Metal-sensing in Salmonella: A model for targeting a network that differentiates metals

Metals have been used to control microbes in agriculture, food handling, domestic hygiene, medicine and more broadly as an additive to preserve perishables. The industrial partner, Procter and Gamble, have on-going programmes to develop new metal-based biocides to replace existing preservatives and to increase the efficacy of current metal-based anti-microbials (ensuring compliance with shifting legislative guidelines). Ionophores can help minimise the amount of metal added to products and there is interest in using more subtle combinations of metals and/or chelators. Historically, the exploitation of metals has been empirical but the discovery of natural metal-based antimicrobial mechanisms and of bacterial systems that sense and adapt to metals, presents opportunities to improve these additives through mimicry and subversion respectively.

Metals are implicated in several natural anti-microbial mechanisms. For example hosts and pathogens have evolved to compete for iron. Copper is pumped into the phagolysosomal compartment of macrophages about eight hours post infection most probably as a biocide to drive the Fenton reaction. Neutrophils are thought to starve microbes of zinc and manganese by releasing the metal-binding protein calprotectin. Bacteria (in common with all other types of cells) have evolved elaborate homeostatic mechanisms to balance the buffered intracellular concentrations of various metals within critical thresholds; critical to ensure that vast numbers of metalloproteins acquire their correct metal-cofactors. Central to such metal homeostasis is a set of metal-sensing proteins that detect when the buffered limits have been exceeded. The sensors trigger expression of proteins that restore the correct metal-balance. Having discovered bacterial metal-sensors and identified properties that determine which metals they 'can' sense in vitro, the next step is to investigate how metal-specificity in vivo (the metals the sensors 'do' sense inside cells) is a shared function of a set of metal-sensors. We will begin to model the network of interactions between the different sensor proteins. This programme will explore a fundamental question central to the cell biology of metals, coincidentally providing insight needed to formulate additives that subvert bacterial metal-sensing networks.

Five transcriptional regulators that detect metals have been characterised in S. Typhimurium (CueR, GolS, Fur, MntR and Zur). Metal-effectors and operator-promoter targets can be predicted for a further five deduced sensors (hypothetically ZntR, NikR, RcnR, ModE and ArsR) exploiting sequence similarity and gene context. Predicted DNA-binding sites and metal-specificities will be validated by experiment, establishing which metals each sensor responds to under steady state conditions (generations of growth under maximum and minimum permissive concentrations of each metal). One additional CsoR/RcnR homologue plus five further MerR-homologues are also encoded in the genome and will be analysed to discover which ones are metal sensors.

Recombinant proteins will be expressed and purified to determine affinities (of the tightest sensory site in the first instance) for the metals that each sensor detects, affinities for the metals detected by the other sensors and affinities for DNA. This part of the programme will be aided by a linked PhD student, also funded by our industrial partners. In addition to advancing knowledge of the individual components of the set of metal sensors in a micro-organism of relevance to food production and processing, this programme will quantify the abundance of each metal sensor in vivo under each steady state metal condition to enable the generation of integrated mathematical models to describe metal-specificity of metal-sensing. An attractive feature is that some intracellular parameters (the buffered available concentration of metals for example) can be treated as common functions for all of the sensors, assisting the mathematical modelling.

The logical next step in our on-going (two decades) basic research programme to understand how bacteria sense and discern metals, is to model interactions between the individual metal-sensors. This is expected to discover why some DNA-binding, metal-responsive, transcriptional regulators are competent to respond (allosterically) to certain metals, but fail to do so in vivo. Coincidentally, this research aims to uncover combinations of treatments which subvert the metal-sensory network. For this reason we are extremely fortunate that an industrial sponsor, Procter and Gamble (P&G), has offered to partly finance this fundamental project in the form of an Industrial Partner Award (IPA). In addition the industrial partner is funding a linked PhD student who will work closely with the research associate appointed to this IPA. Moreover, P&G will supply additional expertise to aid the microbial systems modelling, and the latter stages of the project will use their in-house testing facilities.

P&G already exploit metal and/or metal-chelator-based antimicrobial treatments in consumer products for which they hold substantive market shares; as preservatives, in antibacterial cleaning agents and in personal hygiene products. This research programme has the potential to inform the development of more effective anti-microbial treatments exploiting less metal and/or combinations of metals, chelators and other agents. Durham University and Procter and Gamble have a strategic research relationship in which a Master Collaboration Agreement is already in place to cover Intellectual Property, confidentiality, publication and any subsequent commercial exploitation. Within the provision of any overriding terms of the research grant offer letter, these existing agreed terms will be used to govern impact relating to commercial exploitation. Development of this proposal has involved interaction between the University and Industrial partner in the form of visits, conference calls and of course extensive e-mail traffic to review draft documents. Throughout the programme there will be regular meetings and on-going electronic communication. All of these mechanisms will ensure that discoveries with industrial impact are exploited in the swiftest possible manner.

Other forms of impact, the trained personnel and public engagement activities, are noted in the pathways to impact statement. The publications that result from this research may also assist in the development of metal-based antimicrobial treatments for other purposes, for example in the control of plant pathogens (we have support from Syngenta in this area), and more broadly adding to the arsenal of approaches to protect the UK against disease.