Defining early events underpinning Chlamydia trachomatis entry into mammalian host cells

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 protective 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. Chlamydia use a molecular syringe to deliver effectors that enable the bacteria to enter into host cells and replicate inside a special membrane-bound compartment inside called the 'inclusion'. We have recently described more about how the bacteria enter their host cells. Chlamydia achieve this by interfering with a filament-like network called the actin cytoskeleton that acts like cellular scaffolding. This scaffolding controls the shape of cells, the position of their internal structures and how they divide. Our proposed experiments will examine in detail how chlamydial effectors rearrange this scaffolding to allow the bacteria to successfully enter inside human cells. 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 employ to control their architecture.

Chlamydiae are obligate intracellular bacterial pathogens that have worldwide medical impact. Chlamydiae force their own entry into human host cells where they replicate within a specialised membrane-bound compartment, termed an 'inclusion'. Both bacterial entry and replication require a bacterial type III secretion system (T3SS), a macromolecular machine that deploys virulence effector proteins into the host cytosol, nucleus and the boundary membrane of the inclusion. We have recently examined events that occur during the entry of infectious extracellular C.trachomatis elementary bodies into human cells. We have shown that filopodial capture is a predominant early event during Chlamydia entry. We have described a novel macropinocytosis-like pathway underpinning bacterial entry and demonstrated a pivotal role for the SNX-PX-BAR family protein sorting nexin 9 (SNX9) in filopodial capture. We propose to address important interrelated questions that arise from our findings using established multidisciplinary approaches including emerging genetic techniques, cell biology and bioimaging. We propose to define the major route of actin-dependent entry into target cells involving filopodial capture and macropinocytosis by investigating the T3SS effectors involved in SNX9-dependent signalling, by determining why the actin nucleation promoting factor N-WASP is critical for C.trachomatis entry, and by examining the mechanism of filopodial capture in detail. We will also characterise a novel early trafficking step involving F-actin stress fibres during the biogenesis of early chlamydial-containing vacuoles. Together the data will provide a mechanistic understanding of how this major human pathogen subverts the host actin cytoskeleton to promote its uptake and establish a replicative niche within target cells.

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. 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 proposed research addresses the mechanism by which C.trachomatis forces its own uptake into human cells, and the associated subversion of the actin cytoskeleton by bacterial proteins. Uncoupling these host-pathogen interactions individually or in combination attenuates the bacterial entry rate and subsequent intracellular replication. This project proposes molecular studies to elucidate the mechanisms of these processes, providing insights into potential strategies for future intervention. 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 PhD programmes within the School of Biological Sciences at the University of Cambridge. Seeding well-trained scientists into academia and industry is essential to ensure the competitiveness of the UK science base in the future, particularly in microbiology. This will yield not only academic but also substantial economic and sociological benefits.