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1.
J Immunol Methods ; 464: 47-56, 2019 01.
Article in English | MEDLINE | ID: mdl-30365927

ABSTRACT

X-ray crystallographic studies of class I peptide-MHC molecules (pMHC) continue to provide important insights into immune recognition, however their success depends on generation of diffraction-quality crystals, which remains a significant challenge. While protein engineering techniques such as surface-entropy reduction and lysine methylation have proven utility in facilitating and/or improving protein crystallisation, they risk affecting the conformation and biochemistry of the class I MHC antigen binding groove. An attractive alternative is the use of noncovalent crystallisation chaperones, however these have not been developed for pMHC. Here we describe a method for promoting class I pMHC crystallisation, by exploiting its natural ligand interaction with the immunoregulatory receptor LILRB1 as a novel crystallisation chaperone. First, focussing on a model HIV-1-derived HLA-A2-restricted peptide, we determined a 2.4 ŠHLA-A2/LILRB1 structure, which validated that co-crystallisation with LILRB1 does not alter conformation of the antigenic peptide. We then demonstrated that addition of LILRB1 enhanced the crystallisation of multiple peptide-HLA-A2 complexes, and identified a generic condition for initial co-crystallisation. LILRB1 chaperone-based crystallisation enabled structure determination for HLA-A2 complexes previously intransigent to crystallisation, including both conventional and post-translationally-modified peptides, of diverse lengths. Since both the LILRB1 recognition interface on the HLA-A2 α3 domain molecule and HLA-A2-mediated crystal contacts are predominantly conserved across class I MHC molecules, the approach we outline could prove applicable to a diverse range of class I pMHC. LILRB1 chaperone-mediated crystallisation should expedite molecular insights into the immunobiology of diverse immune-related diseases and immunotherapeutic strategies, particularly involving class I pMHC complexes that are challenging to crystallise.


Subject(s)
Antigens, CD/chemistry , Crystallography, X-Ray/methods , HLA-A2 Antigen/chemistry , Leukocyte Immunoglobulin-like Receptor B1/chemistry , Molecular Chaperones/chemistry , Antigens, CD/immunology , Binding Sites , Crystallization , HIV-1/immunology , HLA-A2 Antigen/immunology , Humans , Immunodominant Epitopes , Leukocyte Immunoglobulin-like Receptor B1/immunology , Ligands , Models, Molecular , Molecular Chaperones/immunology , Phosphorylation , Protein Binding , Protein Conformation , Structure-Activity Relationship
2.
J Biol Chem ; 292(48): 19792-19803, 2017 12 01.
Article in English | MEDLINE | ID: mdl-28982977

ABSTRACT

Bacterial adhesion to host receptors is an early and essential step in bacterial colonization, and the nature of adhesin-receptor interactions determines bacterial localization and thus the outcome of these interactions. Here, we determined the host receptors for the multivalent adhesion molecule (MAM) from the gut commensal Escherichia coli HS (MAMHS), which contains an array of seven mammalian cell entry domains. The MAMHS adhesin interacted with a range of host receptors, through recognition of a shared 3-O-sulfogalactosyl moiety. This functional group is also found in mucin, a component of the intestinal mucus layer and thus one of the prime adherence targets for commensal E. coli Mucin gels impeded the motility of E. coli by acting as a physical barrier, and the barrier effect was enhanced by specific interactions between mucin and MAMHS in a sulfation-dependent manner. Desulfation of mucin by pure sulfatase or the sulfatase-producing commensal Bacteroides thetaiotaomicron decreased binding of E. coli to mucin and increased the attachment of bacteria to the epithelial surface via interactions with surface-localized sulfated lipid and protein receptors. Together, our results demonstrate that the E. coli adhesin MAMHS facilitates retention of a gut commensal by attachment to mucin. They further suggest that the amount of sulfatase secreted by mucin-foraging bacteria such as B. thetaiotaomicron, inhabiting the same niche, may affect the capacity of the mucus barrier to retain commensal E. coli.


