Acinetobacter baylyi regulates type IV pilus synthesis by employing two extension motors and a motor protein inhibitor.

Bacteria use extracellular appendages called type IV pili (T4P) for diverse behaviors including DNA uptake, surface sensing, virulence, protein secretion, and twitching motility. Dynamic extension and retraction of T4P is essential for their function, and T4P extension is thought to occur through the action of a single, highly conserved motor, PilB. Here, we develop Acinetobacter baylyi as a model to study T4P by employing a recently developed pilus labeling method. By contrast to previous studies of other bacterial species, we find that T4P synthesis in A. baylyi is dependent not only on PilB but also on an additional, phylogenetically distinct motor, TfpB. Furthermore, we identify a protein (CpiA) that inhibits T4P extension by specifically binding and inhibiting PilB but not TfpB. These results expand our understanding of T4P regulation and highlight how inhibitors might be exploited to disrupt T4P synthesis.

On-cell saturation transfer difference NMR for the identification of FimH ligands and inhibitors.

We describe the development of an on-cell NMR method for the rapid screening of FimH ligands and the structural identification of ligand binding epitopes. FimH is a mannose-binding bacterial adhesin expressed at the apical end of type 1 pili of uropathogenic bacterial strains and responsible for their d-mannose sensitive adhesion to host mammalian epithelial cells. Because of these properties, FimH is a key virulence factor and an attractive therapeutic target for urinary tract infection. We prepared synthetic d-mannose decorated dendrimers, we tested their ability to prevent the FimH-mediated yeast agglutination, and thus we used the compounds showing the best inhibitory activity as models of FimH multivalent ligands to set up our NMR methodology. Our experimental protocol, based on on-cell STD NMR techniques, is a suitable tool for the screening and the epitope mapping of FimH ligands aimed at the development of new antiadhesive and diagnostic tools against urinary tract infection pathogens. Notably, the study is carried out in a physiological environment, i.e. at the surface of living pathogen cells expressing FimH.

Exploration of Ligand-receptor Binding and Mechanisms for Alginate Reduction and Phenotype Reversion by Mucoid Pseudomonas aeruginosa.

Bacteria in general can develop a wide range of phenotypes under different conditions and external stresses. The phenotypes that reside in biofilms, overproduce exopolymers, and show increased motility often exhibit drug tolerance and drug persistence. In this work, we describe a class of small molecules that delay and inhibit the overproduction of alginate by a non-swarming mucoid Pseudomonas aeruginosa. Among these molecules, selected benzophenone-derived alkyl disaccharides cause the mucoid bacteria to swarm on hydrated soft agar gel and revert the mucoid to a nonmucoid phenotype. The sessile (biofilm) and motile (swarming) phenotypes are controlled by opposing signaling pathways with high and low intracellular levels of bis-(3',5')-cyclic diguanosine monophosphate (cdG), respectively. As our molecules control several of these phenotypes, we explored a protein receptor, pilin of the pili appendages, that is consistent with controlling these bioactivities and signaling pathways. To test this binding hypothesis, we developed a bacterial motility-enabled binding assay that uses the interfacial properties of hydrated gels and bacterial motility to conduct label-free ligand-receptor binding studies. The structure-activity correlation and receptor identification reveal a plausible mechanism for reverting mucoid to nonmucoid phenotypes by binding pili appendages with ligands capable of sequestering and neutralizing reactive oxygen species.

Recent development in the design of small 'drug-like' and nanoscale glycomimetics against Escherichia coli infections.

Glycoconjugates are involved in several pathological processes. Glycomimetics that can favorably emulate complex carbohydrate structures, while competing with natural ligands as inhibitors, are gaining considerable attention owing to their improved hydrolytic stability, binding affinity, and pharmacokinetic (PK) properties. Of particular interest are the families of α-d-mannopyranoside analogs, which can be used as inhibitors against adherent invasive Escherichia coli infections. Bacterial resistance to modern antibiotics triggers the search for new alternative antibacterial strategies that are less susceptible to acquiring resistance. In this review, we highlight recent progress in the chemical syntheses of this family of compounds, one of which having reached clinical trials against Crohn's disease (CD).

