Molecular mechanisms modulating host epithelial integrity in response to bacterial adhesion

We recently found a family of adhesins, called MAMs, and many different bacteria use these sticky proteins to make contact with host tissues during an infection. We have also observed that, if many MAMs are brought together on the host cell surface, this changes signaling processes within the host so that it becomes more prone to infection. We want to understand what host signaling proteins are activated by MAMs and how MAMs can start these signaling processes. There are many benefits to answering these questions:
First, we will be able to turn MAMs into new drugs against bacterial infections, called adhesion inhibitors. These materials work by sticking to host cells and stopping pathogenic bacteria from attaching themselves. If pathogens cannot attach themselves, they are flushed out of the organism without causing an infection. If we can understand what MAM-based molecules have to look like 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 adhesion inhibitors.
Second, in the more distant future, we may be able to use MAMs to make other drugs more effective. Many drugs cannot be taken orally or cannot be used at all because they cannot cross the barrier between the intestine and the blood stream. Some of the properties of MAMs that make an organism more prone to infection may also be turned into something useful. - They can be used to make certain tissues of the body "leaky" for a short period of time so that they are easier accessible for drugs. This will increase the number of useful drugs altogether and the number of drugs that can be taken orally, rather than by injection.

MAMs aid the attachment of bacteria to host tissue, thus facilitating infection by a wide range of pathogens. MAMs consist of tandem arrays of mammalian cell entry (mce) domains, which mediate host receptor binding. Mce domains are an abundant family of lipid binding proteins present in plants and bacteria and different types of lipid ligands have been described so far, ranging from phosphatidic acids to steroids. We recently found that phosphatidic acid binding by Vibrio MAM triggers a host response and rearrangement of actin, leading to epithelial permeabilization. This project will investigate the signaling events triggered by MAM-attachment to host cells and the molecular features of MAMs required to activate host cells.

This will allow us to engineer MAMs with specific binding/RhoA activation characteristics to be used as safe anti-infectives. To this end, we will study the host signaling events leading to MAM-mediated RhoA activation and epithelial permeabilization. We will characterize signaling proteins using pull-down and mass spectrometry as well as targeted characterization of candidate proteins by immunofluorescence microscopy and Western Blotting. We will also investigate how mce domains have to be clustered to cause signaling and if replenishing host PA restores the wild type phenotype (Aim1). We will investigate the impact of MAM adhesion on the activity of other virulence factors, using biochemical and FRET-based assays. We will test the extent of MAM-mediated epithelial permeability and investigate how this phenotype impacts the establishment of systemic disease (Aim2). We will test if MAMs from commensal and pathogenic bacteria differ in their lipid binding specificity and how these differences impact on the composition of mixed bacterial populations and the outcome of infection (Aim3). For our investigations, we will use two complementary infection models, cultured human cells and zebrafish, putting our findings in the context of a living host

The proposed work will allow us to tailor MAM derivatives with a specific set of properties (transient or stable binding to host cells, activation or evasion of host cell responses). This will allow us to use them for two distinct applications, benefiting the biomedical field and basic physiology research:

1) Development of MAMs as anti-infectives (ongoing, although clinical application is not expected within the duration of this grant): 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 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 which are difficult to treat with conventional antibiotics.

2) Development of MAMs as drug absorption enhancers (future potential for the biomedical field, but immediately applicable to the research community): Epithelial barriers, such as the blood-brain barrier or intestinal epithelium, can prevent drugs from reaching their target site. This prevents the use of many drugs altogether and limits the use of others to intravenous delivery. It also poses a serious limitation to many physiology and neuroscience experiments (e.g., molecular imaging using PET radiotracers). The use of MAMs with strong but transient RhoA activation profile and well characterized adhesion properties as non toxic and tissue-specific absorption enhancers, would increase research potential in these areas in the short-term and may increase the spectrum of orally available drugs and treatment options in the long-term.

The proposed research would benefit and impact several areas:
Industry: Our previous work on MAM-based inhibitors is already covered by a patent and development of our prototype inhibitors into novel anti-infective materials 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 lipid-specific probes, which is also covered by the patent, 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 signaling 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. The tools developed through this proposal will also benefit basic research into lipid signaling (e.g., MAM-based probes as lipid markers) and disciplines such as physiology and neurosciences (e.g., use of specifically tailored MAMs to enhance paracellular delivery of large molecules such as fluorescent probes or peptides across epithelial/endothelial barriers, such as the blood-brain barrier).

Education and Public 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, the ongoing development of anti-infectives). 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 (e.g., the IMI Summer School) to connect with these beneficiaries.