Detection by fluorescence microscopy of N-aminopeptidases in bacteria using an ICT sensor with multiphoton excitation: Usefulness for super-resolution microscopy

Abstract

Bacterial proteases are relevant in pathological processes such as the survival, growth and development of microorganisms. In particular alanine amino peptidase (pepN), which is present in Gram (−) bacteria, is the only peptidase responsible for the ATP-independent degradation of cytosolic proteins in E. coli. These peptidases, including pepN, might be useful targets to fight bacterial infections, which are difficult to treat due to the increase in antibiotic resistance, as well as for diagnosis. In this work, we propose a new methodology for the identification of pepN-expressing bacteria by using a specific substrate, namely, DCM−NH−Ala. Substrate hydrolysis by pepN produces a sharp increase in the fluorescence band with peak at 662 nm when excited by a single photon at 480 nm or by two NIR photons (at approximately 800 nm). The emission kinetics are dependent on the intracellular pepN concentrations, which provide a powerful tool for detecting diverse virulent bacteria in a few minutes and with the inherent advantages of two-photon excitation. We resolved the enzymatic kinetics, obtained the Michaelis-Menten parameters (e.g., KM, vmax, and kcatalytic) and studied the photophysics of the released fluorophore DCM–NH2. In addition, DCM–NH2 meets the requirements for use in super-resolution microscopy. In bacteria with high pepN activity, the probe quickly initiates an enzymatic reaction at specific sites located on the bacterial membrane and some structures inside the bacterial body.