Rational Design and Development of HDAC Inhibitors for Breast Cancer Treatment.

BACKGROUND: Breast cancer is the most prevalent cancer amongst females across the globe, and with over 2 million new cases reported in 2018, it poses a huge economic burden to the already dwindling public health. A dearth of therapies in the pipeline to treat triple-negative breast cancers and acquisition of resistance against the existing line of treatments urge the need to strategize novel therapeutics in order to add new drugs to the pipeline. HDAC inhibitors (HDACi) is one such class of small molecule inhibitors that target histone deacetylases to bring about chromosomal remodelling and normalize dysregulated gene expression that marks breast cancer progression. OBJECTIVE: While four HDACi have been approved by the FDA for the treatment of different cancer types, no HDACi is specifically earmarked for clinical management of breast cancer. Owing to the differential HDAC expression pertaining to different types of breast cancers, isoform-selective HDAC inhibitors need to be discovered. CONCLUSION: This review attempts to set the stage for the rational structure-based discovery of isoform-selective HDACi by providing structural insights into different HDACs and their catalytic folds based on their classes and individual landscape. The development of inhibitors in accordance with the differential expression of HDAC isoforms exhibited in breast cancer cells is a promising strategy to rationally design selective and effective inhibitors, adopting a 'personalized-medicine' approach.

Natural Antibodies: Protecting Role of IgM in Glioblastoma and Brain Tumours.

BACKGROUND: Glioblastoma is a grade IV astrocytoma with an average survival span for patients of 18 months after initial diagnosis and no standard treatment protocol is available. Therefore, there is a need to search for novel approaches to target glioblastoma. OBJECTIVES: This review intends to capture the role of immunoglobulin-M in cancer, more specifically in glioblastoma multiforme (GBM), and to compile the latest developments and immunological pathways relevant to glioblastoma. METHODS: Information on glioblastoma, cancer microenvironment, cancer therapeutics, and how to improve the scenario were obtained from scientific literature databases such as Pubmed, Medline, Google Scholar, Science Direct, Springer, Wiley online library, and some data was harvested from regulatory and compliance databases such as clinicaltrials.gov, FDA database, and WHO Globocan. RESULTS AND CONCLUSION: Currently, only a limited number of therapies are approved for GBM, and no standard care is in place in case of disease relapse, necessitating a possible broader perspective in looking at the disease and its underlying mechanisms.

Minimal epitope for Mannitou IgM on paucimannose-carrying glycoproteins.

Paucimannosidic glycans are restricted to the core structure [Man1-3GlcNAc2Fuc0-1] of N-glycans and are rarely found in mammalian tissues. Yet, especially [Man2-3GlcNAc2Fuc1] have been found significantly upregulated in tumors, including in colorectal and liver cancer. Mannitou IgM is a murine monoclonal antibody that was previously shown to recognize Man3GlcNAc2 with an almost exclusive selectivity. Here, we have sought the definition of the minimal glycan epitope of Mannitou IgM, initiated by screening on a newly designed paucimannosidic glycan microarray; among the best binders were Man3GlcNAc2 and its α1,6 core-fucosylated variant, Man3GlcNAc2Fuc1. Unexpectedly and in contrast to earlier findings, Man5GlcNAc2-type structures bind equally well and a large tolerance was observed for substitutions on the α1,6 arm. It was confirmed that any substitution on the single α1,3-linked mannose completely abolishes binding. Surface plasmon resonance for kinetic measurements of Mannitou IgM binding, either directly on the glycans or as presented on omega-1 and kappa-5 soluble egg antigens from the helminth parasite Schistosoma mansoni, showed submicromolar affinities. To characterize the epitope in greater and atomic detail, saturation transfer difference nuclear magnetic resonance spectroscopy was performed with the Mannitou antigen-binding fragment. The STD-NMR data demonstrated the strongest interactions with the aliphatic protons H1 and H2 of the α1-3-linked mannose and weaker imprints on its H3, H4 and H5 protons. In conclusion, Mannitou IgM binding requires a nonsubstituted α1,3-linked mannose branch of paucimannose also on proteins, making it a highly specific tool for the distinction of concurrent human tumor-associated carbohydrate antigens.

