Biomedical subjects

S Hazebrouck

Publications and source records attributed to S Hazebrouck.

6 recordsLinked to original sources

PubMed2006

Constitutive delivery of bovine beta-lactoglobulin to the digestive tracts of gnotobiotic mice by engineered Lactobacillus casei.

The gut microbiota is critical for maturation of the immune system. Recent evidence suggests that early establishment of lactobacilli in the intestinal microbiota, during neonatal colonization or by probiotic supplementation, could prevent the development of allergic disorders. Postnatal maturation of the gut immune system with allergen-producing lactobacilli colonizing the digestive tract could then affect the development of further allergic sensitization. In this paper, we describe construction of a recombinant Lactobacillus casei strain that can constitutively deliver bovine beta-lactoglobulin (BLG), a major cow's milk allergen, to the guts of gnotobiotic mice. The blg gene was inserted into the L. casei chromosome downstream of an endogenous promoter. BLG production was improved by fusing the propeptide LEISSTCDA (LEISS) to the BLG mature moiety. This led to a 10-fold increase in LEISS-BLG production compared to the production obtained without the propeptide and also led to enhanced secretion corresponding to 5% of the total production. After inoculation into germfree C3H/HeN mice, the genetic stability of the recombinant strain and in vivo BLG production were confirmed for at least 10 weeks. BLG stimulation of spleen cells from mice monoassociated with the BLG-producing lactobacilli induced secretion of the Th1 cytokine gamma interferon and, to a lesser extent, the Th2 cytokine interleukin-5. No BLG-specific immunoglobulin G1 (IgG1), IgG2a, or IgA was detected in sera or in fecal samples. These results suggest that gut colonization with allergen-producing lactobacilli could provide a useful model for studying the modulation of allergic disorders.

Animals↗

PubMed2004

Artificially ambiguous genetic code confers growth yield advantage.

A primitive genetic code is thought to have encoded statistical, ambiguous proteins in which more than one amino acid was inserted at a given codon. The relative vitality of organisms bearing ambiguous proteins and the kinds of pressures that forced development of the highly specific modern genetic code are unknown. Previous work demonstrated that, in the absence of selective pressure, enforced ambiguity in cells leads to death or to sequence reversion to eliminate the ambiguous phenotype. Here, we report the creation of a nonreverting strain of bacteria that produced statistical proteins. Ablating the editing activity of isoleucyl-tRNA synthetase resulted in an ambiguous code in which, through supplementation of a limited supply of isoleucine with an alternative amino acid that was noncoding, the mutant generating statistical proteins was favored over the wild-type isogenic strain. Such organisms harboring statistical proteins could have had an enhanced adaptive capacity and could have played an important role in the early development of living systems.

Acylation↗

PubMed2001

Local and spatial factors determining HIV-1 protease substrate recognition.

Insertional mutagenesis of the Escherichia coli thymidylate synthase (TS) was used to address substrate recognition of HIV-1 protease in a well characterized structural context. By modifying the TS conformation while maintaining its enzymic activity, we investigated the influence of protein folding on protease-substrate recognition. A slight destabilization of the TS structure permitted the cleavage of a target site, which was resistant in the native TS. This result supports a dynamic interpretation of HIV-1 protease specificity. Exposure time of the potential cleavage site, which depends on the stability of the global conformation, must be compatible with the cleavage kinetics, which are determined by the local sequence. Cleavage specificity has been described as the consequence of cumulative interactions, globally favourable, between at least six amino acids around the cleavage site. To investigate influence of local sequence, we introduced insertions of variable lengths in two exposed loops of the TS. In both environments, insertion of only two amino acids could determine specific cleavage. We then inserted libraries of dipeptides naturally cleaved by the HIV-1 protease in order to assess the limitations of established classifications of substrates in different conformational contexts.

Amino Acid Sequence↗

PubMed2000

Substituting selenocysteine for catalytic cysteine 41 enhances enzymatic activity of plant phospholipid hydroperoxide glutathione peroxidase expressed in Escherichia coli.

