Anton V. Zavialov

Receptor binding studies disclose a novel class of high‐affinity inhibitors of the Escherichia coli FimH adhesin

Mannose‐binding type 1 pili are important virulence factors for the establishment of Escherichia coli urinary tract infections (UTIs). These infections are initiated by adhesion of uropathogenic E. coli to uroplakin receptors in the uroepithelium via the FimH adhesin located at the tips of type 1 pili. Blocking of bacterial adhesion is able to prevent infection. Here, we provide for the first time binding data of the molecular events underlying type 1 fimbrial adherence, by crystallographic analyses of the FimH receptor binding domains from a uropathogenic and a K‐12 strain, and affinity measurements with mannose, common mono‐ and disaccharides, and a series of alkyl and aryl mannosides. Our results illustrate that the lectin domain of the FimH adhesin is a stable and functional entity and that an exogenous butyl α‐ d‐mannoside, bound in  the  crystal  structures,  exhibits  a  significantly better affinity for FimH (Kd = 0.15 µM) than mannose (Kd = 2.3 µM). Exploration of the binding affinities of α‐ d‐mannosides with longer alkyl tails revealed affinities up to 5 nM. Aryl mannosides and fructose can also bind with high affinities to the FimH lectin domain, with a 100‐fold improvement and 15‐fold reduction in affinity, respectively, compared with mannose. Taken together, these relative FimH affinities correlate exceptionally well with the relative concentrations of the same glycans needed for the inhibition of adherence of type 1 piliated E. coli. We foresee that our findings will spark new ideas and initiatives for the development of UTI vaccines and anti‐adhesive drugs to prevent anticipated and recurrent UTIs.

Early-onset stroke and vasculopathy associated with mutations in ADA2

BACKGROUND We observed a syndrome of intermittent fevers, early-onset lacunar strokes and other neurovascular manifestations, livedoid rash, hepatosplenomegaly, and systemic vasculopathy in three unrelated patients. We suspected a genetic cause because the disorder presented in early childhood. METHODS We performed whole-exome sequencing in the initial three patients and their unaffected parents and candidate-gene sequencing in three patients with a similar phenotype, as well as two young siblings with polyarteritis nodosa and one patient with small-vessel vasculitis. Enzyme assays, immunoblotting, immunohistochemical testing, flow cytometry, and cytokine profiling were performed on samples from the patients. To study protein function, we used morpholino-mediated knockdowns in zebrafish and short hairpin RNA knockdowns in U937 cells cultured with human dermal endothelial cells. RESULTS All nine patients carried recessively inherited mutations in CECR1 (cat eye syndrome chromosome region, candidate 1), encoding adenosine deaminase 2 (ADA2), that were predicted to be deleterious; these mutations were rare or absent in healthy controls. Six patients were compound heterozygous for eight CECR1 mutations, whereas the three patients with polyarteritis nodosa or small-vessel vasculitis were homozygous for the p.Gly47Arg mutation. Patients had a marked reduction in the levels of ADA2 and ADA2-specific enzyme activity in the blood. Skin, liver, and brain biopsies revealed vasculopathic changes characterized by compromised endothelial integrity, endothelial cellular activation, and inflammation. Knockdown of a zebrafish ADA2 homologue caused intracranial hemorrhages and neutropenia - phenotypes that were prevented by coinjection with nonmutated (but not with mutated) human CECR1. Monocytes from patients induced damage in cocultured endothelial-cell layers. CONCLUSIONS Loss-of-function mutations in CECR1 were associated with a spectrum of vascular and inflammatory phenotypes, ranging from early-onset recurrent stroke to systemic vasculopathy or vasculitis. (Funded by the National Institutes of Health Intramural Research Programs and others.).

Structure and biogenesis of the capsular F1 antigen from Yersinia pestis: Preserved folding energy drives fiber formation

Most gram-negative pathogens express fibrous adhesive virulence organelles that mediate targeting to the sites of infection. The F1 capsular antigen from the plague pathogen Yersinia pestis consists of linear fibers of a single subunit (Caf1) and serves as a prototype for nonpilus organelles assembled via the chaperone/usher pathway. Genetic data together with high-resolution X-ray structures corresponding to snapshots of the assembly process reveal the structural basis of fiber formation. Comparison of chaperone bound Caf1 subunit with the subunit in the fiber reveals a novel type of conformational change involving the entire hydrophobic core of the protein. The observed conformational change suggests that the chaperone traps a high-energy folding intermediate of Caf1. A model is proposed in which release of the subunit allows folding to be completed, driving fiber formation.

