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Structural and Binding Properties of the Active Cell Wall Hydrolase RipA from <i>M. tuberculosis</i>, a Promising Biosensing Molecule for Early Warning Bacterial Detection

[ Vol. 29 , Issue. 24 ]


Flavia Squeglia, Daniela Marasco, Alessia Ruggiero, Genni Testa, Luciana Esposito and Rita Berisio*   Pages 4282 - 4292 ( 11 )


Background: Peptidoglycan is an essential component of the cell wall in all bacteria. In particular, the cell walls of Gram-positive bacteria are composed mostly of a thick layer of peptidoglycan. Its accessibility has important implications for their sensing in whole bacterial detection methodologies. Indeed, there is an urgent demand for rapid tests which can identify whole bacteria, e.g., directly at the point of care.

Objective: The aim of this work is to explore the suitability of RipA, a key cell division protein of M. tuberculosis, for whole cell biosensing of Gram-positive bacteria.

Methods: We here conducted Molecular Dynamics (MD) studies aimed at the understanding of the structural and dynamic features of active RipA and at the design of a suitable bioreceptor. Based on these studies, we engineered a RipA variant for covalent oriented immobilisation on golden surfaces and are able to bind peptidoglycan, albeit without degrading it. Surface Plasmon Resonance (SPR) was employed to check the ability of functionalized golden chips to recognize whole bacteria.

Results: MD analyses elucidated the structural details of the active form of RipA and suggested that this enzyme, once inactivated, presents a rigid and well-exposed peptidoglycan recognition cleft. We engineered RipA for proper oriented immobilisation on golden chips for SPR studies. Results show that once chemically coupled to a golden chip, the developed RipA-based bioreceptor is able to detect B. subtilis, used as a model in a concentration-dependent mode.

Conclusion: Results highlight the potential of the engineered molecule to be integrated in the development of early warning biosensors for Gram-positive contamination in clinical diagnosis or food-borne infections.


Protein, peptidoglycan, structure, binding, SPR, RipA.


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