Elucidation of a new anti-bacterial killing system

Diseases caused by bacteria continue to represent a great threat to human health, particularly as multi-antibiotic resistance rises inexorably. An example of problematic bacterial infection, representing a great cause of public concern in the UK, is that of hospital-acquired infections. A class of bacteria which frequently cause antibiotic-resistant hospital-acquired infections are the 'opportunistic Enterobacteria'. The bacterium Serratia marcescens is a member of this class and is known to cause serious hospital-acquired infections, especially in neonatal units. It is also easy to study in the laboratory, so we can use it as a model to understand how all these related bacteria cause disease. If we study and understand the mechanisms by which harmful bacteria survive, grow, overcome the normal defences of humans and cause disease, then we can develop new ways to combat them. Such new ways might be drugs (antibiotics), vaccines or more efficient diagnostic tools.

A process called 'protein secretion' plays a key role in the ability of many different types of pathogenic bacteria to survive and cause disease. Protein secretion is a process in which bacteria move a specific set of their proteins out of the bacterial cell and into their surroundings, sometimes even injecting them directly into human cells. These secreted proteins can then attack and destroy or hijack the cells of the infected host, causing disease. This research project will look at a new type of secretion system, to try and learn how bacteria can use it as a weapon to become successful pathogens. This secretion system, called 'Type VI Secretion', was only discovered recently and little is known about its mode of action. However it is important to know how it works and what it does, since many pathogenic bacteria use this system to help them successfully infect a host organism and cause disease. Some types of bacteria use the system to directly attack the cells of the infected host, whereas some types of bacteria use the system to efficiently kill other bacteria. The use of Type VI secretion systems to attack other bacteria is important for the success of pathogenic bacteria, since killing their rival bacteria (e.g. harmless or 'good' bacteria normally found in the body) leaves the way clear for the pathogen to cause a harmful infection.

In this research project, we aim to learn how Serratia marcescens uses an 'anti-bacterial' Type VI secretion system to kill competitor bacteria and therefore be a successful pathogen. This will help us to understand how all opportunistic Enterobacteria are able to cause hospital-acquired infections. We have identified a Type VI secretion system in Serratia and shown that it is used by Serratia to kill other bacteria. In this project we will primarily investigate the mechanism of this killing, by aiming to answer three questions: First, what are the proteins secreted by the system, in other words what are the damaging 'bullets' fired or 'poisons' injected by the system? Second, what are the unprotected targets in vulnerable bacteria? Third, how does Serratia protect itself from being killed by its own weapon, i.e. by its own Type VI secretion system? Additionally, we will also start to investigate how important this process is to the success of Serratia in a 'real-life' infection situation.

This research will greatly improve our understanding of how this new secretion system is used by pathogenic bacteria to enable them to out-compete their rivals and thus prosper and cause disease in humans. Importantly, we hope to 'learn from the experts' (i.e. from bacteria themselves) how best to kill a bacterium, potentially inspiring future work to develop new anti-bacterial drugs. We will also know more about an important class of hospital infection-causing bacteria. Ultimately the work may contribute towards the search for novel antibiotics and other therapeutic strategies to combat bacterial disease.

Protein secretion systems play a central role in the virulence, host interaction and survival of Gram-negative bacterial pathogens. The recently-identified Type VI secretion system (T6SS) plays a key role in virulence and competitiveness of many bacteria, including important human pathogens, although the mechanisms and roles of T6SSs are not yet well understood. Pathogens can use T6SSs to directly target eukaryotic organisms, as classical virulence factors. Recent work has shown that certain pathogens can also use T6SSs to target other bacterial cells, killing or inhibiting rivals. Such 'anti-bacterial' T6SSs provide a competitive mechanism to allow pathogens to proliferate in polymicrobial infection sites or environmental reservoirs and ultimately cause disease. The mechanisms by which different bacteria use anti-bacterial T6SSs to specifically kill rivals but protect self are currently unknown, something we aim to address using a clinically-relevant and tractable model organism, Serratia marcescens (Sma). Opportunistic enteric bacteria, including Sma, represent an important cause of problematic and antibiotic resistant hospital-acquired infections. We have recently identified a T6SS in Sma Db10 which exhibits potent anti-bacterial killing activity. The primary aim of this project is to elucidate how the model opportunistic pathogen Sma is able to utilise its T6SS to efficiently kill other bacteria. We will identify and characterise anti-bacterial effector proteins secreted by the T6SS, identify self-resistance determinants protecting Sma from killing itself, and determine the cellular target(s) of the Sma T6SS in susceptible bacteria. Additionally we will begin to examine the wider biological relevance and consequences of the process. Our extensive preliminary data, including identification of several candidate T6-secreted effectors, demonstrates the feasibility of the proposal. Our findings may pave the way for future development of new anti-bacterial therapies.

This research will benefit:

1.The academic researchers directly involved in the project by developing their research, professional and transferable skills. This is a varied, ambitious and multi-approach research project and the PDRA will also be assisted and encouraged to develop public engagement, communication and other skills.

2. Academic researchers worldwide, including those interested in bacterial protein secretion, bacterial physiology and inter-bacterial competition, molecular mechanisms of bacterial virulence and host interaction, and the pathogenicity determinants of opportunist Gram-negative pathogens. They will gain from new data, scientific knowledge, methodologies and ideas generated by this research. Our findings will be communicated to the scientific community by means of publication in peer reviewed journals and presentation at national and international scientific meetings. Moreover we will generate, and make available, new tools, data and reagents to assist in other research projects (including mutant strains, proteomic data and antibodies).

3. Commercial private companies and academic scientists aiming to develop novel anti-bacterial diagnostics, vaccines or inhibitory compounds (including antibiotics). Type VI and other secretion systems are critical for the ability of many bacterial pathogens to cause disease and to survive and proliferate in competition with other bacteria. I anticipate that this research, aiming to elucidate the roles, effectors and cellular targets of Type VI secretion systems (T6SSs) in opportunist pathogens, may eventually lead to the identification of novel targets for anti-bacterial strategies. During this project, we will identify and study novel secreted proteins (T6SS-dependent and independent), some of which could turn out to be candidates for the development of vaccines and diagnostics. Additionally, we will unravel the mechanism by which bacteria can efficiently kill other bacteria (via anti-bacterial T6SSs). The cellular target(s) of T6S in the susceptible bacterium represent attractive targets for inhibitor molecule screens, with the long-term aim of developing new anti-bacterial drugs or compounds. Components and substrates of T6SSs may also represent good long-term targets for antimicrobial development. This study will identify secreted substrates, begin to allow the suitability of Type VI secretion for antimicrobial development to be assessed and may generate useful reagents for the drug discovery process. I am very keen to collaborate with parties interested in exploiting our findings at the appropriate time during or after the three year project. I will utilise the highly experienced and well-connected Research and Innovations Office at the University of Dundee to forge timely links with potential academic and commercial partners and to ensure proper protection and management of any Intellectual Property generated.

4. The general public.
A. I will increase the public's awareness and understanding of science, in particular of my field and this research programme. I will actively participate in public engagement activities and will publicise exciting findings and outcomes of our research beyond the academic sphere. I will continue to engage with the local public and schoolchildren in several ways, including the biennial 'Magnificent Microbes' events and 'Doors Open' days in my institution.
B. In the long term, I aim to generate improvements in health, welfare and UK economic strength by contributing research data and improved understanding of bacterial secretion and its role in pathogenicity towards the development of new anti-bacterial strategies to combat disease of humans (and also of livestock and crop plants). To enable this I would interact with companies or other academic groups during or after the three year period (see 3.)