SERS characterization of aggregated and isolated bacteria deposited on silver-based substrates.

Surface-enhanced Raman scattering (SERS), based on the enhancement of the Raman signal of molecules positioned within a few nanometres from a structured metal surface, is ideally suited to provide bacterial-specific molecular fingerprints which can be used for analytical purposes. However, for some complex structures such as bacteria, the generation of reproducible SERS spectra is still a challenging task. Among the various factors influencing the SERS variability (such as the nature of SERS-active substrate, Raman parameters and bacterial specificity), we demonstrate in this study that the environment of Gram-positive and Gram-negative bacteria deposited on ultra-thin silver films also impacts the origin of the SERS spectra. In the case of densely packed bacteria, the obtained SERS signatures were either characteristic of the secretion of adenosine triphosphate for Staphylococcus aureus (S. aureus) or the cell wall and the pili/flagella for Escherichia coli (E. coli), allowing for an easy discrimination between the various strains. In the case of isolated bacteria, SERS mapping together with principal component analysis revealed some variabilities of the spectra as a function of the bacteria environment and the bactericidal effect of the silver. However, the variability does not preclude the SERS signatures of various E. coli strains to be discriminated.

Binding of Gold(III) Porphyrin by the Pro-metastatic Regulatory Protein Human Galectin-3.

Gold(III) porphyrin presents an attractive alternative to the use of, for example, cisplatin in chemotherapy. However, approaches that allow to selectively target cancer cells are highly sought. Many plant and mammalian lectins have been shown to bind oligosaccharide sequences of the aberrant glycosylation pattern found on cancerous tumors. For example human galectin-3, of the galectin family specific for ß-galactoside, is overexpressed in the extracellular matrix of tumorigenous and metastatic tissues. We searched for non-carbohydrate ligands for galectin-3 that can guide a cytotoxic drug to the cancer cells by maintaining its affinity for tumor associated carbohydrate antigens. Previous findings showed that zinc tetrasulfonatophenylporphyrin can bind galectin-3 with sub-micromolar affinity without disturbing lactose binding. Gold(III) porphyrin is not only cytotoxic to cancer cells, it knows also a potential application as photosensitiser in photodynamic therapy. We investigated the binding of gold(III) porphyrin to galectin-3 using different biophysical interaction techniques and demonstrated a low micromolar affinity of human galectin-3 for the cytotoxic compound. Co-crystallization attempts in order to understand the binding mode of gold porphyrin to galectin-3 failed, but molecular docking emphasized a highly populated secondary binding site that does not hinder lactose or Thomsen Friendenreich disaccharide binding. This suggests that gold(III) porphyrin might significantly enhance its concentration and delivery to cancer cells by binding to human galectin-3 that keeps its orientation towards tumor associated carbohydrate antigens.

Mucin modified SPR interfaces for studying the effect of flow on pathogen binding to Atlantic salmon mucins.

Knowledge on host-pathogen interactions contributes to the development of approaches to alleviate infectious disease. In this work, we developed a surface plasmon resonance (SPR) based method for investigating bacteria/mucins interactions. Furthermore, we investigated adhesion of three pathogens, Aeromonas salmonicida, Aeromonas hydrophila and Vibrio harveyi, to Atlantic salmon mucins isolated from different epithelial sites, using SPR and microtiter-based binding assays. We demonstrated that performing bacterial binding assays to mucins using SPR is feasible and has advantages over microtiter-based binding assays, especially under flow conditions. The fluid flow in the SPR is linear and continuous and SPR enables real-time reading of mucin-bacterial bonds, which provides an in vivo-like setup for analysis of bacterial binding to mucins. The variation between technical replicates was smaller using SPR detection compared to the adenosine 5'-triphosphate (ATP) bioluminescence assay in microtiter plates. Furthermore, we demonstrated that the effect of flow on pathogen-mucin interaction is significant and that bacterial adhesion differ non-linearly with flow rates and depend on the epithelial source of the mucin.