The citrus phospholipid hydroperoxide glutathione peroxidase (cit-PHGPx) was the first plant peroxidase demonstrated to exhibit PHGPx-specific enzymatic activity, although it was 500-fold weaker than that of the pig heart analog. This relatively low activity is accounted for the catalytic residue of cit-PHGPx, which was found to be cysteine and not the rare selenocysteine (Sec) present in animal enzymes. Sec incorporation into proteins is encoded by a UGA codon, usually a STOP codon, which, in prokaryotes, is suppressed by an adjacent downstream mRNA stem-loop structure, the Sec insertion sequence (SECIS). By performing appropriate nucleotide substitutions into the gene encoding cit-PHGPx, we introduced bacterial-type SECIS elements that afforded the substitution of the catalytic Cys(41) by Sec, as established by mass spectrometry, while preserving the functional integrity of the peroxidase. The recombinant enzyme, whose synthesis is selenium-dependent, displayed a 4-fold enhanced peroxidase activity as compared with the Cys-containing analog, thus confirming the higher catalytic power of Sec compared with Cys in cit-PHGPx active site. The study led also to refinement of the minimal sequence requirements of the bacterial-type SECIS, and, for the first time, to the heterologous expression in Escherichia coli of a eukaryotic selenoprotein containing a SECIS in its open reading frame.

Amino Acid Sequence↗

PubMed1999

Structural and functional analysis of surface domains unique to bacteriophage T4 thymidylate synthase.

The bacteriophage T4 genome encodes most of its own enzymes for dNTP synthesis, which form a complex in infected Escherichia coli. The T4 thymidylate synthase (TS) and the T4 deoxycytidylate deaminase (CD) catalyze sequential reactions and are physically linked within this complex [McGaughey, K. M., Wheeler, L. J., Moore, J. T., Maley, G. F. , Maley, F., and Mathews, C. K. (1996) J. Biol. Chem. 271, 23037-23042]. From the crystal structure of T4TS [Finer-Moore, J. S., Maley, G. F., Maley, F., Montfort, W. R., and Stroud, R. M. (1994) Biochemistry 33, 15459-15468], it appears that three regions corresponding to insertions relative to E. coli TS lie on one side of the enzyme surface. We have investigated the residual activity of T4TS in response to complete deletion or substitution mutagenesis of these insertions. Two deletions generated in the small domain (residues 70-103) reduced the TS activity to 0.2% and 0.7% of the wild-type activity, with the latter able to complement growth of a thyA- E. coli strain in the absence of thymidine. By insertion of exogenous sequences variable in length and in sequence into these deletion mutants, enzyme activity increased to 44% that of the wild type. Restoration of the TS activity depended mostly on the hydrophobicity of the inserted residues. The sites of insertions also displayed distinct permissiveness for accommodating the exogenous insertions. Deletions and substitutions near the C-terminus resulted in complete inactivation of the T4TS. Proteolysis experiments revealed that the modified surface loops of the small domain were still accessible and flexible for protein-protein interactions. We have used ELISA to detect a physical association between T4TS and T4CD and compared the binding affinity of WT T4TS for two purified insertion mutants of T4CD. The results obtained showed that the native sequences of the small domain inserts are not required for T4TS/T4CD complex formation.

Amino Acid Sequence↗

PubMed1996

Conversion of thymidylate synthase into an HIV protease substrate.

Thymidylate synthase (TS) is an essential enzyme of DNA metabolism. We have carried out an extensive insertional mutagenesis of the Escherichia coli TS gene (thyA) using three different methods. Insertion of exogenous sequences at unique restriction sites or at random positions produced defective mutants, whereas comparison of TS sequences from different species allowed us to identify six zones permissive for insertions of exogenous sequences. The insertion of Human immunodeficiency virus type 1 (HIV-1) protease substrate sequences into the permissive sites converted TS to an HIV-1 protease substrate, and the in vivo cleavage of these insertions by the cloned HIV-1 protease conferred a thymidylate synthase-deficient phenotype in some of our E. coli mutant strains. In agreement with crystallographic data, these results show that the permissive sites are located in regions of the TS protein not essential for enzyme activity and accessible to cleavage by HIV protease. These results also show that it is possible to control a growth phenotype in E. coli through the protease-mediated destruction of an essential metabolic enzyme. Because both wild type and thymidylate synthase-deficient phenotypes are selectable on the appropriate growth medium, these thyA mutants could be used for genetic selections of protease inhibitors and analysis of protease specificities.

Amino Acid Sequence↗