Patrice Filée

Crystal structure and activity of Bacillus subtilis YoaJ (EXLX1), a bacterial expansin that promotes root colonization

We solved the crystal structure of a secreted protein, EXLX1, encoded by the yoaJ gene of Bacillus subtilis. Its structure is remarkably similar to that of plant β-expansins (group 1 grass pollen allergens), consisting of 2 tightly packed domains (D1, D2) with a potential polysaccharide-binding surface spanning the 2 domains. Domain D1 has a double-ψ β-barrel fold with partial conservation of the catalytic site found in family 45 glycosyl hydrolases and in the MltA family of lytic transglycosylases. Domain D2 has an Ig-like fold similar to group 2/3 grass pollen allergens, with structural features similar to a type A carbohydrate-binding domain. EXLX1 bound to plant cell walls, cellulose, and peptidoglycan, but it lacked lytic activity against a variety of plant cell wall polysaccharides and peptidoglycan. EXLX1 promoted plant cell wall extension similar to, but 10 times weaker than, plant β-expansins, which synergistically enhanced EXLX1 activity. Deletion of the gene encoding EXLX1 did not affect growth or peptidoglycan composition of B. subtilis in liquid medium, but slowed lysis upon osmotic shock and greatly reduced the ability of the bacterium to colonize maize roots. The presence of EXLX1 homologs in a small but diverse set of plant pathogens further supports a role in plant–bacterial interactions. Because plant expansins have proved difficult to express in active form in heterologous systems, the discovery of a bacterial homolog opens the door for detailed structural studies of expansin function.

Development of a Chitosan Nanofibrillar Scaffold for Skin Repair and Regeneration.

The final goal of the present study was the development of a 3-D chitosan dressing that would shorten the healing time of skin wounds by stimulating migration, invasion, and proliferation of the relevant cutaneous resident cells. Three-dimensional chitosan nanofibrillar scaffolds produced by electrospinning were compared with evaporated films and freeze-dried sponges for their biological properties. The nanofibrillar structure strongly improved cell adhesion and proliferation in vitro. When implanted in mice, the nanofibrillar scaffold was colonized by mesenchymal cells and blood vessels. Accumulation of collagen fibrils was also observed. In contrast, sponges induced a foreign body granuloma. When used as a dressing covering full-thickness skin wounds in mice, chitosan nanofibrils induced a faster regeneration of both the epidermis and dermis compartments. Altogether our data illustrate the critical importance of the nanofibrillar structure of chitosan devices for their full biocompatibility and demonstrate the significant beneficial effect of chitosan as a wound-healing biomaterial.

New Integrative Method To Generate Bacillus subtilis Recombinant Strains Free of Selection Markers

ABSTRACT The novel method described in this paper combines the use of blaI, which encodes a repressor involved in Bacillus licheniformis BlaP β-lactamase regulation, an antibiotic resistance gene, and a B. subtilis strain (BS1541) that is conditionally auxotrophic for lysine. We constructed a BlaI cassette containing blaI and the spectinomycin resistance genes and two short direct repeat DNA sequences, one at each extremity of the cassette. The BS1541 strain was obtained by replacing the B. subtilis PlysA promoter with that of the PblaP β-lactamase promoter. In the resulting strain, the cloning of the blaI repressor gene confers lysine auxotrophy to BS1541. After integration of the BlaI cassette into the chromosome of a conditionally lys-auxotrophic (BS1541) strain by homologous recombination and positive selection for spectinomycin resistance, the eviction of the BlaI cassette was achieved by single crossover between the two short direct repeat sequences. This strategy was successfully used to inactivate a single gene and to introduce a gene of interest in the Bacillus chromosome. In both cases the resulting strains are free of selection marker. This allows the use of the BlaI cassette to repeatedly further modify the Bacillus chromosome.

The fate of the BlaI repressor during the induction of the Bacillus licheniformis BlaP b-lactamase