Subject(s)
Bacterial Adhesion , Escherichia coli Proteins/metabolism , Escherichia coli/physiology , Galactose/metabolism , Sulfatases/metabolism , Cell Adhesion Molecules/metabolism , Escherichia coli/enzymology , Mucins/metabolism , Vibrio parahaemolyticus/physiology
3.
Oncotarget ; 8(33): 54160-54172, 2017 Aug 15.
Article in English | MEDLINE | ID: mdl-28903331

ABSTRACT

Dysregulated post-translational modification provides a source of altered self-antigens that can stimulate immune responses in autoimmunity, inflammation, and cancer. In recent years, phosphorylated peptides have emerged as a group of tumour-associated antigens presented by MHC molecules and recognised by T cells, and represent promising candidates for cancer immunotherapy. However, the impact of phosphorylation on the antigenic identity of phosphopeptide epitopes is unclear. Here we examined this by determining structures of MHC-bound phosphopeptides bearing canonical position 4-phosphorylations in the presence and absence of their phosphate moiety, and examining phosphopeptide recognition by the T cell receptor (TCR). Strikingly, two peptides exhibited major conformational changes upon phosphorylation, involving a similar molecular mechanism, which focussed changes on the central peptide region most critical for T cell recognition. In contrast, a third epitope displayed little conformational alteration upon phosphorylation. In addition, binding studies demonstrated TCR interaction with an MHC-bound phosphopeptide was both epitope-specific and absolutely dependent upon phosphorylation status. These results highlight the critical influence of phosphorylation on the antigenic identity of naturally processed class I MHC epitopes. In doing so they provide a molecular framework for understanding phosphopeptide-specific immune responses, and have implications for the development of phosphopeptide antigen-specific cancer immunotherapy approaches.

4.
mSphere ; 2(5)2017.
Article in English | MEDLINE | ID: mdl-28959735

ABSTRACT

Foodborne infections with enterohemorrhagic Escherichia coli (EHEC) are a major cause of diarrheal illness in humans and can lead to severe complications such as hemolytic uremic syndrome. Cattle and other ruminants are the main reservoir of EHEC, which enters the food chain through contaminated meat, dairy, or vegetables. Here, we describe the establishment of a vertebrate model for foodborne EHEC infection, using larval zebrafish (Danio rerio) as a host and the protozoan prey Paramecium caudatum as a vehicle. We follow pathogen release from the vehicle, intestinal colonization, microbe-host interactions, and microbial gene induction within a live vertebrate host, in real time, throughout the course of infection. We demonstrate that foodborne EHEC colonizes the gastrointestinal tract faster and establishes a higher burden than waterborne infection. Expression of the locus of enterocyte effacement (LEE), a key EHEC virulence factor, was observed early during infection, mainly at sites that experience fluid shear, and required tight control to enable successful host colonization. EHEC infection led to strain- and LEE-dependent mortality in the zebrafish host. Despite the presence of the endogenous microbiota limiting EHEC colonization levels, EHEC colonization and virulence can be studied either under gnotobiotic conditions or against the backdrop of an endogenous (and variable) host microbiota. Finally, we show that the model can be used for investigation of factors affecting shedding and transmission of bacteria to naive hosts. Overall, this constitutes a useful model, which ideally complements the strengths of existing EHEC vertebrate models. IMPORTANCE Enterohemorrhagic Escherichia coli (EHEC) is a foodborne pathogen which can cause diarrhea, vomiting, and, in some cases, severe complications such as kidney failure in humans. Up to 30% of cattle are colonized with EHEC, which can enter the food chain through contaminated meat, dairy, and vegetables. In order to control infections and stop transmission, it is important to understand what factors allow EHEC to colonize its hosts, cause virulence, and aid transmission. Since this cannot be systematically studied in humans, it is important to develop animal models of infection and transmission. We developed a model which allows us to study foodborne infection in zebrafish, a vertebrate host that is transparent and genetically tractable. Our results show that foodborne infection is more efficient than waterborne infection and that the locus of enterocyte effacement is a key virulence determinant in the zebrafish model. It is induced early during infection, and loss of tight LEE regulation leads to a decreased bacterial burden and decreased host mortality. Overall, the zebrafish model allows us to study foodborne infection, including pathogen release from the food vehicle and gene regulation and its context of host-microbe interactions, as well as environmental shedding and transmission to naive hosts.

