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

Lead Research Organisation: University of Birmingham
Department Name: Sch of Biosciences


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.

Technical Summary

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.

Planned Impact

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.


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publication icon
Alsharif G (2015) Host attachment and fluid shear are integrated into a mechanical signal regulating virulence in Escherichia coli O157:H7. in Proceedings of the National Academy of Sciences of the United States of America

Description Objectives:
1) understand the molecular requirements for MAM-triggered host signalling processes. MAMs consist of tandem arrays of
6-7 mammalian cell entry (mce) domains, which we found can cluster lipid molecules in the host membrane and trigger
downstream signalling processes on the host side, thus promoting infection. We will study which features of MAMs are
required to trigger this host response to better understand this novel type of host -pathogen interaction. This objective has been partially met, in that we have defined several important features defining downstream signaling responses (Lim et al, PLOS Path 2014; AL-Saedi et al, JBC 2017, Roberts et al, PLOS Comp Biol 2018). These data have laid the ground for a further grant application, which has recently been submitted to the NIH (Krachler as a PI).

2) identify the molecular signature for ligand binding specificity of MAMs. Mce domains can bind at least two different types
of ligands; phosphatidic acids and steroids. MAMs from different species are predicted to display varying lipid binding
specificities, so depending on which component of the host cell membrane they recognize, bacteria may trigger different
host responses, thus influencing the fate of infection. We will investigate the molecular repertoire of mce ligands and
identify ligand-specific sequence features of mce domains to be able to predict and engineer the way MAMs interact with
host cells. This objective has been fully met and our findings have been published recently (Al-Saedi, IAI 2016; Al-Saedi, Vaz et al, JBC 2017).

3) identify the mechanism of ligand binding by MAMs. We will use NMR and crystallography to solve the first structure of
this important group of lipid-binding proteins, elucidate the way they bind their lipid ligand and investigate how tandem
arrangements of mce domains communicate ligand binding to achieve cooperativity and thus tight interaction with host
cells. We recently solved 7.5A Cryo-EM structures of both E. coli and V. parahaemolyticus MAMs, as well as ligand-bound E. coli MAM. We are currently working on further refining these structures prior to publication, which will meet objective 3. These data have also laid ground for a further grant application, which has recently been submitted to the NIH (Krachler as a PI).
Exploitation Route looking into anti-adhesive therapies
Sectors Pharmaceuticals and Medical Biotechnology

Description Optimizing Wound Care in the Combat Casualty: Inhibitors of bacterial adhesion as new targets for the prevention and treatment of drug-resistant infections 
Organisation University of Texas Southwestern Medical Center
Country United States 
Sector Academic/University 
PI Contribution We are synthesizing and characterizing adhesion inhibitors, which are evaluated for their in vivo efficacy by our US collaborators.
Collaborator Contribution Our collaborators have established and are now using an in vivo burn wound infection model to test the efficacy of adhesion inhibitors from our group against multidrug-resistant bacterial infections.
Impact DoD grant application, currently under review, Joint manuscript, currently in preparation. This is a multi-disciplinary collaboration, involving medical sciences, biochemistry, microbiology and applied mathematics.
Start Year 2014
Description IMI Summer School 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact Each year, approximately 30 students attended the IMI summer school, which was a week-long activity at the IMI Birmingham. This was an opportunity for students to engage with our microbiology research, and for undergraduate and postgraduate students to participate in science communication.
Year(s) Of Engagement Activity 2014,2015
Description Seminar at University of Tromso 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Delivered two seminars as part of a postgraduate summer school on Infection Biology at the University of Tromso.
Year(s) Of Engagement Activity 2015
Description Talk at Experimental Biology, San Diego 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Delivered an invited talk at Experimental Biology in San DIego, 2014
Year(s) Of Engagement Activity 2014
Description Various Scientific Meeting Presentations 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Oral
• Young Microbiologist Symposium 2016, Dundee: Stones, D.H. "Targeting bacterial adherence suppresses burn wound infection with drug-resistant Pseudomonas aeruginosa"

• Midlands Molecular Microbiology Meeting 2015, Nottingham. " Multivalent adhesion molecules are able to form oligomers in vivo and have a diverse lipid ligand-binding repertoire"
• Microbiology Society annual meeting 2015: " Made to stick: Anti-adhesion therapy as an approach to prevent and treat multidrug-resistant bacterial infections"
Year(s) Of Engagement Activity 2015,2016
Description Various presentations by Krachler group 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Presentations were:
FASEB Research Conference on Small GTPases in Membrane Processes: Trafficking, Autophagy and Disease, VA, 2018
University of Dundee, Division of Microbiology and Biological Chemistry, 2016
University of Texas Rio Grande Valley, 2017
ASM Texas Branch Fall Meeting, Dallas, 2016
Year(s) Of Engagement Activity 2016,2017,2018