Characterization of the clumping phenotype displayed by a Quorum Sensing deficient strain in Brucella melitensis 16M and research of genes involved in its exopolysaccharide biosynthesis

Recently the bacterial pathogen Brucella melitensis 16M has been shown to display a clumping phenotype in liquid culture when affected in its Quorum Sensing system, a bacterial communication system allowing the coordinated gene expression within a population regarding its density and/or environmental properties (diffusion, flow…). During the first part of this work, we found that the overexpression of the AHL-acylase of Brucella (an enzyme able to degrade the signal molecules required for successful Quorum Sensing) leads to a similar clumping phenotype. The characterization of this strain allowed us to demonstrate the presence of extracellular DNA and exopolysaccharide(s) (EPS), two classical components of extracellular matrices, in clumps produced by this strain. We also observed that the production of outer membrane vesicles is strongly increased in this strain. The use of this strain allowed us to purify the EPS and to carry out a structural analysis which gave rise to the composition and the first structural information on the complex polymer produced by B. melitensis 16M. Moreover, a classical biofilm attachment assay and a HeLa cells infection model demonstrated that the clumping strain is more adherent to both surfaces tested than the wild-type one. Taken together, these results bring insights on the matrix composition and reinforce the evidences that B. melitensis could form biofilms in its lifecycle. In the second part of the work, we attempted to identify genes involved in the biosynthesis of B. melitensis EPS. We screened the genome of B. melitensis which allowed us to identify about thirty genes homologous to well-described EPS biosynthesis genes closely conserved among phylogenetically related bacteria. Seven of those genes were selected for deletion in B. melitensis. None of the resulting mutants was found to be impaired in its clumping formation or EPS production abilities in the conditions tested. However, two of these mutants (∆BMEI1177 and ∆BMEII0851) presented a strong and systematic trend to form chains in liquid culture. The observation of this phenotype by time-lapse microscopy allowed us to observe that these chains are formed by clonal growth. Moreover, mice infected with the ∆BMEI1177 or ∆BMEII0851 strains have a lower bacterial charge in their spleen compared to the wild-type strain. This result suggests that Brucella BMEI1177 and BMEII0852 products have an important role in the infectious cycle of B. melitensis.