5.
Sci Rep ; 6: 39341, 2016 12 20.
Article in English | MEDLINE | ID: mdl-27996032

ABSTRACT

Classical antimicrobial drugs target proliferation and therefore place microbes under extreme selective pressure to evolve resistance. Alternative drugs that target bacterial virulence without impacting survival directly offer an attractive solution to this problem, but to date few such molecules have been discovered. We previously discovered a widespread group of bacterial adhesins, termed Multivalent Adhesion Molecules (MAMs) that are essential for initial binding of bacteria to host tissues and virulence. Thus, targeting MAM-based adherence is a promising strategy for displacing pathogens from host tissues and inhibiting infection. Here, we show that topical application of polymeric microbeads functionalized with the adhesin MAM7 to a burn infected with multidrug-resistant Pseudomonas aeruginosa substantially decreased bacterial loads in the wound and prevented the spread of the infection into adjacent tissues. As a consequence, the application of this adhesion inhibitor allowed for vascularization and wound healing, and maintained local and systemic inflammatory responses to the burn. We propose that MAM7-functionalized microbeads can be used as a topical treatment, to reduce bacterial attachment and hence prevent bacterial colonization and infection of wounds. As adhesion is not required for microbial survival, this anti-infective strategy has the potential to treat multidrug-resistant infections and limit the emergence of drug-resistant pathogens.


Subject(s)
Adhesins, Bacterial/pharmacology , Bacterial Adhesion/drug effects , Burns/microbiology , Pseudomonas Infections/prevention & control , Pseudomonas aeruginosa/metabolism , Wound Infection/prevention & control , Adhesins, Bacterial/metabolism , Animals , Anti-Bacterial Agents/pharmacology , Bacterial Load/drug effects , Drug Resistance, Multiple, Bacterial , Humans , Male , Microspheres , Pseudomonas Infections/drug therapy , Pseudomonas Infections/microbiology , Rats , Rats, Sprague-Dawley , Wound Healing/drug effects , Wound Infection/drug therapy , Wound Infection/microbiology
7.
J Vis Exp ; (105)2015 Nov 16.
Article in English | MEDLINE | ID: mdl-26650964

ABSTRACT

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The biochemical and functional characterization of adhesins mediating these initial bacteria-host interactions is often compromised by the presence of other bacterial factors, such as cell wall components or secreted molecules, which interfere with the analysis. This protocol describes the production and use of biomimetic materials, consisting of pure recombinant adhesins chemically coupled to commercially available, functionalized polystyrene beads, which have been used successfully to dissect the biochemical and functional interactions between individual bacterial adhesins and host cell receptors. Protocols for different coupling chemistries, allowing directional immobilization of recombinant adhesins on polymer scaffolds, and for assessment of the coupling efficiency of the resulting "bacteriomimetic" materials are also discussed. We further describe how these materials can be used as a tool to inhibit pathogen mediated cytotoxicity and discuss scope, limitations and further applications of this approach in studying bacterial - host interactions.

8.
Int J Mol Sci ; 16(2): 2626-40, 2015 Jan 23.
Article in English | MEDLINE | ID: mdl-25625516

ABSTRACT

The ability of bacterial species to colonize and infect host organisms is critically dependent upon their capacity to adhere to cellular surfaces of the host. Adherence to cell surfaces is known to be essential for the activation and delivery of certain virulence factors, but can also directly affect host cell signaling to aid bacterial spread and survival. In this review we will discuss the recent advances in the field of bacterial adhesion, how we are beginning to unravel the effects adhesins have on host cell signaling, and how these changes aid the bacteria in terms of their survival and evasion of immune responses. Finally, we will highlight how the exploitation of bacterial adhesins may provide new therapeutic avenues for the treatment of a wide range of bacterial infections.


