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 diseases. Traditionally, the main objective of our projects has been the Gram-positive bacterium Streptococcus pneumoniae (pneumococcus), although we are currently expanding our studies towards other pathogens such as Gram-negative Pseudomonas aeruginosa or Haemophilus influenzae.
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, pneumococcal 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; choline binding proteins; virulence; Gram-negative pathogens; enzybiotics; bacteriophages; structure-function; nanobiotecnology; protein engineering; polihydroxialkanoates; carrier state; biofilms; protein structure, stability and folding
Vázquez R, Seoane-Blanco M, Rivero-Buceta V, Ruiz S, van Raaij MJ, García P. . Monomodular Pseudomonas aeruginosa phage JG004 lysozyme (Pae87) contains a bacterial surface-active antimicrobial peptide-like region and a possible substrate-binding subdomain. Acta Cryst Section D; 78,435-454.
Vázquez R, Díez-Martínez R, Domingo-Calap P, García P, Gutiérrez D, Muniesa M, Ruiz-Ruigómez M, Sanjuán R, Tomás M, Tormo-Mas MA, García P. . Essential topics for the regulatory consideration of phages as clinically valuable therapeutic agents: a perspective from Spain. Microorganisms; 10,717.
Maestro B, Zamora-Carreras H, Jiménez MÁ, Sanz JM. . Inter-hairpin linker sequences determine the structure of the ββ-solenoid fold: a "bottom-up" study of pneumococcal LytA choline-binding module. Int J Biol Macromol;190,679-692
Vázquez R, Caro-León FJ, Nakal A, Ruiz S, Doñoro C, García-Fernández L, Vázquez-Lasa B, San Román J, Sanz J, García P, Aguilar MR. . DEAE-chitosan nanoparticles as a pneumococcus-biomimetic material for the development of antipneumococcal therapeutics. Carbohydr Polym 273,118605.
Vázquez R, García E, García P . Sequence-Function Relationships in Phage-Encoded Bacterial Cell Wall Lytic Enzymes and Their Implications for Phage-Derived Product Design. J. Virol. 95:e00321-21
Vázquez R, Blanco-Gañán S, Ruíz S, García P . Mining of Gram-negative surface-active enzybiotic candidates by sequence-based calculation of physicochemical properties. Front. Microbiol. 12:660403
Vázquez, R., Doménech-Sánchez, A., Ruiz, S., Sempere, J., Yuste, J., Albertí, S., García, P . Improvement of the antibacterial activity of phage lysin-derived peptide P87 through maximization of physicochemical properties and assessment of its therapeutic potential
Bhattacharyya A, Herta T, Conrad C, Frey D, García P, Suttorp N, Hippenstiel S, Zahlten J. . Induction of Krüppel-Like Factor 4 Mediates Polymorphonuclear Neutrophil Activation in Streptococcus pneumoniae Infection. Front. Microbiol. 11:582070
Blanco FG, Hernández N, Rivero-Buceta V, Maestro B, Sanz JM, Mato A, Hernández-Arriaga AM, Prieto MA . From Residues to Added-Value Bacterial Biopolymers as Nanomaterials for Biomedical Applications. Nanomaterials, 11,149
Fuentes-Baile, M.; Bello-Gil, D.; Pérez-Valenciano, E.; Sanz, J.M.; García-Morales, P.; Maestro, B.; Ventero, M.P.; Alenda, C.; Barberá, V.M.; Saceda, M. . CLytA-DAAO, Free and Immobilized in Magnetic Nanoparticles, Induces Cell Death in Human Cancer Cells. Biomolecules. 10, pp. 222
Zamora-Carreras, H.; Maestro, B.; Sanz, J.M.*; Jiménez, M.A.* . Turncoat polypeptides: We adapt to our environment. ChemBioChem. 21, pp. 432 - 441.
Domenech M, García E. . The N-acetylglucosaminidase LytB of Streptococcus pneumoniae is involved in the structure and formation of biofilms. Appl. Environ. Microbiol. 86: e00280-20.
García, P., Vázquez, R. y García, E. . Polypeptides with antibacterial activity. Patent application No. 20382666.4-1118. (Spain)
Roig-Molina, E.; Sánchez-Angulo, M.; Seele, J.; García-Asencio, F.; Nau, R.; Sanz, J.M*.; Maestro, B*. . Searching for antipneumococcal targets: Choline-binding modules as phagocytosis enhancers. ACS Infect. Dis. 6, 954–974
Mato, A.; Blanco, F.G.; Maestro, B.; Sanz, J.M.; Pérez-Gil, J.; Prieto, M. A. . Dissecting the polyhydroxyalkanoate binding domain of the PhaF phasin: rational design of a minimized affinity tag. Appl. Environ. Microbiol.86, pp. e00570-20.
Our research is currently funded by grants from Spanish Ministerio de Ciencia e Innovación MCIN/ AEI/10.13039/501100011033/
- PID2019-105126RB-I00, R&D project in the framework of “FEDER, a way to make Europe”
- TED2021-129747B-C22, R&D project in the framework of “European Union NextGenerationEU/PRTR (Plan de Recuperación, Transformación y Resiliencia de España)”.
We also funded by the Centro de Investigación Biomédica en Red de Enfermedades Respiratorias (CIBERES, Instituto Carlos III)
— MCyT, BMC2000-1002 (2000-2003).
— Fundación Ramón Areces (2000-2003).
— BIO2000-0009-P4-04 (2001-2005).
— MCyT, BMC2003-00074 (2003-2006).
— Ministerio de Sanidad y Consumo, Redes G03/103 y C03/104.
— MCyT, SAF2006-00390 (2006-2009)
— Member of the CIBER of Respiratory Diseases (Instituto de Salud Carlos III)
– CAM, COMBACT Program, S-BIO-0260/2006 (2007-2010)
– MICINN, SAF2009-10824 (2010-2012)
– MICINN, IPT-2011-1337-010000
- MINECO, SAF2012-39444-C02-01 (2013-2015)
- MINECO: BFU2010-17824 (2011-2014)
- BIO2013-47684-R (2014-2016)
- BIO2016-79323-R (2017-2019)
- EU (FP7): HEALTH-F3-2009-2231, 2011
- MINECO, SAF2017-88664-R (2018-2020)