Anti-CfaE nanobodies provide broad cross-protection against major pathogenic enterotoxigenic Escherichia coli strains, with implications for vaccine design.

Enterotoxigenic Escherichia coli (ETEC) is estimated to cause approximately 380,000 deaths annually during sporadic or epidemic outbreaks worldwide. Development of vaccines against ETEC is very challenging due to the vast heterogeneity of the ETEC strains. An effective vaccines would have to be multicomponent to provide coverage of over ten ETEC strains with genetic variabilities. There is currently no vaccine licensed to prevent ETEC. Nanobodies are successful new biologics in treating mucosal infectious disease as they recognize conserved epitopes on hypervariable pathogens. Cocktails consisting of multiple nanobodies could provide even broader epitope coverage at a lower cost compared to monoclonal antibodies. Identification of conserved epitopes by nanobodies can also assist reverse engineering of an effective vaccine against ETEC. By screening nanobodies from immunized llamas and a naïve yeast display library against adhesins of colonization factors, we identified single nanobodies that show cross-protective potency against eleven major pathogenic ETEC strains in vitro. Oral administration of nanobodies led to a significant reduction of bacterial colonization in animals. Moreover, nanobody-IgA fusion showed extended inhibitory activity in mouse colonization compared to commercial hyperimmune bovine colostrum product used for prevention of ETEC-induced diarrhea. Structural analysis revealed that nanobodies recognized a highly-conserved epitope within the putative receptor binding region of ETEC adhesins. Our findings support further rational design of a pan-ETEC vaccine to elicit robust immune responses targeting this conserved epitope.

Anti-FIM and Anti-FHA Antibodies Inhibit Bordetella pertussis Growth and Reduce Epithelial Cell Inflammation Through Bacterial Aggregation.

The pertussis vaccination is highly recommended for infants, children, and pregnant women. Despite a high coverage of vaccination, pertussis continues to be of public health concern as a re-emerging infectious disease. The mechanism by which vaccine-elicited anti-pertussis antibodies mediate direct bactericidal effects is poorly understood. In this study, we showed that the interaction of B. pertussis with A549 epithelial cells induce release of biological factors which enhance bacteria growth. Complement-depleted antisera from vaccine-immunized guinea pigs or monoclonal antibodies targeting FHA and FIM mediate bacteria aggregation and elicit bactericidal effects. Our in vitro results indicated that aggregation of bacteria through anti-FIM and anti-FHA specific antibodies is one of the major biological mechanisms to clear bacterial infections and restore epithelial cell survival in vitro. Our data also indicates that the anti-pertussis antibodies reduce secretion of proinflammatory chemokines and cytokines by preventing interaction of B. pertussis with host cells. The results of this study not only demonstrate mechanism of action of anti-FIM and anti-FHA antibodies, but also opens translational applications for potential therapeutic approaches or development of analytical assays such as in vitro potency assays.

Compounds targeting YadC of uropathogenic Escherichia coli and its host receptor annexin A2 decrease bacterial colonization in bladder.