Subject(s)
Adhesins, Bacterial/metabolism , Bacteria/metabolism , Adhesins, Bacterial/chemistry , Animals , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Fimbriae, Bacterial/chemistry , Fimbriae, Bacterial/metabolism , Host-Pathogen Interactions , Humans , Integrins/metabolism , Signal Transduction
9.
PLoS Pathog ; 10(9): e1004421, 2014 Sep.
Article in English | MEDLINE | ID: mdl-25255250

ABSTRACT

Vibrio parahaemolyticus is an emerging bacterial pathogen which colonizes the gastrointestinal tract and can cause severe enteritis and bacteraemia. During infection, V. parahaemolyticus primarily attaches to the small intestine, where it causes extensive tissue damage and compromises epithelial barrier integrity. We have previously described that Multivalent Adhesion Molecule (MAM) 7 contributes to initial attachment of V. parahaemolyticus to epithelial cells. Here we show that the bacterial adhesin, through multivalent interactions between surface-induced adhesin clusters and phosphatidic acid lipids in the host cell membrane, induces activation of the small GTPase RhoA and actin rearrangements in host cells. In infection studies with V. parahaemolyticus we further demonstrate that adhesin-triggered activation of the ROCK/LIMK signaling axis is sufficient to redistribute tight junction proteins, leading to a loss of epithelial barrier function. Taken together, these findings show an unprecedented mechanism by which an adhesin acts as assembly platform for a host cellular signaling pathway, which ultimately facilitates breaching of the epithelial barrier by a bacterial pathogen.


Subject(s)
Cell Adhesion Molecules/metabolism , Epithelial Cells/pathology , Escherichia coli/metabolism , GTP Phosphohydrolase Activators/metabolism , Host-Pathogen Interactions , Vibrio Infections/pathology , rho GTP-Binding Proteins/metabolism , Adhesins, Bacterial/metabolism , Blotting, Western , Caco-2 Cells , Cell Membrane Permeability , Epithelial Cells/metabolism , Escherichia coli/growth & development , HeLa Cells , Humans , Microscopy, Fluorescence , Signal Transduction , Vibrio Infections/metabolism , Vibrio Infections/microbiology , Vibrio parahaemolyticus/pathogenicity , Virulence
10.
Cell Rep ; 2(2): 270-82, 2012 Aug 30.
Article in English | MEDLINE | ID: mdl-22884366

ABSTRACT

It is clear from epidemiological studies that excess iron is associated with increased risk of colorectal cancer; however, questions regarding the mechanism of how iron increases cancer risk, the source of the excess iron (circulating or luminal), and whether iron reduction represents a potential therapeutic option remain unanswered. In this study, we show that after Apc deletion, the cellular iron acquisition proteins TfR1 and DMT1 are rapidly induced. Conversely, restoration of APC reduces cellular iron due to repression of these proteins. To test the functional importance of these findings, we performed in vivo investigations of the impact of iron levels on intestinal tumorigenesis. Strikingly, depletion of luminal (but not systemic) iron strongly suppressed murine intestinal tumorigenesis, whereas increased luminal iron strongly promoted tumorigenesis. Taken together, our data definitively delineate iron as a potent modifier of intestinal tumorigenesis and have important implications for dietary iron supplementation in patients at high risk of colorectal cancer.


Subject(s)
Adenomatous Polyposis Coli Protein , Cell Transformation, Neoplastic/metabolism , Colorectal Neoplasms/metabolism , Intestinal Mucosa/metabolism , Iron/metabolism , Animals , Cation Transport Proteins/genetics , Cation Transport Proteins/metabolism , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/pathology , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , Colorectal Neoplasms/prevention & control , Dietary Supplements , Gene Deletion , Humans , Intestines/pathology , Iron/pharmacology , Mice , Mice, Knockout , Receptors, Transferrin/genetics , Receptors, Transferrin/metabolism
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