Subject
Bacteria have evolved different strategies to successfully colonize habitats either inside or outside their host tissues. Our laboratory aims to decipher such mechanisms in two alphaproteobacteria: Rhizobium, which establishes symbiotic relationships with legume plants allowing them to assimilate atmospheric nitrogen; and Brucella, a facultative intracellular pathogen responsible for a debilitating disease of mammals known as brucellosis.
We are interested in identifying and characterizing factors involved in the assembly of the extracellular matrix and in bacterial cohesion of rhizobial biofilms. In particular, we attempt to expand our understanding of the role of Rhizobium-adhering proteins (Rap), a family of secreted proteins that share domains of binding to the acidic exopolysaccharide (EPS), a key component of rhizobial biofilms.
The initial interaction with the host cells is a crucial step in the infection process of intracellular pathogens. In Brucella, we focus on addressing the following questions: Which are the protein factors that mediate adhesion of these bacteria to different cell types of the host? How is the proper assembly of these proteins in the bacterial cell envelope ensured? How is the adhesin expression regulated?

Approaches
In order to study factors involved in biofilm formation and in the interaction with the host, we carry out phenotypic, proteomic and functional studies in either mutant strains or in bacteria overexpressing such protein factors, both in vivo and in vitro. To study bacterial cell cohesion, biofilm formation, and determining the extracellular localization of the involved protein factors, we use confocal microscopy. We also perform genomic, transcriptomic analyses, and apply bioinformatic methods to identify transcriptional regulators and environmental stimuli involved in regulation of adhesins. To understand the mode of action of the proteins under study, we carry out structural and functional studies expressing recombinant proteins heterologously.

Advances
Rhizobium biofilms.
In order to establish biofilms and to attach to root tissues or abiotic surfaces, Rhizobium requires to produce acidic exopolysaccharide (EPS) and the exposed part of lipopolysaccharide (LPS), and to secrete Rap proteins through the Type I Secretion System PrsDE. We showed that RapA2, which contains two Ra/CHDL (Ra1-Ra2), presents a calcium-dependent β-sheet cadherin-like structure. Under its native conformation, this protein binds to specific structures of glucuronic acid-rich growing EPS chains. RapA lectins were shown to be involved in development of the Rhizobium biofilm matrix and in bacterial cell cohesion by modulating the size of the EPS chains and the balance between capsular polysaccharide (CPS) and EPS.
Brucella cell envelope and adhesion to the host.
We demonstrated that Brucella expresses five adhesins from the autotransporter family (three monomeric and two dimeric) that play redundant or complementary roles in virulence and in adhesion of the pathogen to the host extracellular matrix in different cell types. These adhesins do not localize randomly but to a restricted region that constitutes a true adhesive pole. Recently, we observed that BmaB, one of the monomeric autotransporters, requires the TAM (translocation and assembly module) machinery for assembly in the bacterial cell envelope and to exert its biological role. Both in Brucella and other alpha-Proteobacteria, TAM is not only necessary for proper autotransporter assembly but also plays a key role in biogenesis and stability of the bacterial cell envelope, in a coordinated manner with cell division. Regarding adhesin regulation, we observed that expression of the trimeric autotransporter BtaE is integrated into a complex transcriptional network involving other virulence factors, and that its regulation directly responds to transcription factors that integrate metabolic and environmental signals. Recently, in collaboration with Pablo Baldi’s group and Mariana Ferrero (IDEHU, FfyB UBA) we assessed the immune response and protection against mucosal challenge induced by nasal vaccination with the soluble domain of recombinant BtaF. Although nasal immunization with BtaF did not protect mice from respiratory challenge with Brucella suis, it conferred a marked protection from the intragastric challenge.