Expression and characterization of pantothenate energy-coupling factor transporters as an anti-infective drug target

Abstract

This study investigates the potential of energy-coupling factor (ECF) transporters as promising anti-infective targets to combat antimicrobial resistance (AMR). ECF transporters, a subclass of ATP-binding cassette (ABC) transporters, facilitate the uptake of B-vitamins across bacterial membranes by utilizing ATP as an energy source. Vitamins are essential cofactors for bacterial metabolism and growth, and they can either be synthesized de novo or absorbed from the environment. These transporters are considered promising drug targets, underscoring the need for further research to harness their medicinal potential and develop selective inhibitors that block vitamin uptake in bacteria. Herein, we focused on the ECF transporter for pantothenate (vitamin B5) from Streptococcus pneumoniae and the ECF transporter for folate (vitamin B9) from Lactobacillus delbrueckii as a reference protein. We also included the energizing module for pantothenate along with both full transporter complexes. Initially, we transformed and purified the transporters, followed by an assessment of their thermal stability under various buffer composition, pH, and salt concentrations. Additionally, we monitored the melting temperature over six days to confirm their stability for further assays. We then measured the binding affinities of six ECF inhibitors using surface plasmon resonance (SPR) and evaluated their inhibitory effects through in vitro assays, including bacterial growth assay, whole-cell uptake, and transport-activity assays. After determining cytotoxicity in two human cell lines, we established an in vivo infection model using Galleria mellonella larvae to further validate our findings.

Keywords: B‐vitamins; Galleria mellonella infection model; Streptococcus pneumoniae; antimicrobial resistance (AMR); energy‐coupling factor (ECF) transporters; pantothenate (vitamin B‐5); surface plasmon resonance.

FIGURE 1

Schematic representation of the ECF transporters subunits and mechanism of transport. The ECF transporters use the integral‐membrane S proteins (EcfS) to bind to a substrate and transport it into the cytoplasm in an ATP‐dependent process (Zhang, ; Zhang et al., 2014). Source: Created with BioRender.com.

FIGURE 2

Assessment of ECF‐PanT stability with and without DMSO. (a) Stability of ECF‐PanT, ECF‐FolT2, and ECF‐module was evaluated by monitoring melting temperatures (T _m) at room temperature over 6 days with 0% and 5% (v/v) DMSO. The data represent the mean and standard deviation from two independent experiments. (b) To assess protein stability, ECF‐PanT samples were incubated at room temperature in an optimal buffer (50 mM KPi pH 7.5, 50 mM NaCl, 0.05% DDM) with or without 5% DMSO. UV absorption at 280 nm was measured using size exclusion chromatography (SEC) to determine the consistency of protein absorbance over time. Measurements were performed in technical duplicate to ensure accuracy.

FIGURE 3

Transport activity assay of reconstituted S. pneumoniae ECF‐PanT with a protein‐to‐lipid ratio of 1:125. (a) Time‐course traces of pantothenate uptake over 4 min, showing transport activity in the presence of inhibitors with 5 mM Mg‐ATP. The controls were 5 mM Mg‐ADP (negative control) and 5 mM Mg‐ATP (positive control) alone. The data demonstrate the uptake rates for each condition and was performed in duplicates. (b) Percentage inhibition of pantothenate uptake in presence of ECF inhibitors 1–6, calculated relative to the activity observed with 5 mM Mg‐ATP.

FIGURE 4

Simple survival analysis (Kaplan–Meier) was performed using GraphPad Prism. (a) The larvae were injected with S. pneumoniae (S.p) at OD₆₀₀ of 1.5 in the absence and presence of compounds 2–4 at 10 μM. The control groups are with no injection and PBS. (b) The larvae were injected with S. pneumoniae (S.p) at OD₆₀₀ of 1.5 in the absence and presence of compounds 2–4 at 50 μM. The control groups are with no injection and PBS.