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

Lead Research Organisation: Durham University
Department Name: Biosciences

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

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Foster AW (2014) Metal preferences and metallation. in The Journal of biological chemistry

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Schilter D (2019) Finding the right match in Nature Reviews Chemistry

 
Description The network of metal sensors has been characterised in Salmonella, providing insight into what enables a DNA-binding metal-sensor to detect a specific metal inside a cell. In turn, these findings informed the creation of a DNA-binding metal-sensor from a transcriptional regulator that normally detects formaldehyde (Journal of Biological Chemistry, 2015).

By measuring metal-affinities, DNA-affinities of apo- and metalated sensors, plus the number of sensor molecules per cell, it has become possible to combine these parameters to model metal specificity of metal-sensing transcriptional-regulators. This established that the bona fide sensor for each metal is simply the most sensitive in the set of Salmonella sensors for that cognate ion (Nature Communications, 2017). However, these data also established that perfect metal specificity only operates when the buffered metal concentrations are fine tuned to within a narrow range (about one order of magnitude). Outside this narrow window of concentrations, other sensors are liable to mal-respond to the wrong metal, and indeed this was shown to be the case when cells were transiently shocked with elevated metal concentrations (Nature Communications, 2017). The order of mal-responses matched the thermodynamic predictions. These data establish that the metal sensory system of bacteria is vulnerable to subversion by combinations of metal chelants and ionophores, offering an explanation for why metal excess and deficiency appears to be a microbial 'Achillees heel' exploited in nutritional immunity and in a range of antimicrobial treatments. Synergistic combinations of metal chelants have been discovered.

Finally, this work has provided major insight into protein metalation. There is a challenge for metalloenzymes to acquire their correct metals because some inorganic elements form more stable complexes with proteins than do others. These preferences can be overcome provided some metals are more available than others. However, while the total amount of cellular metal can be readily measured, the available levels of each metal have been more difficult to define. Metal-sensing transcriptional regulators are tuned to the intracellular availabilities of their cognate ions. In this programme, the standard free energy for metal complex formation to which each sensor, in a set of bacterial metal sensors, is attuned, has been established. This has shown that the less competitive the metal, the less favourable the free energy and hence the greater availability to which the cognate allosteric mechanism is tuned. Crucially, comparing these free energies with values derived from the metal affinities of a metalloprotein reveals the mechanism of correct metalation. This work was published in Nature Chemical Biology in 2019, which was recommended by two members of Faculty 1000 and highlighted by an article in Nature Chemistry, We are working to generate a metalation calculator to allow researchers in academia and industry alike, to determine the metalation state of proteins (and other molecules) inside living cells. For P&G this could include ionophore ingredients with implications for product preservation. For industrial biotechnologists, this could include half of enzymes (the metallo-enzymes) many of which drive critical bioprocesses and bio-transformations.
Exploitation Route Refer to impact section. The work also has relevance to the development of new antimicrobials and to the optimisation of metal supply for metalloenzymes in Industrial Biotechnology.
Sectors Chemicals,Manufacturing, including Industrial Biotechology,Retail

 
Description This BBSRC IPA research grant with Procter and Gamble has formed the foundation for a series of additional related programmes on microbial-metal-systems in Durham, funded to the tune of between £0.5 and £1 million by Procter and Gamble. These programmes have supported four related PhD students and (at least) three research assistants. The insights from this entire body of research are being exploited by the Insdustrial partner in relation to the formulation of fast moving consumer goods. Regular meetings between the Durham metals-team and Procter and Gamble staff from the UK and from Cincinnatti are ongoing (next funding planning meeting scheduled for April 2020). This work underpinned a successful application for a phase II BBSRC NIBB and ongoing work to develop a metalation calculator for widespread use in bioscience and biotechnology. In February 2020, BBSRC have been generating an impact case related to this award for inclusion in a larger publication.
First Year Of Impact 2018
Sector Manufacturing, including Industrial Biotechology
Impact Types Economic

 
Description 12 month JGW GS programme
Amount £112,500 (GBP)
Organisation Procter & Gamble 
Sector Private
Country United States
Start 04/2016 
End 03/2017
 
Description BBSRC NIBB phase II
Amount £1,360,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 03/2019 
End 02/2024
 
Description MB studentship
Amount £64,500 (GBP)
Organisation Procter & Gamble 
Sector Private
Country United States
Start 09/2014 
End 09/2018
 
Description MM studentship
Amount £44,000 (GBP)
Organisation Procter & Gamble 
Sector Private
Country United States
Start 09/2015 
End 09/2019
 
Description MS programme
Amount £318,646 (GBP)
Organisation Procter & Gamble 
Sector Private
Country United States
Start 12/2014 
End 12/2017
 
Description PhD studentship CP
Amount £60,000 (GBP)
Organisation Procter & Gamble 
Sector Private
Country United States
Start 11/2011 
End 10/2015
 
Description RM studentship
Amount £99,000 (GBP)
Organisation Procter & Gamble 
Sector Private
Country United States
Start 10/2012 
End 09/2016
 
