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Modular Construction of Bacteriophage for Diagnostic Systems

Introduction and Aim

Personal medicine and non-invasive biological measurements are the fastest growing areas in biotechnology. Viruses and bacteriophages are central to a number of biotechnology applications spanning diverse fields, from adenovirus for gene therapy in humans to M13 phages for use in lasers and batteries. These simple organisms have also provided us with much of our understanding of gene regulation and replication. The specific interaction that enables a virus to infect a host and carry out its multiplication process is at the heart of many new emerging technologies that have the capacity of providing breakthroughs in multiple fields of research.

Lambda phage is a well-characterised bacteriophage where all steps in the infection and life cycles are known. Multiple biotechnological tools based on it have also been developed (such as recombineering and Gateway® cloning). Our aim is to use the lambda bacteriophage to explore the avenue of producing a modular chassis. For example, broad host phage could be used for biosensing and biotechnological applications and high specificity could be explored for therapy. Host broadening and host reassignment can both (and have been shown to) be achievable by fusing genes from related phages (T4 / lambda) and by directed evolution.

Experimental Approach

Using available data on how different phages infect cells and provided the time restrictions, we will assess the modularity of the infection strategies as follows:

• Bioinformatics analysis of key lambda genes involved in host specificity and infection mechanism will be used to analyse relating phylogeny to known mechanisms, to explore modularity / host reassignment or broadening host range.

• Host specificity factors (i.e. tail fibre protein [stf gene, loci:lambdap27/lambdap28] and tip attachment protein J [J gene, locus: lambdap21]) will be targeted for engineering with a view to increase host infectivity (against a LamB / OmpC E. coli negative mutants) or to alter host specificity (against a second type species – potentially, Photorhabdus luminescens or a Rhizobium type strain as target).

• Engineering of phage cycle regulators will be performed to allow exogenous control of lytic / lysogenic cycle and to allow introduction of a biosensor.

Impact of Findings

We hope to establish lambda phage modularity as a proof-of-principle showing that phages can be engineered as biosensors that are cheap to make, stable for transportation and not requiring specialist knowledge to use. We see this as a potential route towards developing affordable point-of-care assays for pathogen detection.

Previous efforts so far have yielded great results regarding the proof of concept for quickly detecting bacteria using phages, and we would like to take the technology one step further by producing a single robust lambda phage-based multi-purpose chassis.

Policy and practice

Why is our project important?

Many infectious diseases that are common in the developing world receive little research funding because these diseases are rare in developed countries. However, these diseases can cause serious health problems and hundreds of thousands of fatalities a year – especially when coupled with a lack of access to high-quality medical care. It is estimated that over a billion people in developing countries are at risk of such diseases (Bill & Melinda Gates Foundation, 2015).

Over the past two decades, changes in global travel patterns and lifestyles has led to the emergence of new infectious diseases in the developing world and re-emergence of old infectious diseases in the developed world. Coupled with the global spread of antibiotic resistance, this has made the emergence of new infectious diseases a truly global concern.

According to the World Health Organisation, one of the main ways of addressing infectious diseases is through a strong surveillance system to monitor the spread of different diseases (WHO, 2015). Improved detection and surveillance can help in prioritising public health resources and research funding to combat these diseases more efficiently.

Diagnosis of many infectious diseases relies on expensive medical or laboratory technology, which additionally requires trained personnel to operate. In remote areas of the developing world these facilities may be out of reach for the most vulnerable people. This can be due to a lack of effective infrastructure to enable people to travel the often long distances to hospitals that are usually based in urban areas, and also because of the relatively high cost of accessing healthcare. As a result traditional medicine is still heavily relied upon. Not only do these issues prevent people from accessing quick and effective treatment, they also create “blind spots” in the global surveillance of disease transmission.

Application of virus-based diagnostics

The major health challenges faced by the developing world differ considerably from those encountered in developed countries, caused by a combination of factors including lack of funding, poor access to medical care and different environmental conditions. These issues are contributing to the ongoing spread of bacterial diseases including tuberculosis (TB), leprosy, syphilis and yaws. Different problems have emerged in the fight against these diseases: in the cases of leprosy (caused by Mycobacterium leprae or M. lepromatosis), syphilis (Treponema pallidum pallidum) and yaws (T. pallidum pervenue) conclusive diagnosis remains difficult, as these bacteria have not been successfully cultured under lab conditions. TB, caused by the bacterium M. tuberculosis, now displays widespread drug resistance, with some areas even facing multi-drug resistant TB (MDR-TB) – antibiotic resistance has been identified as ‘one of the greatest challenges to global public health today’ (WHO 2015).

M. tuberculosis is notoriously slow-growing, making positive diagnosis of TB a lengthy process, with drug resistance even slower to confirm: although faster diagnostic methods for MDR-TB have been developed in recent years (WHO 2014), these remain expensive and rare, so even in areas with access to modern technology the time to diagnosis is usually 2-4 weeks. In developing nations, and especially in rural communities without access to laboratories, equipment for the growth and analysis of liquid cultures is not available and a significant percentage of drug-resistant TB goes undiagnosed. Therefore, a bacteriophage-based diagnostic tool that does not require advanced training or equipment could significantly improve rates of diagnosis, which could lead to better treatment and improved monitoring of the spread of drug resistance. Such a method was first proposed by Hemvani et al (2012) using plaque assays of mycobacteriophage D29 to identify bacterial viability after exposure to the five most common TB medications. Although this study was carried out on concentrated M. tuberculosis cultures, the rapid and extensive proliferation of bacteriophages in the presence of host cells could enable the test to be performed on sputum samples of affected patients, with diagnosis requiring no more than 48 hours. Furthermore, the addition of a chromoprotein marker to the phage capsid would simplify the process of resistance identification by local medics or healers with minimal training. Such a tool would offer a considerable improvement on current diagnostic methods in much of the developing world.

References

Gates Foundation (2015) Neglected infectious diseases strategy overview [online]. Available at: http://www.gatesfoundation.org/What-We-Do/Global-Health/Neglected-Infectious-Diseases. [Accessed 27 July 2015]

N Hemvani, V. Patidar, D.S. Chitnis (2012) In-house, simple & economical phage technique for rapid detection of rifampicin, isoniazid, ethambutol, streptomycin & ciprofloxacin drug resistance using Mycobacterium tuberculosis isolates, Indian Journal of Medical Research, Volume 135, pp 783-787.

World Health Organisation (2014) Progress in diagnosing multidrug-resistant tuberculosis [online]. Available at: http://www.who.int/mediacentre/news/releases/2014/tb-day/en/. [Accessed 30 July 2015]

World Health Organisation (2015) Worldwide country situation analysis: response to antimicrobial resistance [online]. Available at: http://apps.who.int/iris/bitstream/10665/163473/1/WHO_HSE_PED_AIP_2015.1_eng.pdf?ua=1. [Accessed 30 July 2015]

World Health Organisation (2015) Global infectious disease surveillance [online]. Available at: http://www.who.int/mediacentre/factsheets/fs200/en/. [Accessed 27 July 2015]