The induction of the Staphylococcus aureus BlaZ and Bacillus licheniformis 749/I BlaP β‐lactamases by β‐lactam antibiotics occurs according to similar processes. In both bacteria, the products of the blaI and blaR1 genes share a high degree of sequence homology and act as repressors and penicillin‐sensory transducers respectively. It has been shown in S. aureus that the BlaI repressor, which controls the expression of BlaZ negatively, is degraded after the addition of the inducer. In the present study, we followed the fate of BlaI during β‐lactamase induction in B. licheniformis 749/I and in a recombinant Bacillus subtilis 168 strain harbouring the pDML995 plasmid, which carries the B. licheniformis blaP, blaI and blaR1 genes. In contrast to the situation in B. licheniformis 749/I, β‐lactamase induction in B. subtilis 168/pDML995 was not correlated with the proteolysis of BlaI. To exclude molecular variations undetectable by SDS–PAGE, two‐dimensional gel electrophoresis was performed with cellular extracts from uninduced or induced B. subtilis 168/pDML995 cells. No variation in the BlaI isoelectric point was observed in induced cells, whereas the DNA‐binding property was lost. Cross‐linking experiments with dithiobis(succimidylpropionate) confirmed that, in uninduced recombinant B. subtilis cells, BlaI was present as a homodimer and that this situation was not altered in induced conditions. This latter result is incompatible with a mechanism of inactivation of BlaI by proteolysis and suggests that the inactivation of BlaI results from a non‐covalent modification by a co‐activator and that the subsequent proteolysis of BlaI might be a secondary phenomenon. In addition to the presence of this co‐activator, our results show that the presence of penicillin stress is also required for full induction of β‐lactamase biosynthesis.

The Bacillus licheniformis BlaP β‐lactamase as a model protein scaffold to study the insertion of protein fragments

Using genetic engineering technologies, the chitin‐binding domain (ChBD) of the human macrophage chitotriosidase has been inserted into the host protein BlaP, a class A β‐lactamase produced by Bacillus licheniformis. The product of this construction behaved as a soluble chimeric protein that conserves both the capacity to bind chitin and to hydrolyze β‐lactam moiety. Here we describe the biochemical and biophysical properties of this protein (BlaPChBD). This work contributes to a better understanding of the reciprocal structural and functional effects of the insertion on the host protein scaffold and the heterologous structured protein fragments. The use of BlaP as a protein carrier represents an efficient approach to the functional study of heterologous protein fragments.

Amyloid-Like Fibril Formation by PolyQ Proteins: A Critical Balance between the PolyQ Length and the Constraints Imposed by the Host Protein

Nine neurodegenerative disorders, called polyglutamine (polyQ) diseases, are characterized by the formation of intranuclear amyloid-like aggregates by nine proteins containing a polyQ tract above a threshold length. These insoluble aggregates and/or some of their soluble precursors are thought to play a role in the pathogenesis. The mechanism by which polyQ expansions trigger the aggregation of the relevant proteins remains, however, unclear. In this work, polyQ tracts of different lengths were inserted into a solvent-exposed loop of the β-lactamase BlaP and the effects of these insertions on the properties of BlaP were investigated by a range of biophysical techniques. The insertion of up to 79 glutamines does not modify the structure of BlaP; it does, however, significantly destabilize the enzyme. The extent of destabilization is largely independent of the polyQ length, allowing us to study independently the effects intrinsic to the polyQ length and those related to the structural integrity of BlaP on the aggregating properties of the chimeras. Only chimeras with 55Q and 79Q readily form amyloid-like fibrils; therefore, similarly to the proteins associated with diseases, there is a threshold number of glutamines above which the chimeras aggregate into amyloid-like fibrils. Most importantly, the chimera containing 79Q forms amyloid-like fibrils at the same rate whether BlaP is folded or not, whereas the 55Q chimera aggregates into amyloid-like fibrils only if BlaP is unfolded. The threshold value for amyloid-like fibril formation depends, therefore, on the structural integrity of the β-lactamase moiety and thus on the steric and/or conformational constraints applied to the polyQ tract. These constraints have, however, no significant effect on the propensity of the 79Q tract to trigger fibril formation. These results suggest that the influence of the protein context on the aggregating properties of polyQ disease-associated proteins could be negligible when the latter contain particularly long polyQ tracts.

Specificity inversion of Ochrobactrum anthropi D‐aminopeptidase to a D,D‐carboxypeptidase with new penicillin binding activity by directed mutagenesis

The serine penicillin‐recognizing proteins have been extensively studied. They show a wide range of substrate specificities accompanied by multidomain features. Their adaptation capacity has resulted in the emergence of pathogenic bacteria resistant to β‐lactam antibiotics. The most divergent enzymatic activities in this protein family are those of the Ochrobactrum anthropi D‐aminopeptidase and of the Streptomyces R61 D,D‐carboxypeptidase/transpeptidase. With the help of structural data, we have attempted to identify the factors responsible for this opposite specificity. A loop deletion mutant of the Ochrobactrum anthropi D‐aminopeptidase lost its original activity in favor of a new penicillin‐binding activity. D‐aminopeptidase activity of the deletion mutant can be restored by complementation with another deletion mutant corresponding to the noncatalytic domain of the wild‐type enzyme. By a second step site‐directed mutagenesis, the specificity of the Ochrobactrum anthropi D‐aminopeptidase was inverted to a D,D‐carboxypeptidase specificity. These results imply a core enzyme with high diversity potential surrounded by specificity modulators. It is the first example of drastic specificity change in the serine penicillin‐recognizing proteins. These results open new perspectives in the conception of new enzymes with nonnatural specificities. The structure/specificity relationship in the serine penicillin‐recognizing proteins are discussed.