BACKGROUND: Uropathogenic Escherichia coli (UPEC) is the leading cause of urinary tract infections (UTIs), and fimbrial tip adhesins, play important roles in UPEC colonization. Few fimbrial tip adhesins and their receptors on host cells, which have the potential to be the therapeutic targets, have been identified. METHODS: the UPEC wild-type strain CFT073, ΔyadC and the complemented strain were used to perform assays in vitro and in vivo. The effects of D-xylose targeting YadC on UPEC colonization were evaluated. A YadC receptor was identified by far-western blotting, LC-MS/MS and co-immunoprecipitation. The effects of compounds targeting the receptor on UPEC colonization were tested. FINDINGS: YadC was investigated for its mediation of UPEC adhesion and invasion to bladder epithelial cells in vitro; and its promotion of UPEC colonization in bladder in vivo. D-xylose, targeting YadC, showed prophylactic and therapeutic effects on UPEC colonization. Annexin A2 (ANXA2) was identified as a YadC receptor, involved in UPEC infection. ANXA2 inhibitors attenuated UPEC infections. The yadC gene was widely present in UPEC clinical isolates and phylogenetic analysis of yadC was performed. INTERPRETATION: YadC and its receptor ANXA2 play important roles in UPEC colonization in bladder, leading to novel treatment strategies targeting YadC or ANXA2 for acute UTIs. FUND: This study was supported by grants from the National Natural Science Foundation of China (NSFC) Programs (31670071 and 31970133), the National Key Technologies R&D Program, Intergovernmental international innovation cooperation (2018YFE0102000), Tianjin Science and Technology Commissioner Project (18JCZDJC36000), the Science & Technology Development Fund of Tianjin Education Commission for Higher Education (2017ZD12). The Science Foundation of Tianjin Medical University (2016KY2M08).

Improvement of Aglycone π-Stacking Yields Nanomolar to Sub-nanomolar FimH Antagonists.

Antimicrobial resistance has become a serious concern for the treatment of urinary tract infections. In this context, an anti-adhesive approach targeting FimH, a bacterial lectin enabling the attachment of E. coli to host cells, has attracted considerable interest. FimH can adopt a low/medium-affinity state in the absence and a high-affinity state in the presence of shear forces. Until recently, mostly the high-affinity state has been investigated, despite the fact that a therapeutic antagonist should bind predominantly to the low-affinity state. In this communication, we demonstrate that fluorination of biphenyl α-d-mannosides leads to compounds with perfect π-π stacking interactions with the tyrosine gate of FimH, yielding low nanomolar to sub-nanomolar KD values for the low- and high-affinity states, respectively. The face-to-face alignment of the perfluorinated biphenyl group of FimH ligands and Tyr48 was confirmed by crystal structures as well as 1 H,15 N-HSQC NMR analysis. Finally, fluorination improves pharmacokinetic parameters predictive for oral availability.

Development of Mannopyranoside Therapeutics against Adherent-Invasive Escherichia coli Infections.

Preventing bacterial adhesion to host cells is a provocative and alternative approach to traditional antibiotic treatments given the increasing microbial resistance. A brief overview of common antibiotic treatments is described in light of their respective resistance and remaining susceptibility. This strategy has been seriously considered in the context of adherent-invasive infections in Crohn's disease and urinary tract infections in particular. The adhesions of various pathogenic Escherichia coli strains to host cells are primarily mediated through carbohydrate-protein interactions involving bacterial organelles called fimbriae that can recognize specific glycoconjugate receptors on host cells. Of particular interest are the FimH and PapG fimbriae, which bind to mannosylated glycoproteins and glycolipids of the galabiose series, respectively. Therefore, blocking FimH- and PapG-mediated bacterial adhesion to uroepithelial cells by high-affinity carbohydrate antagonists constitutes a challenging therapeutic target of high interest. This is of particular interest since bacterial adhesion to host cells is a parameter unlikely to be the subject of bacterial mutations without affecting the carbohydrate ligand binding interactions at the basis of the recognition and infection processes. To date, there have been several families of potent FimH antagonists that include natural O-linked as well as unnatural analogues of α-d-mannopyranosides. These observations led to a thorough understanding of the intimate binding site interactions that helped to reveal the so-called "tyrosine gate mechanism" at the origin of the strong necessary interactions with sugar-possessing hydrophobic aglycones. By modification of the aglycones of single monosaccharidic d-mannopyranosides, it was possible to replace the natural complex oligomannoside structure by simpler ones. An appealing and successful series of analogues have been disclosed, including nanomolecular architectures such as dendrimers, polymers, and liposomes. In addition, the data were compared to the above multivalent architectures and confirmed the possibility of working with small sugar candidates. This Account primarily concentrates on the most promising types of FimH inhibitors belonging to the family of α-C-linked mannopyranosides. However, one of the drawbacks associated with C-mannopyranosides has been that they were believed to be in the inverted chair conformation, which is obviously not recognized by the E. coli FimH. To decipher this situation, various synthetic approaches, conformational aspects, and restrictions are discussed using molecular modeling, high-field NMR spectroscopy, and X-ray analysis. These combined techniques pointed to the fact that several α-C-linked mannopyranosides do exist in the required 4C1 chair conformation. Ultimately, recent findings in this growing field of interest culminated in the identification of drug candidates that have reached clinical phase I.

