Isabel Vandenberghe

Characterization of Serracin P, a Phage-Tail-Like Bacteriocin, and Its Activity against Erwinia amylovora, the Fire Blight Pathogen

ABSTRACT Serratia plymithicum J7 culture supernatant displayed activity against many pathogenic strains of Erwinia amylovora, the causal agent of the most serious bacterial disease of apple and pear trees, fire blight, and against Klebsiella pneumoniae, Serratia liquefaciens, Serratia marcescens, and Pseudomonas fluorescens. This activity increased significantly upon induction with mitomycin C. A phage-tail-like bacteriocin, named serracin P, was purified from an induced culture supernatant of S. plymithicum J7. It was found to be the only compound involved in the antibacterial activity against sensitive strains. The N-terminal amino acid sequence analysis of the two major subunits (23 and 43 kDa) of serracin P revealed high homology with the Fels-2 prophage of Salmonella enterica, the coliphages P2 and 168, the φCTX prophage of Pseudomonas aeruginosa, and a prophage of Yersinia pestis. This strongly suggests a common ancestry for serracin P and these bacteriophages.

Granzyme K displays highly restricted substrate specificity that only partially overlaps with granzyme A.

Granzymes are serine proteases stored in cytolytic granules of cytotoxic lymphocytes that eliminate virus-infected and tumor cells. Little is known about the molecular mechanism and function of granzyme (Gr)K. GrK is similar to GrA in that they are the only granzymes that display tryptase-like activity. Both granzymes induce cell death by single-stranded nicking of the chromosomal DNA by cleaving the same components of the endoplasmic reticulum-associated SET complex. Therefore, GrK may provide a backup and failsafe mechanism for GrA with redundant specificity. In the present study, we addressed the question of whether GrK displays identical substrate specificity as GrA. In peptide- and protease-proteomic screens, GrK and GrA displayed highly restricted substrate specificities that overlapped only partially. Whereas GrK and GrA cleave SET with similar efficiencies likely at the same sites, both granzymes cleaved the pre-mRNA-binding protein heterogeneous ribonuclear protein K with different kinetics at distinct sites. GrK was markedly more efficient in cleaving heterogeneous ribonuclear protein K than GrA. GrK, but not GrA, cleaved the microtubule network protein β-tubulin after two distinct Arg residues. Neither GrK cleavage sites in β-tubulin nor a peptide-based proteomic screen revealed a clear GrK consensus sequence around the P1 residue, suggesting that GrK specificity depends on electrostatic interactions between exosites of the substrate and the enzyme. We hypothesize that GrK not only constitutes a redundant functional backup mechanism that assists GrA-induced cell death but that it also displays a unique function by cleaving its own specific substrates.

Gastric epithelial cell death caused by Helicobacter suis and Helicobacter pylori γ‐glutamyl transpeptidase is mainly glutathione degradation‐dependent

Helicobacter (H.) suis is the most prevalent non‐H. pylori Helicobacter species colonizing the stomach of humans suffering from gastric disease. In the present study, we aimed to unravel the mechanism used by H. suis to induce gastric epithelial cell damage. H. suis lysate induced mainly apoptotic death of human gastric epithelial cells. Inhibition of γ‐glutamyl transpeptidase (GGT) activity present in H. suis lysate and incubation of AGS cells with purified native and recombinant H. suis GGT showed that this enzyme was partly responsible for the observed apoptosis. Supplementation of H. suis or H. pylori GGT‐treated cells with glutathione strongly enhanced the harmful effect of both enzymes and resulted in the induction of oncosis/necrosis, demonstrating that H. suis and H. pylori GGT‐mediated degradation of glutathione and the resulting formation of glutathione degradation products play a direct and active role in the induction of gastric epithelial cell death. This was preceded by an increase of extracellular H2O2 concentrations, generated in a cell‐independent manner and causing lipid peroxidation. In conclusion, H. suis and H. pylori GGT‐mediated generation of pro‐oxidant glutathione degradation products brings on cell damage and causes apoptosis or necrosis, dependent on the amount of extracellular glutathione available as a GGT substrate.

A cytochrome c peroxidase from Pseudomonas nautica 617 active at high ionic strength: expression, purification and characterization.