Comparative functional analysis of the human macrophage chitotriosidase

This work analyses the chitin‐binding and catalytic domains of the human macrophage chitotriosidase and investigates the physiological role of this glycoside hydrolase in a complex mechanism such as the innate immune system, especially its antifungal activity. Accordingly, we first analyzed the ability of its chitin‐binding domain to interact with chitin embedded in fungal cell walls using the β‐lactamase activity reporter system described in our previous work. The data showed that the chitin‐binding activity was related to the cell wall composition of the fungi strains and that their peptide‐N‐glycosidase/zymolyase treatments increased binding to fungal by increasing protein permeability. We also investigated the antifungal activity of the enzyme against Candida albicans. The antifungal properties of the complete chitotriosidase were analyzed and compared with those of the isolated chitin‐binding and catalytic domains. The isolated catalytic domain but not the chitin‐binding domain was sufficient to provide antifungal activity. Furthermore, to explain the lack of obvious pathologic phenotypes in humans homozygous for a widespread mutation that renders chitotriosidase inactive, we postulated that the absence of an active chitotriosidase might be compensated by the expression of another human hydrolytic enzyme such as lysozyme. The comparison of the antifungal properties of chitotriosidase and lysozyme indicated that surprisingly, both enzymes have similar in vitro antifungal properties. Furthermore, despite its more efficient hydrolytic activity on chitin, the observed antifungal activity of chitotriosidase was lower than that of lysozyme. Finally, this antifungal duality between chitotriosidase and lysozyme is discussed in the context of innate immunity.

Rapid and easy development of versatile tools to study protein/ligand interactions

The system described here allows the expression of protein fragments into a solvent-exposed loop of a carrier protein, the beta-lactamase BlaP. When using Escherichia coli constitutive expression vectors, a positive selection of antibioresistant bacteria expressing functional hybrid beta-lactamases is achieved in the presence of beta-lactams making further screening of correctly folded and secreted hybrid beta-lactamases easier. Protease-specific recognition sites have been engineered on both sides of the beta-lactamase permissive loop in order to cleave off the exogenous protein fragment from the carrier protein by an original two-step procedure. According to our data, this approach constitutes a suitable alternative for production of difficult to express protein domains. This work demonstrates that the use of BlaP as a carrier protein does not alter the biochemical activity and the native disulphide bridge formation of the inserted chitin binding domain of the human macrophage chitotriosidase. We also report that the beta-lactamase activity of the hybrid protein can be used to monitor interactions between the inserted protein fragments and its ligands and to screen neutralizing molecules.

Analysis of the biochemical properties of, and complex formation between, glycoproteins H and L of the gamma2 herpesvirus bovine herpesvirus-4.

Genes encoding glycoprotein gH and gL homologues were localized in the genome of the gamma-herpesvirus bovine herpesvirus-4 (BHV-4). Both genes were sequenced and glutathione S-transferase fusion proteins were produced and used to immunize rabbits against the translation products of the two genes. The anti-gH serum recognized a protein with an apparent molecular mass (MM) of 110 kDa both in infected cells and in virions. This protein was sensitive to endo-beta-N-acetylglucosaminase-H (endoH) and endoglycosidase F-N-glycosidase F (endoF-PNGaseF) digestion. A protein with the same relative mobility was immunoprecipitated from infected cells radiolabelled with [3H]glucosamine which confirmed that this product (gp110), now designated BHV-4 gH, was glycosylated. Western blotting with the anti-gL serum detected in infected cells a product with an apparent MM ranging from 31-35 kDa and diffusely migrating protein species ranging from 45-65 kDa. Tunicamycin, monensin, endoH or endoF-PNGaseF treatments showed that both the 31-35 kDa and the 45-65 kDa proteins were glycosylated, gp31-35 being a precursor of the 45-65 kDa glycoprotein species. In radioimmunoprecipitation assays, the anti-gL serum immunoprecipitated from infected cells two glycosylated proteins with apparent MMs of 31-35 kDa (gp31-35) and 45-55 kDa (gp45-55). However a third glycoprotein, gp110, was also immunoprecipitated together with gp31-35 and gp45-55. gp110 and gp45-55 were subsequently confirmed to be virion glycoproteins corresponding to mature forms of BHV-4 gH and gL respectively. In addition, the present study clearly demonstrated complex formation between BHV-4 gH and gL both in virions and in infected cells.