Structure and function of the Pseudomonas aeruginosa type VI secretion system: On the bacteriophage trail

Bacterial pathogens infect humans using various strategies. They develop acute infections, which turn fatal to the host in a short period of time, but they also establish chronic infections and persist within the host over a lifetime. That is for example the case for lung infections in cystic fibrosis (CF) patients. At early stages of infection, diverse bacteria can be identified, including Staphylococcus aureus, Burkholderia cepacia or Pseudomonas aeruginosa. At later stages in the CF patient life the sole microorganism left in the lung is P. aeruginosa, which is firmly and chronically established and will lead to patient death.
P. aeruginosa is a potent pathogen involved in many infections, not only in CF patients. It is the 3rd most commonly-isolated nosocomial pathogen accounting for 10% of hospital-acquired infections, with 10,000 cases each year in UK. Infection is life threatening and the range of infection is broad, including urinary tract, respiratory system, dermatitis, soft tissue, bacteremia, bone and joint, intestine and a variety of systemic infections, particularly in immuno-suppressed patients. Treatment of P. aeruginosa infections involves combination therapies, e.g. a beta-lactam plus an aminoglycoside or a fluoroquinolone, but are not as efficient as desired. Alternative drugs (e.g., colistin) are useful against multi-resistant strains, but innovative therapeutic options for the future remain scarce, while attempts to develop vaccines have been unsuccessful.
To develop new therapies it is important to identify molecular targets, which once inhibited will compromise successful infection by P. aeruginosa. The target of choice is usually a major virulence factor. Secretion systems are molecular devices that bacteria use to release enzymes and toxins, which will contribute to colonization, host tissues degradation, persistence or immune system escape. These secretion systems are molecular complexes embedded in the bacterial envelope and are made up of a number of protein components.
The system we study is called the type VI secretion system (T6SS) comprising 13 core components. We selected this system because it is essential for P. aeruginosa to establish chronic infections and it is conserved in important bacterial pathogens. We chose to study 7 of the T6SS components (HsiABCEFGH), which we suspect form the core of the secretion machine. We have evidence that these components share similarities with components of bacterial viruses, i.e. bacteriophages. Since a lot is known on how bacteriophages work, we hope that understanding the correlation between T6SS and bacteriophage will shed light on how the T6SS works. The primary sequences for the phage and T6SS homologues have diverged to such an extent, that it is difficult to identify common ancestors. Although evolution can result in divergent primary sequences for homologous proteins, the structures are often conserved. Our goal is to determine the structures of T6SS proteins by using multidisciplinary approaches such as X-ray crystallography, NMR and cryo-electron microscopy. We will solve the structures of individual components and sub-complexes. The structures will provide molecular insights into how the T6SS machine is organised and will inform site directed mutagenesis studies aimed at assessing T6SS function and assembly. Perturbation in the T6SS machine will be assessed by a unique set of tests allowing us to probe whether the machine is properly assembled, able to transport toxins, and able to deliver toxins into target cells. This knowledge will later be used to design potential small molecule inhibitors that target the T6SS machine and by doing so alter T6SS function and prevent P. aeruginosa colonization and virulence. Since the T6SS is conserved in other bacterial pathogens our research will be applicable in the potential prevention of other bacterial diseases.

The type VI secretion system (T6SS) is conserved in bacteria and is used to subvert host cell response or to kill competing bacteria.
Pseudomonas aeruginosa has 3 T6SS gene clusters. H1-T6SS is induced in mutants lacking the regulatory gene retS and could be studied in vitro. The T6SS is made of 13 core components, of which some display homologies with bacteriophage tail components. The bacteriophage injects DNA in target cells and it is proposed that the T6SS functions by making localized puncture in the bacterial envelope to allow secretion/injection of toxins. It is noteworthy that the few similarities between T6SS and bacteriophage, namely VgrG and Hcp, could only be predicted by 3D structural comparisons. Amino acid sequence comparisons fail to show that these components have greatly diverged in the course of evolution.
We predict that other T6SS components will have similarity with bacteriophage tail components. In order to document the correlation between T6SS and bacteriophage we will solve the structure of 7 T6SS components, HsiABCEFGH. The structures of these components will inform site directed mutagenesis studies which will be conducted to assess the assembly and function of the T6SS machine. Standard protein secretion assay and a newly developed bacterial competition assay will also be used.
The structures of T6SS components will be solved using primarily X-ray crystallography and NMR (where appropriate) and the individual structures are unlikely to fully elucidate the T6SS mechanism. Advances in EM instrumentation and image reconstruction methods now allow the architecture of large macromolecular assemblies to be elucidated. We will therefore use cryo-EM methods to characterize T6SS sub-complexes based on data from our HsiABCEFGH interaction network and combine these EM envelopes with high-resolution models of individual or sub-components to obtain pseudo-atomic models for the T6SS complex.

The beneficiaries of this research are as follows:

1) The UK academic community with interests in molecular microbiology and infection and structural biology of supramolecular assemblies and nanomachines. The insights gained will be of use to academic researchers interested in developing and applying the general principles of bacterial secretion systems to understanding how they contribute to the molecular basis of human infections. Furthermore the evolutionary relationships between the T6SS and bacteriophage tail will be of benefit to evolutionary biologists by providing a clear example of lateral gene transfer and diversification.

2) Pharmaceutical industries and Biotech will also benefit form this research as it will potentially provide a novel therapeutic target (the T6SS) for small molecule intervention in human infections. The T6SS system is conserved within a number of human pathogens and the structural and mechanistic information produced in the research will be applicable to other human pathogens in addition to P. aeruginosa. The structural information will provide atomic resolution models that could form the basis for in silico ligand screening and inhibitor development, whilst the functional research could provide opportunities for assay development to test inhibition of the T6SS by small molecule entities. The potential development and manufacture of novel anti-infective agents by UK-based Pharma or Biotech based on targeting the T6SS will be of direct benefit to the UK economy as such therapeutics if successful would have a world-wide market.

3) Public sector health care professionals will benefit from the research in terms of an improved knowledge about the cause of P. aeruginosa infections and possible new treatment plans. P. aeruginosa is the 3rd most commonly-isolated nosocomial pathogen accounting for 10% of hospital-acquired infections, with 10,000 cases each year in UK. The development of novel therapeutic approaches would improve quality of life and health in the UK. Specialist health care workers treating cystic fibrosis patients would particularly benefit from the work as in late stage CF, the sole microorganism left in CF patient lungs is P. aeruginosa, which is firmly and chronically established and will lead to the patient death.

4) The UK knowledge-based economy will benefit from the interdisciplinary training of the PDRAs working in on the research project. Quantitative approaches will feature highly, and the outcomes arising from the integration of the various data sets will demonstrate the power of the interdisciplinary approach. Such trained RAs (and associated PhD, masters and undergraduate students) are likely to benefit the biotechnology and pharmaceutical industries, as well as the academic base in the UK. We therefore anticipate medium term economic benefits arising from a well-trained UK and international research base, reflected in maintaining internationally competitive research intensive universities and associated industries.