Antibiotic uptake through porins located in the outer membrane of Gram-negative bacteria

ABSTRACT

Introduction: Making selective inhibitors of novel Gram-negative targets is not a substantial challenge – getting them into Gram-negative bacteria to reach their lethal target is the bottleneck. Poor permeability of the antibiotic requires high concentration causing off target activity. The lack of simple experimental techniques to measure antibiotic uptake as well as the local concentration at the target site creates a particular bottleneck in understanding and in improving the antibiotic activity.

Areas covered: Here we recall current approaches to quantify the uptake. For a few antibiotics with known evidence for channel-limited permeation, the flux across a single OmpF or OmpC channel has been measured. For a typical concentration gradient of 1 µM of antibiotics the uptake varies between one up to few hundred molecules per second and per channel.

Expert opinion: The current research effort is on quantifying the flux for a larger list of compounds on a cellular (mass spectra, fluorescence) or at single channel level (electrophysiology). A larger dataset of single channel permeabilities under various condition will be a powerful tool for understanding and improving the activity of antibiotics.

KEYWORDS:

• Antibiotic uptake
• electrophysiology
• fluorescence
• mass spectrometry
• outer membrane proteins
• permeability
• porins

Article highlights

• The poor outer membrane permeability in Gram-negative bacteria is a bottleneck for the activity of small molecule antibiotics.

• Membrane channel-forming proteins in the outer cell wall called porins facilitate the uptake. The diffusion across a channel depend on size, shape and in a complex manner on the interaction between the molecule, the buffer and the channel.

• Mass spectrometry allows to quantify the uptake but requires careful separation of the taken-up fraction from the remaining molecules. On a single cellular level spectro-fluorometry allows to follow the uptake kinetics without separation of fluorescent antibiotics. In both cases equilibrium can be reached within minutes.

• Single channel recording can count the uptake and in combination with molecular modelling reveals the rate-limiting steps. Typical numbers ranging from one to a few hundred per second and per channel which would lead to an instantaneous equilibrium.

• To date only a few transport numbers have been measured. More quantitative numbers in particular for various compartments will help to identify and overcome the rate-limiting steps in antibiotic uptake into bacteria.

This box summarizes key points contained in the article.

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

Declaration of interest

The author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Additional information

Funding

The research leading to these results was conducted as part of the Translocation consortium and has received support from the Innovative Medicines Initiative Joint Undertaking under Grant Agreement No. [115525]; resources which are composed of financial contribution from European Union’s seventh framework program [FP7/2007–2013]; and EFPIA companies. We further received support from the JPIAMR network RESET-ME [BMBF - 01KI1827B]; and the JPIAMR Virtual Institute Translocation-Transfer [BMBF - 01KI1828].