Heptylmannose-functionalized cellulose for the binding and specific detection of pathogenic E. coli.

We developed a chemical method to covalently functionalize cellulose nanofibers and cellulose paper with mannoside ligands displaying a strong affinity for the FimH adhesin from pathogenic E. coli strains. Mannose-grafted cellulose proved efficient to selectively bind FimH lectin and discriminate pathogenic E. coli strains from non-pathogenic ones. These modified papers are valuable tools for diagnosing infections promoted by E. coli, such as cystitis or inflammatory bowel diseases, and the concept may be applicable to other life-threatening pathogens.

Enhanced antibacterial activity of carbon dots functionalized with ampicillin combined with visible light triggered photodynamic effects.

In the last years, carbon-based nanomaterials have attracted considerable attention in a wide range of fields, particularly in biomedicine, owing to their remarkable photo-physical and chemical properties. In this study, we demonstrate that amine-terminated carbon dots (CDs-NH2) functionalized with ampicillin (AMP) offer a new perspective for antibacterial treatment. The amine-functionalized carbon dots were used as a carrier for immobilization and delivery of ampicillin (CDs-AMP) and as a visible light-triggered antibacterial material. Additionally, AMP immobilization on the CDs-NH2 surface improves its stability in solution as compared to free AMP. The AMP conjugated CDs platform combines the antibacterial function of AMP and conserves the intrinsic theranostic properties of CDs-NH2. Therefore, the AMP immobilized onto CDs-NH2 surface together with the generation of moderate quantities of reactive oxygen species under visible light illumination are very effective to inactivate the growth of Escherichia coli.

Oligomannose-Rich Membranes of Dying Intestinal Epithelial Cells Promote Host Colonization by Adherent-Invasive E. coli.

A novel mechanism is revealed by which clinical isolates of adherent-invasive Escherichia coli (AIEC) penetrate into the epithelial cell layer, replicate, and establish biofilms in Crohn's disease. AIEC uses the FimH fimbrial adhesin to bind to oligomannose glycans on the surface of host cells. Oligomannose glycans exposed on early apoptotic cells are the preferred binding targets of AIEC, so apoptotic cells serve as potential entry points for bacteria into the epithelial cell layer. Thereafter, the bacteria propagate laterally in the epithelial intercellular spaces. We demonstrate oligomannosylation at two distinct sites of a glycoprotein receptor for AIEC, carcinoembryonic antigen related cell adhesion molecule 6 (CEACAM6 or CD66c), on human intestinal epithelia. After bacterial binding, FimH interacts with CEACAM6, which then clusters. The presence of the highest-affinity epitope for FimH, oligomannose-5, on CEACAM6 is demonstrated using LC-MS/MS. As mannose-dependent infections are abundant, this mechanism might also be used by other adherent-invasive pathogens.

The potential of FimH as a novel therapeutic target for the treatment of Crohn's disease.

INTRODUCTION: Crohn's disease (CD) is a life-long chronic disorder characterized by intestinal inflammation. Current treatments for CD are directed towards abnormal immune responses rather than the intestinal bacteria that trigger intestinal inflammation. Areas covered: Adherent-Invasive Escherichia coli (AIEC) bacteria abnormally colonize the ileal mucosa in a subgroup of CD patients. They can promote or perpetuate chronic inflammation and are therefore an interesting therapeutic target. Various strategies that target these E. coli strains have been developed to promote their intestinal clearance. Here, we review current AIEC-targeted strategies, especially anti-adhesive strategies, that are based on the development of FimH antagonists. We discuss their potential as personalized microbiota-targeted treatments for CD patients abnormally colonized by AIEC. Expert opinion: A large panel of mannose-derived FimH antagonists were tested for their ability to inhibit E. coli adhesion to host cells. Documented reports suggest that monovalent mannosides are promising candidates that could represent a complementary therapeutic strategy to prevent intestinal inflammation in the E. coli-colonized CD patient subgroup. Ongoing research continues to improve the pharmacokinetic properties of mannosides, and hopefully, clinical trials will be performed in CD patients in the near future.