Cytochrome c peroxidase was expressed in cells of Pseudomonas nautica strain 617 grown under microaerophilic conditions. The 36.5 kDa dihaemic enzyme was purified to electrophoretic homogeneity in three chromatographic steps. N-terminal sequence comparison showed that the Ps. nautica enzyme exhibits a high similarity with the corresponding proteins from Paracoccus denitrificans and Pseudomonas aeruginosa. UV-visible spectra confirm calcium activation of the enzyme through spin state transition of the peroxidatic haem. Monohaemic cytochrome c(552) from Ps. nautica was identified as the physiological electron donor, with a half-saturating concentration of 122 microM and allowing a maximal catalytic centre activity of 116,000 min(-1). Using this cytochrome the enzyme retained the same activity even at high ionic strength. There are indications that the interactions between the two redox partners are mainly hydrophobic in nature.

Crystal structure of a cold-adapted class C beta-lactamase

In this study, the crystal structure of a class C β‐lactamase from a psychrophilic organism, Pseudomonas fluorescens, has been refined to 2.2 Å resolution. It is one of the few solved crystal structures of psychrophilic proteins. The structure was compared with those of homologous mesophilic enzymes and of another, modeled, psychrophilic protein. The elucidation of the 3D structure of this enzyme provides additional insights into the features involved in cold adaptation. Structure comparison of the psychrophilic and mesophilic β‐lactamases shows that electrostatics seems to play a major role in low‐temperature adaptation, with a lower total number of ionic interactions for cold enzymes. The psychrophilic enzymes are also characterized by a decreased number of hydrogen bonds, a lower content of prolines, and a lower percentage of arginines in comparison with lysines. All these features make the structure more flexible so that the enzyme can behave as an efficient catalyst at low temperatures.

Characterization of the in vitro adhesion of Actinobacillus pleuropneumoniae to swine alveolar epithelial cells

Actinobacillus pleuropneumoniae biovar 1 serotypes 2, 5a, 9 and 10 strains were tested for their ability to adhere to alveolar epithelial cells in culture. For the serotypes 5a, 9 and 10 strains, optimal adherence was observed after growth of bacterial cells in a NAD-restricted medium (0.001% NAD). This condition was also associated with the expression of a 55 kDa outer membrane protein (OMP) and of fimbriae. For the serotype 2 strain, adherence and expression of fimbriae and a 55 kDa OMP was less influenced by the growth conditions. The N-terminal amino acid sequence of the 55 kDa OMP had no homology with any known sequence, suggesting that it is an as yet unknown protein. Adherence capabilities were significantly reduced following treatment of the bacteria with proteolytic enzymes or heat. These findings suggest that proteins are involved in adhesion. The hydrophobic bond-breaking agent tetramethylurea was unable to inhibit the adherence of A. pleuropneumoniae to alveolar epithelial cells. Treatment of the bacteria with sodium metaperiodate resulted in lower adhesion scores for the serotypes 2 and 9 strains but the inhibition of adhesion was clearly lower than after treatment with proteolytic enzymes. This indicates that, besides proteins, carbohydrates might also be involved in adhesion of A. pleuropneumoniae to alveolar epithelial cells. The finding that inhibition of adhesion was very high when bacteria were treated with a combination of sodium metaperiodate and pronase also suggests that more than one adhesin is involved.

Amino Acid Sequences and Distribution of High-Potential Iron–Sulfur Proteins That Donate Electrons to the Photosynthetic Reaction Center in Phototropic Proteobacteria

High-potential iron-sulfur protein (HiPIP) has recently been shown to function as a soluble mediator in photosynthetic electron transfer between the cytochrome bc1 complex and the reaction-center bacteriochlorophyll in some species of phototrophic proteobacteria, a role traditionally assigned to cytochrome c2. For those species that produce more than one high-potential electron carrier, it is unclear which protein functions in cyclic electron transfer and what characteristics determine reactivity. To establish how widespread the phenomenon of multiple electron donors might be, we have studied the electron transfer protein composition of a number of phototrophic proteobacterial species. Based upon the distribution of electron transfer proteins alone, we found that HiPIP is likely to be the electron carrier of choice in the purple sulfur bacteria in the families Chromatiaceae and Ectothiorhodospiraceae, but the majority of purple nonsulfur bacteria are likely to utilize cytochrome c2. We have identified several new species of phototrophic proteobacteria that may use HiPIP as electron donor and a few that may use cytochromes c other than c2. We have determined the amino acid sequences of 14 new HiPIPs and have compared their structures. There is a minimum of three sequence categories of HiPIP based upon major insertions and deletions which approximate the three families of phototrophic proteobacteria and each of them can be further subdivided prior to construction of a phylogenetic tree. The comparison of relationships based upon HiPIP and RNA revealed several discrepancies.

