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Estudios estructurales y funcionales de la lisozima Cpl-7 del bacteriógrafo Cp-7 de neumococo

AutorDíez-Martínez, Roberto
DirectorGarcía, Pedro
Palabras claveNeumococo
Modelo infección
Pez cebra
Fecha de publicación8-jul-2014
EditorCSIC - Centro de Investigaciones Biológicas (CIB)
Universidad Complutense de Madrid
ResumenSUMMARY INTRODUCTION: The Gram-positive bacterium Streptococcus pneumoniae (the pneumococcus) is a common asymptomatic resident of the human nasopharynx and is also an important etiologic agent in several diseases like pneumonia, otitis media, meningitis, and sepsis. Current numbers of morbidity and mortality impose a significant burden in healthcare costs in developed and non-developed countries. Therapeutics against pneumococcus is hampered by insufficient vaccine coverage and an increase in antibacterial resistance, which makes urgent the identification of new targets and development of new antimicrobials. Bacteriophage endolysins constitute an alternative (or complementary) approach to classic antibiotics in the search for novel therapeutic strategies for fighting invasive diseases. Endolysins are phage murein-hydrolases that cleave the major bond types in the peptidoglycan and specifically break the host cell wall, provoking cellular death. This bactericidal effect of the endolysins is now fostering to use them as exogenous enzymes to kill susceptible bacteria and is also named “enzybiotics”. Most murein-hydrolases reported so far in the pneumococcal system, from either host or phage origin, are choline-binding proteins (CBPs) that depend on their attachment to the choline moieties of pneumococcal teichoic acids for activity. There is a single exception to this rule, the Cpl-7 lysozyme, encoded by the lytic pneumococcal phage Cp-7, whose cell-wall binding module (CWBM) is built by three identical CW_7 repeats that are sequentially and structurally unrelated to the choline-binding motifs of the CBPs. In contrast, its N-terminal catalytic module is 85.6% identical to that of Cpl-1 lysozyme. PRINCIPAL FINDINGS: In this Thesis, it has been completely sequenced the Cp-7 genome, a 19 741 bp linear DNA that harbors a terminal protein covalently linked to their 5’-ends. A comparison between the modular arrangement of different open reading frames of Cp-1 and Cp-7 has been made and major emphasis has focused on the lytic enzymes of the lysis cassette, the lysozymes Cpl-1 and Cpl-7, respectively. Both enzymes had similar specific activities when tested on purified pneumococcal cell walls, but their bactericidal effects were very different when added exogenously on pneumococcal cells resuspended in PBS. After a careful analysis of physico-chemical differences between Cpl-1 and Cpl-7, the sign of the charge of the CWBM of Cpl-7 was inverted (from −14.93 to +3.0 at neutral pH). The resulting engineered variant, Cpl-7S, harbors 15 amino acid changes (5 in each CW_7 repeat) and it showed an increased lytic activity against S. pneumoniae (including different encapsulated and multiresistant strains), Streptococcus pyogenes, and other pathogens. Furthermore, a combined action of 0.01% carvacrol (an essential oil) and Cpl-7S, a protocol designed to destabilize the outer membrane of Gram-negative bacteria, was capable to render Escherichia coli and Pseudomonas putida susceptible to the killing activity of the lysozyme, an effect not described previously.
To check whether Cpl-7S could bind to a variety of bacteria, a fusion protein between Green Fluorescent Protein (GFP) and CWBM of Cpl-7S was constructed, GFP-CWBM7S. Addition of this fusion protein to Gram-positive bacteria or a mixture of GFP-CWBM7S and carvacrol to Gram-negative bacteria demonstrated a fluorescent signal, which suggested that Cpl-7S interacted with several bacteria. Complementary experiments carried out with the entire Cpl-7S added to susceptible bacteria allowed measuring the bactericidal effect of this lysozyme against each pathogen. In order to search a better enzyme related to a stronger killing effect, different chimeric proteins were constructed using the well defined structural elements of Cpl- 1 and Cpl-7S lysozymes: the two modules involved in the catalysis and substrate recognition, and linkers connecting both modules. Thus, Cpl-711, Cpl-771, Cpl-117, and Cpl-177 enzymes were produced in E. coli and subsequently purified. All these new enzymes were assayed as before, on purified cell walls and on living bacteria, and Cpl-711 was found to be the most active one, even more potent than Cpl-1 that, until now, was the more lethal weapon against different pneumococcal strains. Validation of the above in vitro results has been carried out using two animal models of infection. Initially, a zebrafish embryo was chosen to test Cpl-7S. In these experiments a single 25-μg of Cpl-7S significantly increased the survival rate of such embryos infected with pneumococcus or S. pyogenes, confirming the killing effect of Cpl-7S in vivo. This is the first example to use zebrafish embryos as a successful animal model to measure the protective effect of an antibacterial compound against an infection provoked by two important pathogens, pneumococcus and S. pyogenes. Also, a mouse model was set up to assay the bactericidal effects of Cpl-7S and Cpl- 711, with two versions of infection. Cpl-7S was checked in an intranasal colonization of mixed infection of S. pneumoniae and S. pyogenes demonstrating that was also capable to reduce a significant population of both bacteria, although was more effective against S. pyogenes. On the other hand, chimeric Cpl-711 endolysin was tested in a sepsis model by intraperitoneal injection of the enzyme, once established the bacteremia with the pneumococcal D39_IU strain. Results were also very promising, as protection of 50% of mice was achieved with as little as 50 μg of Cpl- 711. CONCLUSIONS: All these results allow the conclusion that modulation of the net charge of cell wall-binding motifs might be a general way of improving the enzymatic efficiency and selectivity of putative or actual enzybiotics, thereby expanding the range of susceptible pathogens. In this sense, currently available results support the notion that even lysins with a wider range of antimicrobial activity would exert a less dramatic effect on the normal microbiota than conventional antibiotics. Moreover, swapping structural elements of phage lysozymes have led to the construction of new chimeric enzymes with noticeable bactericidal effects, compared to their parental enzymes.
Descripción185 p.-47 fig.-15 tab.-2 videos.
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