Physiochemical Tuning of Potent Escherichia coli Anti-Adhesives by Microencapsulation and Methylene Homologation.

Thiazolylaminomannosides (TazMan) are FimH antagonists with anti-adhesive potential against adherent-invasive Escherichia coli (AIEC) promoting gut inflammation in patients with Crohn's disease. The lead TazMan is highly potent in vitro, but shows limited in vivo efficiency, probably due to low pH stability and water solubility. We recently developed a second generation of stable TazMan, but the anti-adhesive effect was lower than the first. Herein we report a co-crystal structure of the lead TazMan in FimH, revealing that the anomeric NH group and the second thiazole moiety provide a positive hydrogen bonding interaction with a trapped water molecule, and π-stacking with Tyr48 of FimH, respectively. Consequently, we developed NeoTazMan homologated with a methylene group for low-pH and mannosidase stability with a conserved NH group and bearing various heterocyclic aglycones. Microencapsulation of the lead NeoTazMan in γ-cyclodextrin dramatically improved water solubility without disrupting the affinity for FimH or the anti-adhesive effect against AIEC isolated from patients with Crohn's disease.

On demand electrochemical release of drugs from porous reduced graphene oxide modified flexible electrodes.

Despite the advantages of an electrochemical control for drug release, only a handful of electrochemical-based release systems have been developed so far. We report herein on the development of an electrochemically activatable platform for on-demand delivery of drugs. It is based on flexible gold thin film electrodes coated with porous reduced graphene oxide (prGO) nanosheets onto which the drug of interest has been integrated beforehand. Two different drugs are investigated here: ondansetron hydrochloride (ODS), a selective 5-HT3 receptor antagonist used for preventing nausea and vomiting caused by chemotherapy and radiotherapy, and ampicillin (AMP), an antibiotic to prevent and treat a number of bacterial infections such as respiratory tract infections, urinary tract infections, and meningitis. In the case of ODS, application of a negative potential bias of -0.8 V results in a sustained slow ODS release with an ODS flux of 47 µg cm-2 h-1. In the case of AMP, we show that polyethyleneimine modified prGO (prGO/PEI) is an extremely efficient matrix. Upon the application of +0.8 V, 24% of AMP could be released from the electrical interface in a time span of 2 h. The released AMP kept its antibacterial activity as demonstrated by antimicrobial tests. These examples illustrate the major benefits of the developed approach for biomedical applications.

The Antiadhesive Strategy in Crohn's Disease: Orally Active Mannosides to Decolonize Pathogenic Escherichia coli from the Gut.

Blocking the adherence of bacteria to cells is an attractive complementary approach to current antibiotic treatments, which are faced with increasing resistance. This strategy has been particularly studied in the context of urinary tract infections (UTIs), in which the adhesion of pathogenic Escherichia coli strains to uroepithelial cells is prevented by blocking the FimH adhesin expressed at the tips of bacteria organelles called fimbriae. Recently, we extended the antiadhesive concept, showing that potent FimH antagonists can block the attachment of adherent-invasive E. coli (AIEC) colonizing the intestinal mucosa of patients with Crohn's disease (CD). In this work, we designed a small library of analogues of heptyl mannoside (HM), a previously identified nanomolar FimH inhibitor, but one that displays poor antiadhesive effects in vivo. The anomeric oxygen atom was replaced by a sulfur or a methylene group to prevent hydrolysis by intestinal glycosidases, and chemical groups were attached at the end of the alkyl tail. Importantly, a lead compound was shown to reduce AIEC levels in the feces and in the colonic and ileal mucosa after oral administration (10 mg kg(-1) ) in a transgenic mouse model of CD. The compound showed a low bioavailability, preferable in this instance, thus suggesting the possibility of setting up an innovative antiadhesive therapy, based on the water-soluble and non-cytotoxic FimH antagonists developed here, for the CD subpopulation in which AIEC plays a key role.

