Deciphering the role of the host endoplasmic reticulum in Chlamydia inclusion biogenesis

Many 'friendly' bacteria live passively in the environment or engage in mutually 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 detect how these syringes and effectors operate. Remarkably, the results so far are not only beginning to tell us how many bacteria cause disease, but they are also providing astonishing new insights into how our own cells function, as the bacteria have been perfecting their armaments for millions of years. Paradoxically, these bacterial effectors therefore also provide exquisite and exciting new tools to study cell biology.

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 difficult to study Chlamydia in the laboratory as they cannot grow outside host cells at all and unlike other bacteria they cannot yet be genetically manipulated. Chlamydia use a molecular syringe to deliver effectors that enable the bacteria to replicate inside a special membrane-bound compartment inside host cells called the 'inclusion'. We have recently shown that the inclusion interacts with an important host membrane system called the endoplasmic reticulum (ER), which manufactures host proteins. We showed that this novel interaction is critical to bacterial survival and infectivity. In fact, the ER contacts the inclusion at the points where the bacteria assemble their syringes, structures we call 'pathogen synapses'. Our proposed experiments will examine why this interaction with the ER is so important and how it might relate to the delivery of effectors through the syringe. The findings will not only provide important new information about how Chlamydia cause disease, which might eventually lead to new treatments, it might also illuminate the fundamental pathways cells use to control their intracellular compartments and the synthesis of their own proteins.

Chlamydiae are obligate intracellular bacterial pathogens that have worldwide medical impact. Chlamydiae replicate within a specialised compartment, termed an 'inclusion'. Inclusion biogenesis requires a bacterial type III secretion system (T3SS), a macromolecular machine that deploys effector proteins into the inclusion boundary membrane and beyond into the host cytosol and nucleus. Chlamydial inclusion proteins (Incs) are hydrophobic T3SS substrates that localise to the inclusion membrane from where they subvert host functions. The inclusion membrane is therefore a critical host-pathogen interface, yet the extent of its interaction with cellular organelles and the origin of this membrane remain poorly defined. We have shown that the host rough endoplasmic reticulum (rER) is recruited to inclusions, and that generation of infectious bacterial progeny requires an intact rER. Electron tomography of Chlamydia-infected cells revealed 'pathogen synapses' where ordered arrays of T3SSs connect to the inclusion membrane only at rER contact sites. We propose to address important interrelated questions that arise from our findings using established approaches including protein biochemistry, cell biology, confocal microscopy and electron tomography. We will investigate whether the T3SS directs synapse formation by using effectors to recruit the rER. We wish to establish why the rER is recruited to the inclusion, specifically by investigating whether the translocon is hijacked during chlamydial infection. Our screening of predicted Incs in the genome revealed potential rER-retention signals. We will investigate whether these interact with rER-based machinery and how they might determine Inc partitioning within the inclusion membrane. We will establish how rER morphology influences pathogen synapse formation and the shape and positioning of the inclusion. Together the data will provide a mechanistic understanding of the role of the rER in Chlamydia inclusion biogenesis.

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 the 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. The latter remains particularly important as the vast majority of sexually transmitted Chlamydia infections remain asymptomatic and go undetected, particularly in the teenage population.

Our research has already demonstrated that disrupting the interaction of the host endoplasmic reticulum with the chlamydial inclusion can stall inclusion biogenesis and disrupt the integrity of this bacteria-filled compartment, leading to an associated reduction in bacterial infectivity. Stalling of inclusion biogenesis in this way has rarely been described, and our work in this proposal seeks to directly analyse the molecular basis for this event, which in the longer term might enable the development of targeted interventions. Our research also addresses the function of the chlamydial type III secretion system (T3SSs), a molecular nanomachine that injects virulence proteins into target cells. T3SSs represent a viable conserved target for future antimicrobials given their conservation amongst a wide variety of bacterial pathogens. Our research has enabled assembled T3SSs to be visualised in situ for the first time in contact with the inclusion boundary membrane and the host endoplasmic reticulum. This offers an exciting new opportunity to decipher the structure and function of these critical bacterial weapons. 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 contribute to the training of talented scientists, both at postdoctoral level and in support of the Wellcome Trust-, BBSRC- and recently awarded 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.