Molecular and functional characterization of protein-lipid interactions at the bacterial host interface

Currently available antibiotics for the treatment of bacterial infections rely on killing harmful bacteria or stopping them from multiplying. Unfortunately, many antimicrobials are becoming ineffective against infections because bacteria have come up with ways to resist drugs which once were effective. An alternative way of treating bacterial infections is the use of anti-adhesion inhibitors, molecules that stop disease-causing bacteria from sticking to host tissues. Most bacteria have to attach themselves to the host to cause infections and they use sticky proteins (adhesins) on their surface to do this. If the binding sites they stick to are already taken up by other sticky molecules (called anti-adhesion inhibitors), they cannot attach themselves and are flushed out of the organisms without causing infection. We use this strategy to design such sticky anti-adhesion inhibitors as new drugs to prevent and treat infections.

We have started using molecules derived from bacterial adhesins as anti-adhesion inhibitors - when they are used to "treat" host cells, they stick and prevent pathogenic bacteria from causing infection. Recently we found a family of adhesins, called MAMs, which are used by many different bacteria to stick to the host. This means that anti-adhesion inhibitors based on these adhesins will be useful in fending off a wide range of different pathogenic bacteria (similar to broad-spectrum antibiotics) that would stick to the same sites. We want to study exactly how these proteins stick to the host cells (i.e. how can they recognize the host surface) and how, in the context of bacteria, these molecules can change the host cell so that it becomes more prone to infection.

If we can understand how MAM-based molecules manage to bind to the host really tightly (so they are better at fending off pathogens) but without causing harm to the cells themselves, we will be able to make new drugs which can be used instead of antibiotics. The advantage will be that they will be effective for a long time to come, because bacteria cannot easily become resistant against anti-adhesion therapy.

Multivalent Adhesion Molecules (MAMs) aid the initial attachment of bacteria to host tissue, thus facilitating infection by a wide range of Gram-negative pathogens. MAMs consist of tandem arrays of mammalian cell entry (mce) domains, which mediate binding to host receptors. Mce domains are an abundant family of lipid binding proteins present in plants and bacteria and two different types of mce ligands have been described so far; phosphatidic acids and steroids. We have recently characterized the Vibrio parahaemolyticus MAM, MAM7 and found that phosphatidic acid binding by MAM7 triggers a host response and rearrangement of actin, which aids epithelial penetration and infection of deeper tissues. We also showed that MAM-derived synthetic inhibitors are efficient inhibitors of pathogen attachment and have potential to be used as anti-infectives. Our ultimate goal is to understand the basic biology of MAM-mediated host cell binding and host responses and the molecular basis of ligand binding by mce domains.

To this end, we will study the molecular requirements for MAM-triggered host signalling. We will use synthetic "bacteriomimetic" constructs and genetically engineered bacterial strains and evaluate their effect on polarized epithelial cells, measuring transepithelial electrical resistance and analyzing cellular phenotypes by fluorescence microscopy (Aim 3).

To lay the ground work for this aim, we will first identify the molecular signature for ligand binding by mce domains. This will be achieved by studying the ligand binding specificity and affinity of mce domains using in silico analysis, followed by a range of biochemical and biophysical experiments on recombinant purified proteins and lipid ligands (Aim 1).

In Aim 2, we will solve the first ever structure of an mce protein and identify the mechanism of ligand binding and basis of cooperativity between mce domains using a combination of NMR and X-ray crystallography.

Antibiotic resistance is an increasing problem with tremendous societal and economic impact. Alternative approaches to prevent and treat bacterial infections are urgently required and one such approach is anti-adhesion therapy. The proposed work on MAMs, a family of bacterial adhesins, will significantly inform and improve our ability to develop MAM-based inhibitors for anti-adhesion therapy. Because of their broad-spectrum efficacy, MAM-based inhibitors will be useful for prophylactic and, once further developed, therapeutic use against a range of bacterial diseases both in animals and humans.

We have already demonstrated the efficacy of MAM inhibitors against a number of zoonotic pathogens (e.g., Yersinia pseudotuberculosis, Vibrio parahaemolyticus), making this a viable option for preventing pathogen colonization of animals and especially lifestock raised for human consumption. Not only will this significantly enhance food security, it could also present an alternative to the prophylactic use of antibiotics in food animals, which escalates the problem of bacterial antibiotic resistance. Development of MAM-based inhibitors will also help to drive the area of nanotechnology, as we develop synthetic strategies to present and increase the avidity of MAM-derived molecules, e.g. through multivalent surface display. Our research will also impact the area of lifelong health and wellbeing: We have shown the potential use of MAM-inhibitors against a range of multidrug-resistant bacteria isolated from wounds. Chronic wounds a major cause of morbidity worldwide and a burden to the health care system. Chronic wounds are increasingly a problem because underlying conditions contributing to a delay in wound healing, such as obesity, age or systemic diseases, such as diabetes and arthritis, are on the rise and treatment is increasingly complicated by the emergence of multidrug-resistance, often over the course of treatment. We have shown the potential of MAM-based anti-adhesion inhibitors against e.g. Klebsiella, Acinetobacter and Pseudomonas, which often give rise to wound infections and prevent colonized wounds from healing.

Industry: Our previous work on MAM-based inhibitors is already covered by a patent (UTSD.P2412US.P1) and development of our prototype inhibitors into pharmaceutical compounds for anti-adhesion treatment has large potential to benefit the pharmaceutical and healthcare industry. In addition, exploitation of these molecules, which bind to mammalian membrane lipids with high specificity and affinity, as tools for cell biology (e.g. lipid-specific probes) will benefit both the biotech industry as well as basic cell biology research.

Basic Science: Although it is well established that bacterial adhesion is crucial to infection, how adhesion can directly manipulate host signalling pathways and how this impacts infection is currently not well understood. Our approach of using a combination of synthetic biology and microbiology will provide the methodology to study the influence of pathogen adhesion to host infection in more detail and thus impact basic research on host-pathogen interactions.

Students/Outreach: Our work provides a good example of how transdisciplinary research (e.g. a combination of biochemical, genetic and structural biology approaches) can create a tangible output (in this case, development of prototype anti-adhesion compounds). Students and the public alike appreciate this link between basic research and application and this project will be used to feed into ongoing teaching and outreach activities to connect with these beneficiaries.