Development of Heptylmannoside-Based Glycoconjugate Antiadhesive Compounds against Adherent-Invasive Escherichia coli Bacteria Associated with Crohn's Disease.

UNLABELLED: The ileal lesions of Crohn's disease (CD) patients are colonized by adherent-invasive Escherichia coli (AIEC) bacteria. These bacteria adhere to mannose residues expressed by CEACAM6 on host cells in a type 1 pilus-dependent manner. In this study, we investigated different antagonists of FimH, the adhesin of type 1 pili, for their ability to block AIEC adhesion to intestinal epithelial cells (IEC). Monovalent and multivalent derivatives of n-heptyl α-d-mannoside (HM), a nanomolar antagonist of FimH, were tested in vitro in IEC infected with the AIEC LF82 strain and in vivo by oral administration to CEACAM6-expressing mice infected with LF82 bacteria. In vitro, multivalent derivatives were more potent than the monovalent derivatives, with a gain of efficacy superior to their valencies, probably owing to their ability to form bacterial aggregates. Of note, HM and the multi-HM glycoconjugates exhibited lower efficacy in vivo in decreasing LF82 gut colonization. Interestingly, HM analogues functionalized with an isopropylamide (1A-HM) or ß-cyclodextrin pharmacophore at the end of the heptyl tail (1CD-HM) exerted beneficial effects in vivo. These two compounds strongly decreased the amount of LF82 bacteria in the feces of mice and that of bacteria associated with the gut mucosa when administered orally at a dose of 10 mg/kg of body weight after infection. Importantly, signs of colitis and intestinal inflammation induced by LF82 infection were also prevented. These results highlight the potential of the antiadhesive compounds to treat CD patients abnormally colonized by AIEC bacteria and point to an alternative to the current approach focusing on blocking proinflammatory mediators. IMPORTANCE: Current treatments for Crohn's disease (CD), including immunosuppressive agents, anti-tumor necrosis factor alpha (anti-TNF-α) and anti-integrin antibodies, focus on the symptoms but not on the cause of the disease. Adherent-invasive Escherichia coli (AIEC) bacteria abnormally colonize the ileal mucosa of CD patients via the interaction of the mannose-specific adhesin FimH of type 1 pili with CEACAM6 mannosylated proteins expressed on the epithelial cell surface. Thus, we decided to develop an antiadhesive strategy based on synthetic FimH antagonists specifically targeting AIEC bacteria that would decrease intestinal inflammation. Heptylmannoside (HM)-based glycocompounds strongly inhibit AIEC adhesion to intestinal epithelial cells in vitro. The antiadhesive effect of two of these compounds of relatively simple chemical structure was also observed in vivo in AIEC-infected CEACAM6-expressing mice and was associated with a reduction in the signs of colitis. These results suggest a new therapeutic approach for CD patients colonized by AIEC bacteria, based on the development of synthetic FimH antagonists.

Graphene-coated surface plasmon resonance interfaces for studying the interactions between bacteria and surfaces.

A variety of physical and chemical parameters are of importance for adhesion of bacteria to surfaces. In the colonization of mammalian organisms for example, bacterial fimbriae and their adhesins not only seek particular glycan sequences exposed on diverse epithelial linings, they also enable the bacteria to overcome electrostatic repulsion exerted by their selected surfaces. In this work, we present a new technique based on simplified model systems for studying the adhesion strength of different Escherichia coli strains. For this purpose, gold-based surface plasmon resonance (SPR) interfaces were coated with thin films of reduced graphene oxide (rGO) through electrophoretic deposition. The rGO matrix was post-modified with polyethyleneimine (PEI), poly(sodium 4-styrenesulfonate) (PSS), mannose, and lactose through π-stacking and/or electrostatic interactions by simple immersion of the SPR interface into their respective aqueous solutions. The adhesion behaviors of one uropathogenic and two enterotoxigenic Escherichia coli clinical isolates, that each express structurally characterized fimbrial adhesins, were investigated. It was found that the UTI89 cystitis isolate that carries the mannose-binding FimH adhesin was most attracted to the PEI- and mannose-modified surfaces, whereas the att25 diarrhoeal strain with the N-acetylglucosamine-specific F17a-G adhesin disintegrated the lactose-modified rGO. The highly virulent 107/86 strain interacted strongly with the PSS-modified graphene oxide, in agreement with the polybasic surroundings of the ABH blood group-binding site of the FedF adhesin, and showed a linear SPR response in a concentration range between 1 × 10(2) and 1 × 10(9) cfu/mL.