Structure and antagonism of the receptor complex mediated by human TSLP in allergy and asthma.

The pro-inflammatory cytokine thymic stromal lymphopoietin (TSLP) is pivotal to the pathophysiology of widespread allergic diseases mediated by type 2 helper T cell (Th2) responses, including asthma and atopic dermatitis. The emergence of human TSLP as a clinical target against asthma calls for maximally harnessing its therapeutic potential via structural and mechanistic considerations. Here we employ an integrative experimental approach focusing on productive and antagonized TSLP complexes and free cytokine. We reveal how cognate receptor TSLPR allosterically activates TSLP to potentiate the recruitment of the shared interleukin 7 receptor α-chain (IL-7Rα) by leveraging the flexibility, conformational heterogeneity and electrostatics of the cytokine. We further show that the monoclonal antibody Tezepelumab partly exploits these principles to neutralize TSLP activity. Finally, we introduce a fusion protein comprising a tandem of the TSLPR and IL-7Rα extracellular domains, which harnesses the mechanistic intricacies of the TSLP-driven receptor complex to manifest high antagonistic potency.

Isolation and characterization of SAP and CRP, two pentraxins from Pangasianodon (Pangasius) hypophthalmus

From the serum of Pangasianodon hypophthalmus, two proteins were isolated by affinity chromatography on Sepharose and phosphorylcholine-Sepharose. Their binding on the affinity matrices critically depends on the presence of Ca2+ ions. N-terminal sequencing and sequencing of internal tryptic peptides identified the proteins as pentraxins and from their binding properties they are identified as SAP (serum amyloid P component) and CRP (C-reactive protein). Per ml serum, 36 microg SAP and 56 microg CRP was purified. Upon gel filtration, both the SAP and CRP elute as trimers of respectively 24 kDa and 28 kDa subunits. Both proteins are devoid of inter-chain disulfide bonds. Both SAP and CRP are glycosylated and agglutinate rabbit erythrocytes and pathogenic bacteria Edwardsiella ictaluri and Aeromonas hydrophila, but not Micrococcus lysodeikticus or Escherichia coli. Haemagglutination of SAP and CRP is inhibited by galactose (MIC = 1 mM) and by phosphorylcholine (MIC = 1-2 mM), respectively. Circular dichroism studies revealed that antiparallel beta-pleated sheets are dominating the secondary structure. Upon removing the Ca(2+) ions by EDTA, slight structural changes are observed by CD spectroscopy in the near-UV region. Immunodiffusion shows that P. hypophthalmus SAP and CRP do not cross-react.

Activation mechanism of recombinant Der p 3 allergen zymogen: contribution of cysteine protease Der p 1 and effect of propeptide glycosylation.

The trypsin-like protease Der p 3, a major allergen of the house dust mite Dermatophagoides pteronyssinus, is synthesized as a zymogen, termed proDer p 3. No recombinant source of Der p 3 has been described yet, and the zymogen maturation mechanism remains to be elucidated. The Der p 3 zymogen was produced in Pichia pastoris. We demonstrated that the recombinant zymogen is glycosylated at the level of its propeptide. We showed that the activation mechanism of proDer p 3 is intermolecular and is mediated by the house dust mite cysteine protease Der p 1. The primary structure of the proDer p 3 propeptide is associated with a unique zymogen activation mechanism, which is different from those described for the trypsin-like family and relies on the house dust mite papain-like protease Der p 1. This is the first report of a recombinant source of Der p 3, with the same enzymatic activity as the natural enzyme and trypsin. Glycosylation of the propeptide was found to decrease the rate of maturation. Finally, we showed that recombinant Der p 3 is inhibited by the free modified prosequence TP1R.