Molecular mechanisms modulating host epithelial integrity in response to bacterial adhesion

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


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.

Technical Summary

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

Planned Impact

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.


10 25 50

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

publication icon
O'Donoghue EJ (2016) Mechanisms of outer membrane vesicle entry into host cells. in Cellular microbiology

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 Will generate knowledge of anti-adhesives
Sectors Pharmaceuticals and Medical Biotechnology

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 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