Thiazolylaminomannosides as potent antiadhesives of type 1 piliated Escherichia coli isolated from Crohn's disease patients.

Adherent-invasive Escherichia coli (AIEC) have previously been shown to induce gut inflammation in patients with Crohn's disease (CD). We developed a set of mannosides to prevent AIEC attachment to the gut by blocking the FimH bacterial adhesin. The crystal structure of the FimH lectin domain in complex with a lead thiazolylaminomannoside highlighted the preferential position for pharmacomodulations. A small library of analogues showing nanomolar affinity for FimH was then developed. Notably, AIEC attachment to intestinal cells was efficiently prevented by the most active compound and at around 10000-fold and 100-fold lower concentrations than mannose and the potent FimH inhibitor heptylmannoside, respectively. An ex vivo assay performed on the colonic tissue of a transgenic mouse model of CD confirmed this antiadhesive potential. Given the key role of AIEC in the chronic intestinal inflammation of CD patients, these results suggest a potential antiadhesive treatment with the FimH inhibitors developed.

Nicotinamide dependence of uropathogenic Escherichia coli UTI89 and application of nadB as a neutral insertion site.

NAD and NADP are ubiquitous in the metabolism of Escherichia coli K-12. NAD auxotrophy can be rendered by mutation in any of the three genes nadB, nadA and nadC. The nadB and nadA genes were defined as antivirulence loci in Shigella spp., as a mutation (mainly in nadB) disrupting the synthesis of quinolinate is required for virulence. Uropathogenic E. coli (UPEC) isolates from acute cystitis patients, exhibiting nicotinamide auxotrophy, were of serotype O18 : K1 : H7. E. coli UTI89, the model uropathogenic and O18 : K1 : H7 strain, requires nicotinamide or quinolinate for growth. A mutation in the nadB gene, encoding L-aspartate oxidase, was shown to be responsible for the nicotinamide requirement of UTI89. This was further confirmed by complementation of UTI89 with a recombinant plasmid harbouring the nadB gene of E. coli K-12. An Ala28Val point mutant of the recombinant plasmid failed to support the growth of UTI89 in minimal medium. This proves that the Ala28Val mutation in the NadB gene of UTI89 completely impedes de novo synthesis of nicotinamide. In spontaneous prototrophic revertants of UTI89, the nadB gene has a Val28Ala mutation. Both analyses implicate that the nicotinamide auxotrophy of UTI89 is caused by a single Ala28Val mutation in NadB. We showed that the same mutation is also present in other NAD auxotrophic E. coli O18 strains. No significant differences were observed between the virulence of isogenic NAD auxotrophic and prototrophic strains in the murine ascending urinary tract infection model. Considering these data, we applied the nadB locus as a neutral site for DNA insertions in the bacterial chromosome. We successfully restored the parental phenotype of a fimH mutant by inserting fimH, with a synthetic em7 promoter, into the nadB gene. This neutral insertion site is of significance for further research on the pathogenicity of UPEC.

Synthetic multimeric heptyl mannosides as potent antiadhesives of uropathogenic Escherichia coli.

