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Contribution of the catalytic and cell wall binding modules of LytA, Pal and Skl N-acetylmuramoyl-L-alanine amidases to their anti-pneumococcal activity

AuthorsGallego-Páramo, Cristina; Martínez-Buey, R; Hernández, N; Menéndez, M
Issue Date6-Nov-2018
Citation2nd International Symposium on Antimicrobial Hydrolytic Enzymes (2018)
AbstractThe endolysins Pal and Skl are encoded by the Dp-1 bacteriophage of Streptococcus pneumoniae and the ¿SK137 bacteriophage of Streptococcus mitis, an opportunistic pathogen closely related to S. pneumoniae. Like LytA, the major pneumococcal autolysin, Pal and Skl are N-acetylmuramoyl-L-alanine amidases and all three share a homologous choline-binding domain (CBD) located at the C-terminus. Conversely, the N-terminal catalytic module (EAD) belong to different families; namely, Amidase_5 (Pal), CHAP (Skl) and Amidase_2 (LytA). The bacteriolytic activity of Skl on S. pneumoniae is rather low compared to the antimicrobial activity of LytA and Pal. This disparity of behavior could arise from differences in pneumococcal cell wall recognition by respective CBDs, including choline-induced dimerization, the intrinsic activities of respective EADs, and/or a joint effect of their precise module combinations. To investigate the contribution of these factors, we have studied in a comparative way the affinity for choline as well as the quaternary structure and domain disposition in the free enzymes and the complexes formed with choline by different biophysical approaches. Besides, the impact of EAD intrinsic activities and modular combination was investigated by domain shuffling. Sequence comparison showed a high conservation of the CBDs among the three amidases, including full conservation of the residues involved in choline recognition, according to the crystallographic structure of LytA in complex with choline. However, the affinity for choline is much lower for Skl than for LytA and Pal, and the same occurs with the propensity to form dimers upon choline binding, which could at least partially explain the distinct killing activities. The characterization of the solution structures of Pal and Skl using small angle X-ray scattering (SXAS) in combination with in-silico 3D modeling of the isolated domains disclosed a similar disposition of the two domains within the monomers of the three amidases, as well as of the monomers within the dimers formed at saturating concentrations of choline. Moreover, the EADs showed a similar size and type of folding, which altogether excludes a strong impact of the lysin overall structure on the activity. We built next two chimeras (QSLA and QLAS) by swapping of Skl and LytA domains, and confirmed that the native features of the wild-type domains were preserved in the chimeras. Outstandingly, evaluation of their bactericidal activities showed that lethality of QSLA -made of the EAD of Skl and the CBD of LytA¿ on different pneumococcal strains excess that of LytA by orders of magnitude. By contrary, the chimera QLAS -made of the EAD of LytA and the CBD of Skl- was barely active at the same lysin concentration. Importantly, the chimeras showed comparable specific activities when acting on purified cell walls, which were in the order of LytA activity. These results seem compatible with the notion that QSLA and LytA might preferentially act on different locus of the cell wall when acting on bacterial cells, in spite of breaking the same type of bond and having the same CBD. Such selectivity would arise from either the nature of the EAD or the whole network of interactions created by a given combination of EAD and CBD with the intact cell wall. In conclusion, the study provides new clues on the crucial role played by module combination on the anti-infective potential of lysins, and how the fusion of the EAD from a hardly active lysin with the appropriate CBDs can produce extremely efficient enzybiotics.
DescriptionThe 2nd International Symposium on Antimicrobial Hydrolytic Enzymes (The Lysin Meeting) New York, 6-8 of Novembre (2018) .- https://www.thelysinmeeting.com/symposium-information/#event-organizers
Appears in Collections:(CIB) Comunicaciones congresos
(IQFR) Comunicaciones congresos
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