Molecular strategy for blocking isopeptide bond formation in nascent pilin proteins.

Bacteria anchor to their host cells through their adhesive pili, which must resist the large mechanical stresses induced by the host as it attempts to dislodge the pathogens. The pili of gram-positive bacteria are constructed as a single polypeptide made of hundreds of pilin repeats, which contain intramolecular isopeptide bonds strategically located in the structure to prevent their unfolding under force, protecting the pilus from degradation by extant proteases and oxygen radicals. Here, we demonstrate the design of a short peptide that blocks the formation of the isopeptide bond present in the pilin Spy0128 from the human pathogen Streptococcus pyogenes, resulting in mechanically labile pilin domains. We use a combination of protein engineering and atomic-force microscopy force spectroscopy to demonstrate that the peptide blocks the formation of the native isopeptide bond and compromises the mechanics of the domain. While an intact Spy0128 is inextensible at any force, peptide-modified Spy0128 pilins readily unfold at very low forces, marking the abrogation of the intramolecular isopeptide bond as well as the absence of a stable pilin fold. We propose that isopeptide-blocking peptides could be further developed as a type of highly specific antiadhesive antibiotics to treat gram-positive pathogens.

Targeting Dynamical Binding Processes in the Design of Non-Antibiotic Anti-Adhesives by Molecular Simulation-The Example of FimH.

Located at the tip of type I fimbria of Escherichia coli, the bacterial adhesin FimH is responsible for the attachment of the bacteria to the (human) host by specifically binding to highly-mannosylated glycoproteins located on the exterior of the host cell wall. Adhesion represents a necessary early step in bacterial infection and specific inhibition of this process represents a valuable alternative pathway to antibiotic treatments, as such anti-adhesive drugs are non-intrusive and are therefore unlikely to induce bacterial resistance. The currently available anti-adhesives with the highest affinities for FimH still feature affinities in the nanomolar range. A prerequisite to develop higher-affinity FimH inhibitors is a molecular understanding of the FimH-inhibitor complex formation. The latest insights in the formation process are achieved by combining several molecular simulation and traditional experimental techniques. This review summarizes how molecular simulation contributed to the current knowledge of the molecular function of FimH and the importance of dynamics in the inhibitor binding process, and highlights the importance of the incorporation of dynamical aspects in (future) drug-design studies.

Synthesis and evaluation of thiomannosides, potent and orally active FimH inhibitors.

FimH is a type I fimbrial lectin located at the tip of type-1 pili of Gram-negative uropathogenic Escherichia coli (UPEC) guiding its ability to adhere and infect urothelial cells. Accordingly, blocking FimH with small molecule inhibitor is considered as a promising new therapeutic alternative to treat urinary tract infections caused by UPEC. Herein, we report that compounds having the S-glycosidic bond (thiomannosides) had improved metabolic stability and plasma exposures when dosed orally. Especially compound 5h showed the potential to inhibit biofilm formation and also to disrupt the preformed biofilm. And compound 5h showed prophylactic effect in UTI model in mice.

KinITC-One Method Supports both Thermodynamic and Kinetic SARs as Exemplified on FimH Antagonists.

