Subject
The laboratory is divided into five main areas:
Structural and functional studies on Brucella environmental sensing proteins

The biosynthetic pathway of riboflavin as target for development of antibiotics

Immunological and protein engineering studies on Brucella lumazine synthase (BLS)

Molecular basis of the red/far red light pathway involved in bacterial infection

Generation of nanobodies as a tool for the crystallization of complex macromolecules of therapeutic interest and for development of diagnostic techniques.

Approach
Our conceptual approach is to identify protein targets involved in bacterial sensory or metabolic pathways important in the infectious process and to describe their function both at the atomic level and at the host-microbe interaction scale. For that aim, we functionally characterize these targets in vitro and in vivo. For in vitro characterizations, we utilize X-ray crystallography, NMR spectroscopy, Cryo-EM and a wide variety of biophysical, biochemical and cellular techniques and methods (i.e. SEC-HPLC-SLS, DLS, CD/fluorescence/UV-Vis absorption spectroscopy, FACS, primary cellular culture, ELISA, mAbs, Western Blot, fluorescence and confocal microscopy, radioactive and non-radioactive phosphorylation assays, radioactive vitamin transport assays, enzyme kinetics studies, site directed mutagenesis for functionality evaluation in proteins, etc). For bacterial in vivo characterization, we evaluate the biological relevance of our targets in the host-microbe interaction: we use genome site directed mutagenesis, homologous and heterologous complementation with wild-type and mutant versions of the proteins, classical microbiological determination assays (i.e. biofilm formation, EPS content, flagellar apparatus production, motility, survival to stresses, growth, etc). For immunological studies, we use different animal and cellular models (i.e. knock-out mice, OT-1 and OT-2 transgenic mice, adoptive transfer of immunity, models of protective and therapeutic vaccine against tumors, in vivo analysis of antigen persistence in cell and tissues). We work with different bacterial models:

Mammal pathogens: Brucella and Mycobacterium spp.

Plant symbiont: Rhizobium leguminosarum

Plant pathogen: Xanthomonas campestris

Advances
We have set up a general pipeline to obtain nanobodies against different immunogens, particularly those of clinical relevance. The strategy includes the use of phage display technology on ELISA and nickel coated plates that allows to spatially orient the target protein. As a result, after two or three rounds of selection, specific nanobodies are obtained and expressed in the periplasmic space for biophysical applications.