Urinary tract infections caused by uropathogenic Escherichia coli presents a serious communal and nosocomial health problem initiated by bacterial adhesion to the bladder cells. E. coli expresses fimbriae with a mannose-binding adhesin, FimH, at the tip. Heptyl alpha-D-mannoside (HM) is a nanomolar inhibitor of this lectin, preventing adhesion of type 1-piliated E. coli and reducing bacteria levels in a murine cystitis model. Herein, we described the synthesis of multimeric heptyl-mannosides with valencies ranging from one to four by copper-catalyzed azide alkyne cycloaddition (CuAAC). Biological evaluation of the multivalent compounds revealed an increase in potency compared to HM. Inhibition of bladder cell binding highlighted a promising tetravalent derivative with inhibitory concentrations 6000- and 64-fold lower than mannose and HM respectively.

Intervening with urinary tract infections using anti-adhesives based on the crystal structure of the FimH-oligomannose-3 complex.

BACKGROUND: Escherichia coli strains adhere to the normally sterile human uroepithelium using type 1 pili, that are long, hairy surface organelles exposing a mannose-binding FimH adhesin at the tip. A small percentage of adhered bacteria can successfully invade bladder cells, presumably via pathways mediated by the high-mannosylated uroplakin-Ia and alpha3beta1 integrins found throughout the uroepithelium. Invaded bacteria replicate and mature into dense, biofilm-like inclusions in preparation of fluxing and of infection of neighbouring cells, being the major cause of the troublesome recurrent urinary tract infections. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate that alpha-D-mannose based inhibitors of FimH not only block bacterial adhesion on uroepithelial cells but also antagonize invasion and biofilm formation. Heptyl alpha-D-mannose prevents binding of type 1-piliated E. coli to the human bladder cell line 5637 and reduces both adhesion and invasion of the UTI89 cystitis isolate instilled in mouse bladder via catheterization. Heptyl alpha-D-mannose also specifically inhibited biofilm formation at micromolar concentrations. The structural basis of the great inhibitory potential of alkyl and aryl alpha-D-mannosides was elucidated in the crystal structure of the FimH receptor-binding domain in complex with oligomannose-3. FimH interacts with Man alpha1,3Man beta1,4GlcNAc beta1,4GlcNAc in an extended binding site. The interactions along the alpha1,3 glycosidic bond and the first beta1,4 linkage to the chitobiose unit are conserved with those of FimH with butyl alpha-D-mannose. The strong stacking of the central mannose with the aromatic ring of Tyr48 is congruent with the high affinity found for synthetic inhibitors in which this mannose is substituted for by an aromatic group. CONCLUSIONS/SIGNIFICANCE: The potential of ligand-based design of antagonists of urinary tract infections is ruled by the structural mimicry of natural epitopes and extends into blocking of bacterial invasion, intracellular growth and capacity to fluxing and of recurrence of the infection.

Chloroplasts assemble the major subunit FaeG of Escherichia coli F4 (K88) fimbriae to strand-swapped dimers.

F4 fimbriae encoded by the fae operon are the major colonization factors associated with porcine neonatal and postweaning diarrhoea caused by enterotoxigenic Escherichia coli (ETEC). Via the chaperone/usher pathway, the F4 fimbriae are assembled as long polymers of the major subunit FaeG, which also possesses the adhesive properties of the fimbriae. Intrinsically, the incomplete fold of fimbrial subunits renders them unstable and susceptible to aggregation and/or proteolytic degradation in the absence of a specific periplasmic chaperone. In order to test the possibility of producing FaeG in plants, FaeG expression was studied in transgenic tobacco plants. FaeG was directed to different subcellular compartments by specific targeting signals. Targeting of FaeG to the chloroplast results in much higher yields than FaeG targeting to the endoplasmic reticulum or the apoplast. Two chloroplast-targeted FaeG variants were purified from tobacco plants and crystallized. The crystal structures show that chloroplasts circumvent the absence of the fimbrial assembly machinery by assembling FaeG into strand-swapped dimers. Furthermore, the structures reveal how FaeG combines the structural requirements of a major fimbrial subunit with its adhesive role by grafting an additional domain on its Ig-like core.