Affinity data, such as dissociation constants (KD ) or inhibitory concentrations (IC50 ), are widely used in drug discovery. However, these parameters describe an equilibrium state, which is often not established in vivo due to pharmacokinetic effects and they are therefore not necessarily sufficient for evaluating drug efficacy. More accurate indicators for pharmacological activity are the kinetics of binding processes, as they shed light on the rate of formation of protein-ligand complexes and their half-life. Nonetheless, although highly desirable for medicinal chemistry programs, studies on structure-kinetic relationships (SKR) are still rare. With the recently introduced analytical tool kinITC this situation may change, since not only thermodynamic but also kinetic information of the binding process can be deduced from isothermal titration calorimetry (ITC) experiments. Using kinITC, ITC data of 29 mannosides binding to the bacterial adhesin FimH were re-analyzed to make their binding kinetics accessible. To validate these kinetic data, surface plasmon resonance (SPR) experiments were conducted. The kinetic analysis by kinITC revealed that the nanomolar affinities of the FimH antagonists arise from both (i) an optimized interaction between protein and ligand in the bound state (reduced off-rate constant koff ) and (ii) a stabilization of the transition state or a destabilization of the unbound state (increased on-rate constant kon ). Based on congeneric ligand modifications and structural input from co-crystal structures, a strong relationship between the formed hydrogen-bond network and koff could be concluded, whereas electrostatic interactions and conformational restrictions upon binding were found to have mainly an impact on kon .

Interrupting oral infection of Porphyromonas gingivalis with anti-FimA antibody attenuates bacterial dissemination to the arthritic joint and improves experimental arthritis.

Rheumatoid arthritis (RA) is a chronic autoimmune disease that typically results in strong inflammation and bone destruction in the joints. It is generally known that the pathogenesis of RA is linked to cardiovascular and periodontal diseases. Though rheumatoid arthritis and periodontitis share many pathologic features such as a perpetual inflammation and bone destruction, the precise mechanism underlying a link between these two diseases has not been fully elucidated. Collagen-induced arthritis (CIA) mice were orally infected with Porphyromonas gingivalis (Pg) or Pg preincubated with an anti-FimA antibody (FimA Ab) specific for fimbriae that are flexible appendages on the cell surface. Pg-infected CIA mice showed oral microbiota disruption and increased alveolar bone loss and had synovitis and joint bone destruction. However, preincubation with FimA Ab led to a significant reduction in the severity of both oral disease and arthritis. Moreover, FimA Ab attenuated bacterial attachment and aggregation on human gingival and rheumatoid arthritis synovial fibroblasts. In addition, we discovered bacteria may utilize dendritic cells, macrophages and neutrophils to migrate into the joints of CIA mice. These results suggest that disrupting Pg fimbriae function by FimA Ab ameliorates RA.

Phytochemicals As Uropathognic Escherichia Coli FimH Antagonist: In Vitro And In Silico Approach.

BACKGROUND: Urinary tract infection (UTI) is caused by uropathogenic Escherichia coli (UPEC). The UPEC initiate pathogenesis by expressing type 1 pili, which attach to membrane receptors on the uroepithelial cells. Inhibition of attachment can provide a valuable target for prophylaxis in symptom-free milieu. METHODS: The antibacterial efficacy of alcoholic, hydroalcoholic and aqueous extracts of four plants namely Achyranthes aspera, Andrographis paniculata, Artemissia vulgaris and Glycyrrhiza glabra was evaluated against seven isolated bacterial strains and procured E. coli (UTI89/UPEC) strain. Screening of isolated strains was based on morphological characteristics and biofilm forming ability followed by physiological and biochemical analysis. RESULTS: The hydroalcoholic extracts of G. glabra at 50 µg/ml showed an impending antioxidant (DPPH) effect of 95.65% compared to ascorbic acid. The MIC values of all the plant extracts against selected bacterial strains ranged between 125 to 1000 µg/ml. In silico molecular docking performed to make out the antiadhesive role of 115 documented phytochemicals from selected plants identified quercetin-3-glucoside, ethyl caffeate, liquiritoside, liquiritin and isoliquiritigenin as potential phytochemicals. Molecular dynamics simulation performed by PTRAJ module of Amber11 package to monitor the stability of hydrogen bond showed that quercetin-3-glucoside and ethyl caffeate are potential phytochemicals as antiadhesive forming H-bonds with the FimH protein ligand. CONCLUSIONS: Aforesaid phytochemicals demonstrate effective antibacterial activity through the anti-adhesion mechanism.

