Identification of a candidate CO2 sensor from Helicobacter pylori

Abstract

Carbon dioxide (CO2) is a ubiquitous metabolite. It is fundamental in pathogenesis, involved in the activation of signalling pathways. Several species of human pathogens harbour CO2 sensing mechanisms that allow for the coupling of virulence factor expression to host CO2 levels, facilitating colonisation and persistence. Helicobacter pylori is an example of a human pathogen for which CO2 is especially important in its biology – requiring CO2 for growth and generating CO2 as a result of urease activity. However, what remains unclear are the details of the mechanisms underpinning CO2 sensing and the identity of CO2 sensors.

A candidate mechanism by which CO2 sensing may occur is the direct modification of regulatory proteins, termed carbamylation, entailing the nucleophilic attack of a neutral N-terminal α-amino group or ε-amino group of Lys onto CO2, forming a carbamate. While carbamates observed to date, such as those in haemoglobin and RuBisCO, have significant functional roles, their systematic identification has been limited by their lability and the lack of a tool for their direct investigation, until recently.

This thesis sought to assess the validity of the H. pylori nickel-responsive regulator (HpNikR) as a CO2 sensor, using carbamylation as a mechanism to achieve this, given its pleiotropic regulatory activity, the importance of Lys residues in its DNA binding, and its responsiveness to the environmental cues of Ni(II) availability and pH. Carbamylation would affect DNA binding, which in turn would affect gene expression and allow H. pylori to adapt to fluctuating CO2 levels, potentially in accordance with urease activity. Modification of a conserved Lys was detected, DNA binding assays showed this modification reduced DNA affinity, and this effect was reduced in a mutant insensitive to carbamate formation. This suggests that HpNikR may indeed behave as a CO2 sensor, potentially coupling gene transcription to this additional environmental cue.