Description Research Fellowships - Royal Commission 1851 - to TRY
Amount £250,000 (GBP)
Organisation Royal Commission for the Exhibition of 1851 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2019 
End 09/2022
 
Title Calculating in vivo metalation from the sensitivities of metal-sensors. 
Description Equations, software and spreadsheets to calculate the sensitivities of metal sensors and in turn to determine metal availability inside a cell. This enables the calculation of metalation inside living cells with implications for engineering a half of the reactions of life. Includes: 1. Excel Spreadsheet (with instructions) to enable calculation of fractional DNA occupancy. 2. MATLAB codes (with instructions), to determine the buffered metal concentration from given value(s) of ?D or ?DM. 3. Supplementary equations and unique Supplementary Note 2 references in support of the above. 
Type Of Material Technology assay or reagent 
Year Produced 2019 
Provided To Others? Yes  
Impact Too early 
URL https://www.nature.com/articles/s41589-018-0211-4.pdf
 
Title Clones encoding proteins of metal homeostasis 
Description Clones distributed in 2014 include pETInrS, pETNmtR, pETKmtR, pETCucA, pBAD24-ssCucA-GFP-SsrA 
Type Of Material Biological samples 
Year Produced 2014 
Provided To Others? Yes  
Impact Supplied to reseachers in Bologna, UMass Boston, Universite de Riems 
 
Title Computational method to determine DNA occupancy by metal sensors 
Description Computational method to determine DNA occupancy by metal sensors 
Type Of Material Technology assay or reagent 
Year Produced 2017 
Provided To Others? Yes  
Impact Ongoing industrial collaboration. 
URL https://www.nature.com/articles/s41467-017-02085-z#Sec23
 
Title Crystal coordinates of FrmR 
Description Crystal coordinates of FrmR PDB: 5LCY 
Type Of Material Database/Collection of data 
Year Produced 2016 
Provided To Others? Yes  
Impact PDB entry: 5LCY 
URL http://www.rcsb.org/pdb/explore.do?structureId=5lcy
 
Description Chelation Therapy in the Washing Machine 
Organisation Procter & Gamble
Country United States 
Sector Private 
PI Contribution We funded and administered a Business Interaction Voucher in a collaboration with Nick Jakubovics, Newcastle University (BIVMiB012)
Collaborator Contribution Biofilms are a major problem in all sorts of industrial settings, including bioprocessing facilities. Mechanical biofilm removal is not always possible due to the chemical and physical properties of the contaminated surface and the use of chemical agents is the most appropriate approach for biofilm control. This proposal involves working with an industrial partner (P&G) to develop novel chemical technologies for biofilm removal at low temperatures on a complex surface (laundry). We envisage that successful outcomes can be translated to biofilm control in many different settings including bioprocessing plants.
Impact Project on-going
Start Year 2016
 
Description Interaction with Industry partner 
Organisation Procter & Gamble
Country United States 
Sector Private 
PI Contribution Regular teleconferences and exchange visits, exchange of data and materials.
Collaborator Contribution Regular teleconferences and exchange visits, exchange of data and funding of multiple PhD students and other staff with only an approximate value given above relating to new activities in the most recent ca one year.
Impact Joint publication in 2014 and in subsequent years. Other outcomes are mostly subject to NDA's.
Start Year 2014
 
Description Interaction with industrial sponsor 
Organisation Procter & Gamble
Country United States 
Sector Private 
PI Contribution Regular teleconference meetings (in excess of 50 over 24 months including all forms of interaction) with industrial collaborator Reciprocal exchange of materials and biologics with industrial collaborator Reciprocal visits with industrial collaborator (associated PhD students and academic staff etc) Analytical services provided for industrial partner and others, and vice versa
Collaborator Contribution See above
Impact Ongoing and confidential
Start Year 2012
 
Description Author profile associated with Journal of Biological Chemistry Review. 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Refer to URL to see the item

See next response
Year(s) Of Engagement Activity 2014
URL http://www.jbc.org/content/289/41/28095/suppl/DCAuthor_profile
 
Description BBSRC showcase event, Edinburgh 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Poster sparked discussion.

Further interaction with industrial partner
Year(s) Of Engagement Activity 2012
 
Description EuroBIC bioinorganic chemistry conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Disseminated knowledge about the cell biology of metals which sparked questions and discussion.
Year(s) Of Engagement Activity 2018
URL https://www.birmingham.ac.uk/facilities/mds-cpd/conferences/eurobic/index.aspx
 
Description FASEB, Lake Tahoe, Trace Metals in Health and Disease 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Dissemination of knowledge about the cell biology of metals which sparked questions and discussion.
Year(s) Of Engagement Activity 2018
 
Description Invited lecture by RA Deenah Osman on Metals in Biology in Hawaii 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Invited lecture on the generation of a metals sensor given by RA Deenah Osman.
Year(s) Of Engagement Activity 2015
 
Description Invited lecture, Newcastle University 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact Invited lecture on metals in biology
Year(s) Of Engagement Activity 2015
 
Description Invited lecture, Umea, Sweden 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Public lecture on metals in biology
Year(s) Of Engagement Activity 2015
 
