Bacterial pathogenesis: dissecting the early stages of cellular invasion by the obligate intracellular bacterium Orientia tsutsugamushi

Scrub typhus is one of the leading causes of hospitalisation from high fever in many rural parts of Southeast Asia. The disease is caused when a patient is bitten by a small biting arthropod called a mite, which transmits a dangerous bacterium called Orientia tsutsugamushi. Infection with this bacterium causes a range of symptoms that include high fever, rash and muscle pain. If the infection is not treated quickly and correctly it can result in multiple organ failure and death. It is estimated that 1 billion people are at risk and 1 million people are infected with this disease every year. Because the symptoms are similar to many other tropical infectious diseases - such as dengue fever and other bacterial or viral infections - patients are often misdiagnosed and therefore not treated properly. This risks severe complications for the individual patient and it also contributes to the growing problem of antibiotic resistance due to misuse of antibiotic treatments. Given the high incidence and severity of this disease, it is very important that we study the bacteria that causes it in detail as this will help us to develop better diagnostics, vaccines, and treatments in the future.

In order to understand fully an infectious disease, and therefore improve patient care, it is necessary to understand the basic biology of the organism that causes the disease. In particular, it is important to understand exactly how that organism grows inside humans, and why it makes those humans sick. Humans have around 100 trillion bacteria growing on and inside them all the time, and the vast majority of them cause no ill effects and are actually beneficial. The study of 'pathogenicity' explores how and why a small minority of bacteria cause disease in humans. In the case of O. tsutsugamushi, the bacteria that causes scrub typhus, the first step in its pathogenicity is its entry to the inside of human cells. Most bacteria can survive either inside human cells or free in the environment: O. tsutsugamushi cannot. O. tsutsugamushi can only survive and replicate when it is physically located inside another cell. Therefore the more we can understand about how this bacteria gains initial access into human cells, and then avoids being killed by them, the more we can explore ways to block this essential process and therefore eliminate the bacteria from the patient.

In this study we will explore the detailed mechanisms by which O. tsutsugamushi gets into humans cells and what it does in the first 24 hours after entry. Human cells have developed sophisticated strategies to eliminate most bacteria that get inside them, but O. tsutsugamushi has learned how to avoid these. We want to understand exactly how it does this. We will use a high-throughput screening approach to study these early events of pathogenesis. This means that we will systematically look at all the genes in the human genome to try and find the ones that the bacteria hijacks in order to get into the cells and survive there. Once we have identified these important human genes we will do more experiments to understand the details of exactly how the bacteria uses them in order to gain entry and then survive within the human cells.

The outcome of this study will be a more detailed understanding of exactly how the bacterium O. tsutsugamushi gets into human cells, and how it avoids being killed by them. This will help us develop new strategies to block these processes in the future.

Scrub typhus is a leading cause of non-malarial fever in rural parts of Southeast Asia. Characterised in the early stages by fever, rash and myalgia, delayed or inappropriate treatment can lead to disseminated intravascular coagulation, multiple organ failure and mortality. Whilst highly responsive to tetracycline antibiotic therapy, rapid and unambiguous diagnosis remains a major challenge and an obstacle to high quality patient care.

Scrub typhus is caused by the obligate intracellular bacteria Orientia tsutsugamushi that is carried and transmitted by Leptotrombidium mites. O. tsutsugamushi is a member of the Rickettsiaceae family, occupying its own genus to reflect significant differences in genome structure, cellular organisation and intracellular lifecycle compared with other Rickettsias.

Pathogenicity requires entry into and propagation within mammalian host cells, and we plan to study the early stages of this process. Bacterial attachment to host cells is mediated by specific bacterial surface proteins, and is followed by a clathrin-mediated endocytosis-like mechanism. Bacteria exit from the late endosomal pathway, then induce and escape from host autophagy, and finally employ microtubule-directed motility to move through the cytosol.

In this study we will use a combination of systems biology, cell biology and biochemistry approaches to study these early stages of bacterial infection. Specifically, we will use a genome-wide RNAi screen to identify host cell factors required for (i)entry, (ii)escape to the cytosol and (iii)bacterial motility. We will then perform a range of specific experiments to validate and dissect these molecular pathways. Finally, we will use biochemical approaches to identify specific host receptors that bind O. tsutsugamushi and induce bacterial internalisation.

The outcome of our research will be a better understanding of the molecular pathways exploited by O. tsutsugamushi in the early stages of host cell internalisation.

1. In the long term this research will benefit the poor, rural Thai population that is most at risk of developing the disease scrub typhus. Our research will yield insights into the mechanisms by which the bacteria O. tsutsugamushi invades human cells and causes disease, and this will support ongoing efforts to develop improved diagnostics and treatment options. This research will be physically performed in Bangkok, Thailand, facilitating rapid and frequent interactions with local colleagues currently engaged in these applied aspects of scrub typhus research.

2. Although scrub typhus is a leading cause of severe febrile illness in Thailand and other Southeast Asian nations, it is not widely known amongst the public, policy makers and even researchers in the wider scientific community. Our research will raise the profile of this important human pathogen and this will benefit policy makers in Thailand and beyond, as a greater awareness of the incidence and severity of this disease will support appropriate public policy interventions such as public awareness campaigns.

3. This research will have a positive impact on the Thai and UK researchers that are both directly and indirectly involved in this research. One of the specific aims of this funding call is to support development through capacity building within Thailand. This will be achieved by supporting the two exceptional and internationally trained Thai PIs on the project. This will support the development of their careers in Thailand and will contribute to retention of highly skilled talent within the country. In addition, this project will result in the training of three Thai technicians directly involved in the project as well as indirect exposure of other colleagues working at the two research institutes where the research will be performed. The research will also support the UK PIs involved in the project, and will strengthen links between UK and Thai scientists.

4. Finally, the successful completion of this project will have a positive impact on the Thai scientific community, as it will contribute to raising the international profile of Thai universities and Thai scientists. It is expected that the outcome of this research will be several peer-reviewed publications in high-impact journals, and this will lead to invitations to present research and ideas at important international conferences. Presence at these meetings will have a strong positive impact on the international profile of Thai scientific research.