# Functional analysis of thiamine pyrophosphate-responsive riboswitches in human bacterial pathogens of the ESKAPE group using a dual-luciferase reporter gene assay

## TL;DR

This study explores how TPP riboswitches in dangerous bacteria can be targeted for new antibiotics, using a reporter gene assay to test their function and response to molecules like pyrithiamine.

## Contribution

The paper introduces a dual-luciferase reporter gene assay to functionally analyze TPP riboswitches in ESKAPE pathogens and identifies structural reasons for pyrithiamine resistance.

## Key findings

- Most predicted TPP riboswitches in ESKAPE bacteria are functional and respond to TPP.
- Pyrithiamine does not block all TPP riboswitches, unlike in Escherichia coli.
- Site-directed mutagenesis revealed structural bases for pyrithiamine resistance in K. pneumoniae.

## Abstract

Thiamine pyrophosphate (TPP)-responsive riboswitches are genetic elements in bacteria that regulate the expression of genes coding for proteins involved in the biosynthesis and transport of thiamine (vitamin B1). Following uptake, cytoplasmic thiamine is converted to TPP, which serves as a cofactor for enzymes of central metabolic pathways such as glycolysis, the tricarboxylic acid cycle, and the pentose phosphate pathway, and it is the level of TPP (and not thiamine) that is sensed by TPP riboswitches. TPP riboswitches are the most widespread riboswitches in bacteria. Their key roles in metabolism combined with their absence in humans make them potential targets for antibiotics, whereby the focus of the present study was pathogenic bacteria of the ESKAPE group: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. As a first step toward the development of novel TPP riboswitch-targeting antimicrobials to treat infections caused by ESKAPE organisms, we characterized various TPP riboswitches present in these bacteria. We developed a dual-luciferase reporter gene assay to monitor riboswitch activity and found that most of the predicted TPP riboswitches indeed were functional regulators and responded to TPP. In contrast to the Escherichia coli thiC TPP riboswitch, TPP riboswitches from ESKAPE bacteria were found not to respond to the synthetic thiamine analog pyrithiamine. One TPP riboswitch of K. pneumoniae was examined in detail with regard to the effect of pyrithiamine. Site-directed mutagenesis experiments identified specific nucleotides responsible for the non-response to pyrithiamine, and this should be useful in developing novel TPP riboswitch-targeting antimicrobials.

Riboswitches are RNA molecules that control important processes in bacteria. Infections with pathogens of the ESKAPE group are common, and we are trying to find new ways to fight these bacteria. Small molecules can be designed to bind to riboswitches and optimally block their activity. In the present work, we have analyzed the thiamine pyrophosphate (TPP) riboswitches of ESKAPE pathogens with respect to small molecule binding. For this purpose, we developed a dual-luciferase reporter gene assay. Most of the predicted TPP riboswitches were indeed functional regulators and are thus targets for new anti-infectives. The small molecule pyrithiamine does not block all TPP riboswitches tested, and we found a structural basis for this behavior.