly to the WT strain in macrophage infection. Also, no morphological impairment could be observed for the t:iopp strain. Further investigations are required to explore the roles that could be carried out by this oligopeptide transporter, and to understand why ialB and opp mutant were scored as attenuated in the original Tn-seq experiment.

la date de réponse	2017
langue originale	Anglais
L'institution diplômante	Universite de Namur
Superviseur	Xavier De Bolle (Promoteur)