Identification and characterization of novel AMPs/bacteriocins
Listeria monocytogenes is a ubiquitous, Gram-positive soil bacterium and a frequent contaminant of processed food products. Its abilities to withstand and grow under a wide range of environmental conditions (stress resistance to high/low temperature, pH, salt, etc.) and to form biofilms on different surfaces make L. monocytogenes a serious concern in the food processing industries. Moreover, L. monocytogenes is able to cause life-threatening infections in immuno-compromised persons and serves as a widely used model organism for intracellular pathogenesis.
In recent years, our group was involved in a European research consortium that has investigated the stress response L. monocytogenes to high pressure processing (HPP) used for food preservation. This allowed identification a number of genes as potential targets for pretreatment targets to improve the efficiency of high-pressure treatment (Duru et al., 2021a, 2021b, 2020; Nikparvar et al., 2021a, 2021b). Interestingly, one group of genes differentially regulated by HPP are mannose-specific phosphotransferase systems, which are known as receptors for AMPs belonging to class IIa bacteriocins of the pediocin family. As part of this research consortium, we have constructed sensor strains of human pathogens such as L. monocytogenes (Crauwels et al., 2018) and related non-pathogenic model organisms (e.g. Listeria innocua) that allow to monitoring of intracellular pH (Figure 1). The measurements are based on expression of fluorescent proteins that show changes in the fluorescent properties in a pH-dependent manner. Thus, rapid changes of the intracellular (neutral) pH of the sensor bacteria to the external pH in a more acidic or alkaline environment/buffer are indicative of a disruption of membrane integrity. For example, these sensor strains can be used to screen supernatants of bacterial strain collections for AMPs with pore-forming activity as recently demonstrated for a small library of raw milk isolates (Figure 1 and Desiderato et al., 2021).
In different projects involving both academic and industrial partners, these sensor bacteria are used to screen larger collections of bacteria, e.g. starter cultures for food production, to identify producers of (novel) bacteriocins. Alternatively, gene clusters for biosynthesis of bacteriocins and corresponding resistance mechanisms can be identified in genome and metagenome sequences databases using predictive software tools (Goldbeck et al., 2021b). Once identified, the bacteriocins produced by these bacteria, their mechanisms of action and receptors can be analyzed and characterized again using the developed sensor bacteria.
Figure: (A) Fluorescence imaging of the sensor strain Listeria innocua pNZ-Phelp-pHluorin (pHin; right) or the empty vector control strain (-: left). (B) Relative fluorescence units (RFU; excitation 350–490 nm; emission at 510 nm) of L. innocua pNZ-Phelp-pHluorin in buffer at the indicated pH with 0.005% CTAB for permeabilization. The ratio of fluorescence intensity (emission at 510 nm) after excitation at 400 or 470 nm can be used to determine membrane damaging activity of purified AMPs (C; pediocin PA-1 as an example) or in supernatants of a collection of bacterial isolates from bovine raw milk (D).