Tudor staphylococcal nuclease is an evolutionarily conserved component of the programmed cell death degradome

Programmed cell death (PCD) is executed by proteases, which cleave diverse proteins thus modulating their biochemical and cellular functions. Proteases of the caspase family and hundreds of caspase substrates constitute a major part of the PCD degradome in animals. Plants lack close homologues of caspases, but instead possess an ancestral family of cysteine proteases, metacaspases. Although metacaspases are essential for PCD, their natural substrates remain unknown. Here we show that metacaspase mcII-Pa cleaves a phylogenetically conserved protein, TSN (Tudor staphylococcal nuclease), during both developmental and stress-induced PCD. TSN knockdown leads to activation of ectopic cell death during reproduction, impairing plant fertility. Surprisingly, human TSN (also known as p100 or SND1), a multifunctional regulator of gene expression, is cleaved by caspase-3 during apoptosis. This cleavage impairs the ability of TSN to activate mRNA splicing, inhibits its ribonuclease activity and is important for the execution of apoptosis. Our results establish TSN as the first biological substrate of metacaspase and demonstrate that despite the divergence of plants and animals from a common ancestor about one billion years ago and their use of distinct PCD pathways, both have retained a common mechanism to compromise cell viability through the cleavage of the same substrate, TSN.

The affinity of the FimH fimbrial adhesin is receptor-driven and quasi-independent of Escherichia coli pathotypes.

Type‐1 fimbriae are important virulence factors for the establishment of Escherichia coli urinary tract infections. Bacterial adhesion to the high‐mannosylated uroplakin Ia glycoprotein receptors of bladder epithelium is mediated by the FimH adhesin. Previous studies have attributed differences in mannose‐sensitive adhesion phenotypes between faecal and uropathogenic E. coli to sequence variation in the FimH receptor‐binding domain. We find that FimH variants from uropathogenic, faecal and enterohaemorrhagic isolates express the same specificities and affinities for high‐mannose structures. The only exceptions are FimHs from O157 strains that carry a mutation (Asn135Lys) in the mannose‐binding pocket that abolishes all binding. A high‐mannose microarray shows that all substructures are bound by FimH and that the largest oligomannose is not necessarily the best binder. Affinity measurements demonstrate a strong preference towards oligomannosides exposing Manα1‐3Man at their non‐reducing end. Binding is further enhanced by the β1‐4‐linkage to GlcNAc, where binding is 100‐fold better than that of α‐d‐mannose. Manα1‐3Manβ1‐4GlcNAc, a major oligosaccharide present in the urine of α‐mannosidosis patients, thus constitutes a well‐defined FimH epitope. Differences in affinities for high‐mannose structures are at least 10‐fold larger than differences in numbers of adherent bacteria between faecal and uropathogenic strains. Our results imply that the carbohydrate expression profile of targeted host tissues and of natural inhibitors in urine, such as Tamm‐Horsfall protein, are stronger determinants of adhesion than FimH variation.

Human adenosine deaminase 2 induces differentiation of monocytes into macrophages and stimulates proliferation of T helper cells and macrophages

ADAs play a pivotal role in regulating the level of adenosine, a signaling molecule controlling a variety of cellular responses by binding to and activating four ADRs. Two enzymes, ADA1 and ADA2, are known to possess ADA activity in humans. Although the structure of ADA1 and its role in lymphocytic activation have been known for a long time, the structure and function of ADA2, a member of ADGF, remain enigmatic. Here, we found that ADA2 is secreted by monocytes undergoing differentiation into macrophages or DCs and that it binds to the cell surface via proteoglycans and ADRs. We demonstrate that ADA1 and ADA2 increase the rate of proliferation of monocyte‐activated CD4+ T cells independently of their catalytic activity. We also show that ADA2 induces T cell‐dependent differentiation of monocytes into macrophages and stimulates macrophage proliferation. Our discovery of the growth factor‐like activity of ADA2 explains clinical observations and suggests that this enzyme could be used as a drug candidate to modulate the immune responses during inflammation and cancer.

