Protein engineering against antimicrobial resistance

   Pathogenic bacteria are increasingly resistant to antibiotics. This can lead to a post-antibiotic scenario in which some studies predict, in the mid-term, an increase in mortality that might overcome other diseases such as cancer. This has led us to focus our work towards the search for new antimicrobials that prevent cases of resistance, concentrating on the pathogens that cause respiratory and oral diseases. Traditionally, the main objective of our projects has been the Gram-positive bacterium Streptococcus pneumoniae (pneumococcus) and Gram-negative Pseudomonas aeruginosa or Haemophilus influenzae, although we are currently expanding our studies towards oral pathogens which cause dental diseases (caries) such as Streptococcus mutans.

   Among the most promising alternatives to antibiotics are phage-encoded endolysins, mostly modular enzymes that hydrolyze the bacterial peptidoglycan. These phage lytic enzymes may be added exogenously to act as bactericidal agents with great specificity, and because of their use as therapeutic agents they are also called enzybiotics. In our laboratory we test both wild-type and chimeric endolysins, engineered from the fusion of different functional domains. In recent times, we have built specific chimeric lysins against pneumococcus as well as other enzymes with a broader host range. Likewise, we have verified its synergistic action with certain antibiotics, or with enzymes that target different bonds. All these lytic enzymes have been shown to be effective against susceptible bacteria, both in planktonic cultures and in biofilms, and the results have been validated in animal models, such as mice or zebrafish.

   On the other hand, bacterial surface proteins play an essential role in bacterial viability and virulence and, until now, have not been considered with sufficient attention as targets for the development of new antibiotics. In our group we have developed a collection of molecules, from small organic compounds to peptides and polypeptides, that interfere with the function of these proteins. Furthermore, using the multivalence concept, we design and test nanoparticles that contain several copies of our active compounds, which results in an exponential increase in their antimicrobial activity.

The biophysical studies carried out on the proteins mentioned above, as well as on those domains that bind natural biopolymers such as the cell wall peptidoglycane or natural polyesters (polyhydroxyalkanoates), have allowed us to obtain, using protein engineering, variants of such proteins with important biotechnological applications derived from their molecular recognition properties, e.g. enzyme immobilization systems and construction of enzymatic bioreactors.

Keywords: Streptococcus pneumoniae, Pseudomonas aeruginosa, Streptococcus mutans; choline binding proteins; virulence; Gram-negative pathogens; enzybiotics; bacteriophages; structure-function; nanobiotecnology; protein engineering; polihydroxialkanoates; carrier state; biofilms; protein structure, stability and folding