Adhesion to host cell membrane microdomains in cornea as an antimicrobial target to prevent corneal ulceration

All over the world, there is a growing problem with antibiotics: there have been years of misuse by patients, doctors and even farmers seeking greater yields from livestock, with the result that microbes have learned to live with these once potent drugs. This problem is particularly acute in India, where access to professional healthcare is often limited and so ineffective antibiotics are bought and used by patients. For agricultural and domestic labourers, minor damage to the surface of the eye frequently results in bacterial and fungal infections; when not properly treated, this can lead to corneal ulceration and, eventually, to blindness. In a working-age person, the economic consequences can be disastrous for them and thier families. Given these issues, we are now looking for an alternative route to protect eyes against microbes. We have formed a partnership with a leading eye hospital, LV Prasad Eye Institute. LVPEI have developed a pyramidal structure so that research developed in the advanced research and surgery centre in Hyderabad can rapidly be disseminated throughout their organisation. They have a major drive to develop therapies that can benefit patients in rural villages.

The approach that we have taken came originally from a study of how our cells stick together to form tissues such as skin. There are structures on the surfaces of cells that resemble Velcro, highly organised patches of adhesive molecules that enable cells to cling tightly together. In some cases, these are long-lasting and static; in other cases they are short-lived and dynamic, allowing mobility, for instance when our white blood cells are travelling through tissues to get to the site of an infection. We discovered that some types of bacteria and yeasts can 'hijack' the dynamic sites, to allow them to stick to our tissues even when our natural defences try to dislodge them with tears and blinking. This is the starting point of an infection, when a colony of microorganisms attaches and starts to grow, often then penetrating deeper into tissues to cause serious disease. Even superficial infections can cause problems, leading to ulceration of the surface of the eye and eventually blindness if not properly treated.

Different types of microbes use different types of human molecules to cling to: targeting all of them would be very expensive. Our approach is not to target the molecular hooks themselves, but the material in which they are held to form the sticky patches. On Velcro, hooks are embedded in a baselayer of woven material but on our cells this is formed by a sort of molecular raft called a microdomain. We have discovered how to weaken one type of microdomain, in a way that is analagous to stretching Velcro, pulling the molecular hooks further apart and significantly lowering the stickiness of the patches on cells. By addressing only one type of microdomain, we have found that we do not affect the normal behaviour of our cells but we do make bacteria and fungi much easier to wash away.

Unlike antibiotics that directly target microbes, our treatment targets human cells and so should not lead to new forms of antibiotic resistance. If we can show that it is both safe and effective on the types of microbes that cause eye infections in India, we aim to produce a cheap and simple treatment for people who have injured their eyes, or who have early stage infections. This could be administered by patients or by a network of trained volunteers, easily and quickly. By helping to prevent infection, or giving more time to seek professional diagnosis, we hope to reduce the rate of a common cause of blindness in young people in India.

Clinicians at the LVPrasad Eye Institute identified a vital need for effective first line therapy for patients in rural India with early stage eye infections. Without appropriate treatment, infections can quickly progress into irreversible corneal ulceration with loss of sight. LVPEI alone see 1200 cases of corneal infection per year: 30% will experience vision loss. In LMIC, many eye infections result in corneal ulceration and 10,000 patients per year lose eyes that might otherwise be saved
Inhibiting the adhesion of bacteria to the cornea is an innovative, low-risk strategy to prevent ulceration. Pathogens use a variety of means to adhere but all are dependent on interactions with molecules on the host cell surface. These are frequently located within highly organised microdomains, allowing control of the strength of adhesive interactions, allowing them to resist mechanical removal by mechanisms such as tear production and blinking. Human tetraspanin family proteins form microdomains that organise adhesion receptors such as integrins. Several species of pathogens use these microdomains for tight attachment to host cells. Treatment with tetraspanin-derived peptides weakens the attachment of a range of microbes and facilitates removal by natural defences. Importantly, an interaction occurs between the peptides and antibiotics that will allow the continued use of common antibiotics.
We aim to apply this treatment to microbial keratitis in India, exploring the effect of the peptides on common infections and the underlying mechanism, to promote the development of a safe and inexpensive topical treatment. The targeting of a broad range of pathogens allows treatment without diagnosis. This will benefit economically active people who damage their eyes during agricultural or domestic labour. Loss of sight from eye infection exists in other LMIC and our treatment will be appropriate for corneal infections in other areas lacking an integrated healthcare system.

This proposal seeks to validate the use of a small peptide as a first-line defence in the prevention and treatment of early-stage infection. It would be applied immediately after corneal damage to prevent microbial adhesion and infection that can lead to corneal ulceration and loss of sight. The output will be validation of a new host-targeted anti-microbial strategy, providing a major step towards a simple, safe way of preventing acute corneal infections developing into corneal ulcerations in LMIC.

The activities we will undertake to foster the impacts identified in the impact summary can be grouped into five main categories: 1. Demonstrating the mechanism of a disruptor of membrane microdomains; 2. Taking a product to market in India. ; 3. Maximising patient take up of the treatment; 4. Dissemination beyond the project region; 5. Governance, monitoring and evaluation.