Resolving the energy paradox of chaperone/usher-mediated fibre assembly

Periplasmic chaperone/usher machineries are used for assembly of filamentous adhesion organelles of Gram-negative pathogens in a process that has been suggested to be driven by folding energy. Structures of mutant chaperone-subunit complexes revealed a final folding transition (condensation of the subunit hydrophobic core) on the release of organelle subunit from the chaperone-subunit pre-assembly complex and incorporation into the final fibre structure. However, in view of the large interface between chaperone and subunit in the pre-assembly complex and the reported stability of this complex, it is difficult to understand how final folding could release sufficient energy to drive assembly. In the present paper, we show the X-ray structure for a native chaperone-fibre complex that, together with thermodynamic data, shows that the final folding step is indeed an essential component of the assembly process. We show that completion of the hydrophobic core and incorporation into the fibre results in an exceptionally stable module, whereas the chaperone-subunit pre-assembly complex is greatly destabilized by the high-energy conformation of the bound subunit. This difference in stabilities creates a free energy potential that drives fibre formation.

Donor strand complementation mechanism in the biogenesis of non-pilus systems.

The F1 antigen of Yersinia pestis belongs to a class of non‐pilus adhesins assembled via a classical chaperone–usher pathway. Such pathways consist of PapD‐like chaperones that bind subunits and pilot them to the outer membrane usher, where they are assembled into surface structures. In a recombinant Escherichia coli model system, chaperone–subunit (Caf1M:Caf1n) complexes accumulate in the periplasm. Three inde‐pendent methods showed that these complexes are rod‐ or coil‐shaped linear arrays of Caf1 subunits capped at one end by a single copy of Caf1M chaperone. Deletion and point mutagenesis identified an N‐terminal donor strand region of Caf1 that was essential for polymerization in vitro, in the periplasm and at the cell surface, but not for chaperone–subunit interaction. Partial protease digestion of periplasmic complexes revealed that this region becomes buried upon formation of Caf1:Caf1 contacts. These results show that, despite the capsule‐like appearance of F1 antigen, the basic structure is assembled as a linear array of subunits held together by intersubunit donor strand complementation. This example shows that strikingly different architectures can be achieved by the same general principle of donor strand complementation and suggests that a similar basic polymer organization will be shared by all surface structures assembled by classical chaperone–usher pathways.

Adhesive organelles of Gram‐negative pathogens assembled with the classical chaperone/usher machinery: structure and function from a clinical standpoint

This review summarizes current knowledge on the structure, function, assembly and biomedical applications of the superfamily of adhesive fimbrial organelles exposed on the surface of Gram-negative pathogens with the classical chaperone/usher machinery. High-resolution three-dimensional (3D) structure studies of the minifibers assembling with the FGL (having a long F1-G1 loop) and FGS (having a short F1-G1 loop) chaperones show that they exploit the same principle of donor-strand complementation for polymerization of subunits. The 3D structure of adhesive subunits bound to host-cell receptors and the final architecture of adhesive fimbrial organelles reveal two functional families of the organelles, respectively, possessing polyadhesive and monoadhesive binding. The FGL and FGS chaperone-assembled polyadhesins are encoded exclusively by the gene clusters of the γ3- and κ-monophyletic groups, respectively, while gene clusters belonging to the γ1-, γ2-, γ4-, and π-fimbrial clades exclusively encode FGS chaperone-assembled monoadhesins. Novel approaches are suggested for a rational design of antimicrobials inhibiting the organelle assembly or inhibiting their binding to host-cell receptors. Vaccines are currently under development based on the recombinant subunits of adhesins.

Structural Basis for the Growth Factor Activity of Human Adenosine Deaminase ADA2

Two distinct adenosine deaminases, ADA1 and ADA2, are found in humans. ADA1 has an important role in lymphocyte function and inherited mutations in ADA1 result in severe combined immunodeficiency. The recently isolated ADA2 belongs to the novel family of adenosine deaminase growth factors (ADGFs), which play an important role in tissue development. The crystal structures of ADA2 and ADA2 bound to a transition state analogue presented here reveal the structural basis of the catalytic/signaling activity of ADGF/ADA2 proteins. In addition to the catalytic domain, the structures discovered two ADGF/ADA2-specific domains of novel folds that mediate the protein dimerization and binding to the cell surface receptors. This complex architecture is in sharp contrast with that of monomeric single domain ADA1. An extensive glycosylation and the presence of a conserved disulfide bond and a signal peptide in ADA2 strongly suggest that ADA2, in contrast to ADA1, is specifically designed to act in the extracellular environment. The comparison of catalytic sites of ADA2 and ADA1 demonstrates large differences in the arrangement of the substrate-binding pockets. These structural differences explain the substrate and inhibitor specificity of adenosine deaminases and provide the basis for a rational design of ADA2-targeting drugs to modulate the immune system responses in pathophysiological conditions.