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.

Target-directed Dynamic Combinatorial Chemistry: A Study on Potentials and Pitfalls as Exemplified on a Bacterial Target.

Target-directed dynamic combinatorial chemistry (DCC) is an emerging technique for the efficient identification of inhibitors of pharmacologically relevant targets. In this contribution, we present an application for a bacterial target, the lectin FimH, a crucial virulence factor of uropathogenic E. coli being the main cause of urinary tract infections. A small dynamic library of acylhydrazones was formed from aldehydes and hydrazides and equilibrated at neutral pH in presence of aniline as nucleophilic catalyst. The major success factors turned out to be an accordingly adjusted ratio of scaffolds and fragments, an adequate sample preparation prior to HPLC analysis, and the data processing. Only then did the ranking of the dynamic library constituents correlate well with affinity data. Furthermore, as a support of DCC applications especially to larger libraries, a new protocol for improved hit identification was established.

Selective depletion of uropathogenic E. coli from the gut by a FimH antagonist.

Urinary tract infections (UTIs) caused by uropathogenic Escherichia coli (UPEC) affect 150 million people annually. Despite effective antibiotic therapy, 30-50% of patients experience recurrent UTIs. In addition, the growing prevalence of UPEC that are resistant to last-line antibiotic treatments, and more recently to carbapenems and colistin, make UTI a prime example of the antibiotic-resistance crisis and emphasize the need for new approaches to treat and prevent bacterial infections. UPEC strains establish reservoirs in the gut from which they are shed in the faeces, and can colonize the periurethral area or vagina and subsequently ascend through the urethra to the urinary tract, where they cause UTIs. UPEC isolates encode up to 16 distinct chaperone-usher pathway pili, and each pilus type may enable colonization of a habitat in the host or environment. For example, the type 1 pilus adhesin FimH binds mannose on the bladder surface, and mediates colonization of the bladder. However, little is known about the mechanisms underlying UPEC persistence in the gut. Here, using a mouse model, we show that F17-like and type 1 pili promote intestinal colonization and show distinct binding to epithelial cells distributed along colonic crypts. Phylogenomic and structural analyses reveal that F17-like pili are closely related to pilus types carried by intestinal pathogens, but are restricted to extra-intestinal pathogenic E. coli. Moreover, we show that targeting FimH with M4284, a high-affinity inhibitory mannoside, reduces intestinal colonization of genetically diverse UPEC isolates, while simultaneously treating UTI, without notably disrupting the structural configuration of the gut microbiota. By selectively depleting intestinal UPEC reservoirs, mannosides could markedly reduce the rate of UTIs and recurrent UTIs.

Urinary Tract Infection: Which Conformation of the Bacterial Lectin FimH Is Therapeutically Relevant?

Frequent antibiotic treatment of urinary tract infections has resulted in the emergence of antimicrobial resistance, necessitating alternative treatment options. One such approach centers around FimH antagonists that block the bacterial adhesin FimH, which would otherwise mediate binding of uropathogenic Escherichia coli to the host urothelium to trigger the infection. Although the FimH lectin can adopt three distinct conformations, the evaluation of FimH antagonists has mainly been performed with a truncated construct of FimH locked in one particular conformation. For a successful therapeutic application, however, FimH antagonists should be efficacious against all physiologically relevant conformations. Therefore, FimH constructs with the capacity to adopt various conformations were applied. By examining the binding properties of a series of FimH antagonists in terms of binding affinity and thermodynamics, we demonstrate that depending on the FimH construct, affinities may be overestimated by a constant factor of 2 orders of magnitude. In addition, we report several antagonists with excellent affinities for all FimH conformations.