Description Invited seminar by RA (Deenah Osman) at Procter and Gamble, Ohio USA 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Lecture on metals in biology to the Procter and Gamble Mason Business Center in Ohio, USA, given by Deenah Osman and followed by a series of smaller meetings and discussions.
Year(s) Of Engagement Activity 2015
 
Description Invited speaker, BBSRC NIBB BioProNET 6th annual science meeting, Manchester 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact This aim of the event was to promote collaboration between industry and academia and advertsie the opportunities arising from the E3B BBSRC NIBB.
Year(s) Of Engagement Activity 2019
URL http://biopronetuk.org/6th-annual-science-meeting/
 
Description Invited talk at International Conference on BioInorganic Chemistry, Interlakken, Switzerland 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact The results of our research were described which sparked questions and discussions immediately afterwards and ongoing by e-mail.
Year(s) Of Engagement Activity 2019
URL https://www.chem.uzh.ch/dam/jcr:d809e5d0-e81b-42d0-a1c9-175c8e13e958/ICBIC19_ScientificProgram_v5.pd...
 
Description Metal-Related Antimicrobials Workshop 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact We organised a workshop to bring together academics and industry to discuss opportunities in collaborative reseach: http://prospect.rsc.org/MiB_NIBB/wp-content/uploads/2015/11/Final-programme.pdf

Led to new collaborations and a volume on "Microbiology of Metals Ions" 2016 volume 70: https://www.elsevier.com/books/microbiology-of-metal-ions/author/978-0-12-812386-7
Year(s) Of Engagement Activity 2015
URL http://prospect.rsc.org/MiB_NIBB/wp-content/uploads/2017/01/Metals-in-Biology_Elements-of-the-bioeco...
 
Description Metal-related antimicrobials BBSRC NIBB workshop (November 2015) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact BBSRC NIBB event with representatives from agritechnology business, consumer goods industry, pharmaceutical companies, policy and standards agencies, industrial biotechnology companies to explore the opportunities for metal-related antimicrobials (PI and RA attended).
Year(s) Of Engagement Activity 2015
 
Description Metals in Biology Community Event 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact We organised a workshop involving academic and industry to show case funded projects as exemplars to instigate future collaborations.
Year(s) Of Engagement Activity 2016
URL http://prospect.rsc.org/MiB_NIBB/metals-in-biology-network-community-meeting/
 
Description Metals in Bioprocessing multiple BBSRC NIBB event (Hexham) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact Interaction with Bio-processing industries. PI and RA both attended and PI gave a presentation.
Year(s) Of Engagement Activity 2015
 
Description NJR Bangalore 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Invited keynote speaker, 6th International conference on Metals in Genetics, Chemical Biology and Therapeutics, Bangalore, India.
Year(s) Of Engagement Activity 2016
 
Description NJR Biometals Dresden Invited Talk 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Invited speaker, Biometals Conference 2016, Dresden, Germany.
Year(s) Of Engagement Activity 2016
 
Description NJR Biophysical Society Invited Lecture 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Invited speaker, British Biophysical Society 2016 Biennial Meeting "Metals in Biology" microsymposium, Liverpool.
Year(s) Of Engagement Activity 2016
 
Description NJR GRC Vermont 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Dissemination of knowldege about the Cell Biology of Metals which sparked questions and discussion.
Year(s) Of Engagement Activity 2017
URL https://www.grc.org/cell-biology-of-metals-conference/2017/
 
Description NJR Queen Mary University of London 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Undergraduate students
Results and Impact Delivered the Wills lecture. This sparked questions afterwards followed by requests for information including ICP-MS analyses (for example).
Year(s) Of Engagement Activity 2017
URL http://www.sbcs.qmul.ac.uk/research/researchseminars/speciallectureseries/#4
 
Description NJR San Diego ACS conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Invited speaker, American Chemical Society National Meeting Spring 2016, San Diego California.
Year(s) Of Engagement Activity 2016
 
Description NJR TUM Munich 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Delivered lecture and interacted with postgraduate students and staff at a series of meetings.
Year(s) Of Engagement Activity 2017
 
Description National Institutes of Health, Wshington DC, presentation 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Disseminated knowledge of the cell biology of metals which sparked questions and discussion.
Year(s) Of Engagement Activity 2018
 
Description Nature Microbiology Community Blog 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Invited blog linked to a paper in Nature Communications
Year(s) Of Engagement Activity 2017
URL https://naturemicrobiologycommunity.nature.com/users/71254-deenah-osman/posts/24964-sensing-the-diff...
 
Description Penn State Summer Symposium in Molecular Biology 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Disseminated knowledge of the cell biology of metals which sparked questions and discussion.
Year(s) Of Engagement Activity 2018
 
Description RSC Inorganic Biochemistry Discussion Group including industrial uses of metalloenzymes (York, April 2015) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Discussion of metaloenzymes and their uses (RA attended).
Year(s) Of Engagement Activity 2015
 
Description Tetrapyrroles GRC, Rhode Island 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Disseminated knowledge of the cell biology of metals which sparked questions and discussion.
Year(s) Of Engagement Activity 2018
URL https://www.grc.org/chemistry-and-biology-of-tetrapyrroles-conference/2018/