Capturing functional states of type III secretion systems from Chlamydia in situ

Many 'friendly' bacteria live passively in the environment or engage in beneficial associations with plants and animals, for example by fixing nutrients or assisting digestion. Other bacteria have gained the ability to live inside more complex organisms where they survive and multiply. Although able to expend these 'host' organisms for their own advantage, these aggressive bacteria also cause damage to the host, which results in disease. Doctors have treated such bacterial infections in man and animals with antibiotics, yet recently bacteria are increasingly developing resistance to these drugs. Consequently, it is crucial to understand how different bacteria cause disease at a molecular level, as this will provide clues to new ways to treat patients and develop vaccines. It has emerged that many of these bacteria deploy a sophisticated weapon that acts like a minute syringe to inject host cells with a cocktail of bacterial proteins called 'effectors'. An important focus of current biomedical research is to decipher how these syringes and effectors operate.

One of these disease-causing bacteria called Chlamydia is responsible for serious infections. It is the main bacterial cause of sexually transmitted disease and infertility in the U.K. and other developed countries, and of a widespread form of blindness called 'trachoma', which is designated as a neglected tropical disease by the World Health Organisation. It is very challenging to study Chlamydia in the laboratory as they cannot grow outside host cells at all. Chlamydia use a molecular syringe to deliver effectors that enable the bacteria to force their own entry into host cells and to replicate inside. We have recently used electron microscopy to examine these infectious bacteria in great detail. We have been able to see the bacteria in the process of entering cells when their molecular syringe first contacts the host surface and see in much more detail how the bacteria enter into our cells. Many scientists have studied the syringes by isolating them, but in the process important parts are lost. Our proposed experiments will examine the structure of the intact syringe while it is in the bacterial membrane and when it contacts the host cell. The findings will not only provide important new information about how Chlamydia cause disease, which might eventually lead to new treatments, but also other disease-causing bacteria that have very similar syringes.

Chlamydiae are obligate intracellular bacterial pathogens that have worldwide medical impact. Initial chlamydial entry into host cells and subsequent intracellular replication require a type III secretion system (T3SS), a molecular nanosyringe conserved among many medically important pathogens. T3SSs translocate virulence effector proteins into the host that hijack key cellular processes. The conserved T3SS apparatus is an attractive target for the development of molecular inhibitors, which are becoming vital considering ever-increasing bacterial resistance to conventional antibiotics. We have applied cryo-electron tomography to study the structure of infectious chlamydial elementary bodies (EB). Our data revealed important insights into polarised EB architecture, mapped the global arrangement of T3SSs on the EB surface, and revealed early endocytic structures formed during EB interaction with host cells. We propose to study the intact macromolecular structure of the C.trachomatis T3SS in different functional states by cryo-electron microscopy and sub-tomogram averaging. We will determine the intact membrane-embedded structure of the T3SS in situ. Our preliminary data reveal a substantial cytosolic substructure not previously visualised in detergent-solubilise core complexes. We will examine T3SS structure in contact with host membranes, potentially allowing the visualisation of the enigmatic host membrane-embedded translocon complex, and the conformational and compositional changes associated with T3SS activity. We will exploit transformed chlamydial strains to identify T3SS components using fusion proteins and epitope tags. We will study T3SS disassembly following EB internalisation and early vacuole architecture using focussed ion beam milling. The structural data arising from our studies of Chlamydia has the potential to directly contribute to the development of novel therapeutics targeting T3SS-dependent virulence effector secretion in bacterial pathogens.

Our research is of wider benefit to the public. Chlamydia remains the leading bacterial cause of sexually transmitted disease and associated infertility in the UK and the wider Western world. In developing nations, Chlamydia cause a specific form of blindness called trachoma, which remains designated as a neglected tropical disease by the World Health Organisation. Although these chlamydial diseases can be currently treated with antibiotics, it is unclear how long such therapies will remain viable given the alarming evidence for increasing resistance to these compounds, and the economic impact of their application particularly in developing nations. Our work seeks to decipher the fundamental mechanisms by which this pathogen causes disease, yielding new insights into the molecular basis of sexually transmitted infection and trachoma, and revealing potential new targets for therapeutics, vaccines and diagnostics.

This application specifically addresses the structure and function of the chlamydial type III secretion system (T3SS), a molecular nanomachine that injects virulence proteins into host target cells. T3SSs represent a viable target for future antimicrobials given their conservation amongst a wide variety of bacterial pathogens. Our recent research on infectious chlamydial elementary bodies (EBs) has demonstrated how to visualise a large number of assembled T3SSs in situ by cryo-electron tomography both in the absence and presence of the host cell. This provides a unique opportunity to derive structural information by sub-tomogram averaging to reveal the molecular detail of the T3SS, and critically how it interacts with the host cell and the conformational and compositional changes that might accompany this process. This offers an exciting and timely opportunity to decipher the intact structure of these critical bacterial weapons, which are of relevance not only to Chlamydia but also many other medically important pathogens. The analysis of host-pathogen interaction has also frequently revealed unexpected new insights into the control of fundamental cellular processes, which are of broader relevance to physiology and disease.

The proposed research will also contribute to the training of talented scientists, both at postdoctoral level and in support of the Wellcome Trust-, BBSRC- and MRC-funded PhD programmes at the Institute of Structural and Molecular Biology. Seeding well-trained scientists into academic and industry is essential to ensure the competitiveness of the UK science base in the future, particularly in microbiology. This yields not only academic but also substantial economic and sociological benefits.