A novel mechanism of protein uptake in Gram-negative bacteria

Feeding a rapidly growing world population is one of this century's key scientific challenges. Bacteria are responsible for 20-30% of the > £100 billion per annum lost worldwide to disease and spoilage of crops caused by plant pathogens. For example, the soft rot pathogens Pectobacterium atrosepticum and Dickeya spp., the most damaging bacterial pathogens of potato in Europe, are responsible for the greatest losses and down-grading of stocks of any pathogen in this crop. Traditional measures for reducing losses include the breeding and introduction of resistant potato varieties, chemical treatments and plant hygiene measures aimed at minimising losses due to spoilage during storage. However, for these bacterial pathogens, no good resistant varieties are available and there are no effective chemical treatments. Climate change will only make the problem more severe as these bacteria thrive in warmer moister conditions. It is essential, therefore, that new methods of control are investigated
Despite the economic importance of bacterial plant pathogens, we lack understanding of some of the key mechanisms utilised by these bacteria during infection and so are limited in the interventions that are available to us. Our work aims to address this problem by investigating a key mechanism of iron acquisition from the host during infection. The availability of iron can be a deciding factor in the success or failure of microbial infection.
We have discovered that some bacterial soft rot pathogens are able to acquire iron directly from an iron-containing host protein called ferredoxin. We have identified the protein on the bacterial cell surface that is responsible for this as well as other proteins that are likely to be involved in the process of iron acquisition. The aim of this work is to understand the mechanism through which these bacterial proteins are able to bind ferredoxin and acquire iron from it. If we can fully understand this mechanism then it should be possible to create disease resistant plants that possess an engineered ferredoxin variant to which bacteria cannot bind or from which they cannot acquire iron.

The colicin-like bacteriocins, potent multidomain protein antibiotics, have evolved to efficiently cross the outer membrane (OM) of Gram-negative bacteria by parasitizing existing nutrient uptake pathways that enable bacteria to actively transport large substrates such as iron-siderophores. As such these uptake systems represent an Achilles' heel in the bacterial OM that could be exploited for antibiotic development. It is a central dogma of colicin biology that after binding tightly to an OM receptor, colicins are able to exploit specific uptake pathways by threading an intrinsically unstructured translocation domain (IUTD) through the barrel of a co-receptor. This enables the delivery a binding epitope to the periplasm which functions to recruit the Tol or Ton complexes that mediate translocation across the OM. We have discovered a novel group of bacteriocins, active against Pectobacterium spp., which do not possess an IUTD and therefore must translocate across the OM through a fundamentally different mechanism. Interestingly, these bacteriocins, pectocin M1 and M2 exploit an OM receptor through which the soft-rot phytopathogens Pectobacterium atrosepticum and Pectobacterium carotovorum bind and obtain iron from the major iron-containing plant protein ferredoxin. These novel bacteriocins consist of a plant-like ferredoxin domain linked to an enzymatic colicin M-like cytotoxic domain that kills cell through the cleavage of lipid II and the subsequent arrest of cell wall synthesis. The ferredoxin domain of these bacteriocins substitutes for the portion of colicin M required for receptor binding and translocation, presumably fulfilling its role by parasitizing an existing system for ferredoxin uptake into the periplasm. The aim of this project is therefore to structurally and functionally characterise a novel protein uptake pathway in Gram-negative bacteria and determine if this pathway is required for virulence.

The aims and objectives of the proposed research will contribute to advances in knowledge and understanding at the fundamental level. However, in the medium and long term this fundamental knowledge could have significant economic and societal impact in the following areas:
1. Development of novel antibiotics.
The major beneficiaries of our findings in this in this area will be biotechnology and pharmaceutical and companies who will have access to novel strategies for developing next generation antibiotics against Gram-negative bacteria for which there are few therapeutic options. Direct economic and societal benefits will derive from the development of novel antibiotics, which will reduce morbidity and mortality from bacterial infections and will reduce costs associated with failed antibiotic treatment. The true economic cost of not having effective antibiotics is difficult to estimate, since this would not only affect our ability to treat primary bacterial infections, but would also impinge on our ability to treat of a range of chronic conditions where antibiotics form an integral part of the treatment regime. For 'difficult to treat' Gram-negative bacteria such as Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae and Acinetobacter baumannii where the presence of an outer membrane severely limits the effectiveness of whole classes of antibiotics, there is an urgent need to develop novel antibiotics.
2. Development of disease resistant crops.
Biotechnology and agricultural companies will benefit from the availability of novel strategies to produce soft-rot-resistant potato varieties. As an indication of the potential market in novel soft-rot-resistant potato varieties, the farm gate value of potatoes in the UK is approximately £780 million. UK seed potato industry production (which is predominantly located in Scotland, with export markets worldwide) was valued last year at approx. £80-100 million. Potato growers, wholesalers retailers and processors will benefit from reduced losses from disease in the field and in storage (black-leg, soft rot etc.) Potato breeders, seed companies, biotechnology companies etc., will benefit from commercial exploitation in UK, European and particularly worldwide markets. Direct benefits to consumers (UK and worldwide) derive from reduced spoilage (waste). Food security considerations identify potential for enhanced food output in response to population growth.

To achieve maximum impact from our work useful intellectual property will be referred to the University IP Manager and the Business Development Manager. This will be evaluated for both patentability and commercial value and brought to the attention of the early stage biotech funders and possible commercial partners such as specialist biotech investor groups, of which there are several active in the UK, or UK or foreign biotech and pharmaceutical companies. We will seek arrangements that will maximise the chances of any such developments being successfully exploited. After appropriate IP protection, the data obtained will be made available through primary publications in open access scientific journals, presentations at conferences and in invited review articles. Many pharmaceutical companies are now actively pursuing collaborations with academics, for example GSK though their Discovery Partnerships with Academia scheme. We will engage with pharmaceutical companies directly through such schemes. In addition, the College of Medical, Veterinary and Life Sciences at the University of Glasgow holds and annual Industry day where potentially commercializable research is showcased to Biotechnology companies and other SMEs and commercial organisations. We will participate in this event to showcase our research to Biotechnology companies.