Carbon Monoxide and Carbon Monoxide-Releasing Molecules: Impacts on Enterobacterial Physiology

The emergence of carbon monoxide (CO) as the third ‘gasotransmitter’ (along with the more reactive nitric oxide (NO) and hydrogen sulfide (H2S)) in biology has led to a vast array of studies highlighting the anti-inflammatory, anti-proliferative and vasodilatory effects of the molecule, especially in mammalian studies of disease. The advent of CO-releasing molecules (CORMs) as means to safely deliver CO to biological systems has brought scope for the use of these molecules in microbiology, in particular, as antimicrobial compounds. It has been presumed that these activities are mediated by the release of CO. The antimicrobial activities of a number of CORMs have been demonstrated, mostly through the use of ruthenium-containing CORMs, such as CORM-3. Although these are effective antimicrobials, the use of a biologically foreign metal species renders these compounds less desirable for use in the clinic. In this thesis, a novel manganese-containing CORM, [Mn(CO)4{S2CNMe(CH2CO2H)}], CORM-401, is introduced and its actions against the gram-negative bacterium Escherichia coli are characterised. The compound has greater CO release characteristics that ruthenium CORMs, yet when introduced to bacterial cultures, proves less effective at killing. The CORM (inferred from the presence of manganese in excess of normal cellular pools) is shown to accumulate to high levels inside cells. CO is shown to bind to respiratory oxidases, but inhibition of respiration by CORM-401 is not observed; instead, stimulation of respiration in whole cells is seen. Transcriptomic analysis shows perturbation of cell stress responses and levels of cell-stress response proteins are elevated. CORM-401 leads to gross changes in ion and metal gradients across the cell membrane. It is hypothesised that the metal species of CORM compounds mediate the majority of the toxic effects reported in the literature. Further evidence that the interaction of CO released from CORMs with conventional haem targets cannot wholly explain the antimicrobial effects is also demonstrated in this thesis by the effects of CORM-3 on a haem-deficient mutant (hemA) of E. coli. This thesis also provides the first multi-level analysis of the effect of CO, without the intervention of CORMs, on E. coli cells. Transcriptomic analysis indicates that CO perturbs central metabolism and amino acid metabolism; in particular, gross perturbation of iron homeostasis is described, an effect confirmed by quantification of elevated siderophore production and sensitivity to iron-chelating compounds. Finally, the mechanism(s) of CORM-401 transport are investigated. Transport of CORMs into bacterial cells is an important aspect of their antimicrobial action that is often overlooked. Analysis of uptake is challenging, since radiolabelled CORMs are not available. However, assays of cellular uptake of CORM-401 (by metal detection) hint at a diffusion-based mechanism, although active transport systems cannot be disregarded at this time.