Bruno H. Muller

Improving Escherichia coli Alkaline Phosphatase Efficacy by Additional Mutations inside and outside the Catalytic Pocket

We describe a strategy that allowed us to confer on a bacterial (E. coli) alkaline phosphatase (AP) the high catalytic activity of the mammalian enzyme while maintaining its high thermostability. First, we identified mutations, at positions other than those occupied by essential catalytic residues, which inactivate the bacterial enzyme without destroying its overall conformation. We transferred concomitantly into the bacterial enzyme four residues of the mammalian enzyme, two being in the catalytic pocket and two being outside. Second, the gene encoding the inactive mutant was submitted to random mutagenesis. Enzyme activity was restored upon the single mutation D330N, at a position that is 12 Å away from the center of the catalytic pocket. Third, this mutation was combined with other mutations previously reported to increase AP activity slightly in the presence of magnesium. As a result, at pH 10.0 the phosphatase activity of both mutants D330N/D153H and D330N/D153G was 17‐fold higher than that of the wild‐type AP. Strikingly, although the two individual mutations D153H and D153G destabilize the enzyme, the double mutant D330N/D153G remained highly stable (Tm=87 °C). Moreover, when combining the phosphatase and transferase activities, the catalytic activity of the mutant D330N/D153G increased 40‐fold (kcat=3200 s−1) relative to that of the wild‐type enzyme (kcat=80 s−1). Due to the simultaneous increase in Km, the resulting kcat/Km value was only increased by a factor of two. Therefore, a single mutation occurring outside a catalytic pocket can dramatically control not only the activity of an enzyme, but also its thermostability. Preliminary crystallographic data of a covalent D330N/D153G enzyme–phosphate complex show that the phosphate group has significantly moved away from the catalytic pocket, relative to its position in the structure of another mutant previously reported.

Molecular engineering of antibodies for therapeutic and diagnostic purposes

During the past ten years, monoclonal antibodies (mAbs) have taken center stage in the field of targeted therapy and diagnosis. This increased interest in mAbs is due to their binding accuracy (affinity and specificity) together with the original molecular and structural rules that govern interactions with their cognate antigen. In addition, the effector properties of antibodies constitute a second major advantage associated with their clinical use. The development of molecular and structural engineering and more recently of in vitro evolution of antibodies has opened up new perspectives in the de novo design of antibodies more adapted to clinical and diagnostic use. Thus, efforts are regularly made by researchers to improve or modulate antibody recognition properties, to adapt their pharmacokinetics, engineer their stability, and control their immunogenicity. This review presents the latest molecular engineering results on mAbs with therapeutic and diagnostic applications.

Phage Amplification and Immunomagnetic Separation Combined with Targeted Mass Spectrometry for Sensitive Detection of Viable Bacteria in Complex Food Matrices

We have developed and describe here for the first time a highly sensitive method for the fast and unambiguous detection of viable Escherichia coli in food matrices. The new approach is based on using label-free phages (T4), obligate parasites of bacteria, which are attractive for pathogen detection because of their inherent natural specificity and ease of use. A specific immunomagnetic separation was used to capture the progeny phages produced. Subsequently, T4 phage markers were detected by liquid chromatography coupled to targeted mass spectrometry. Combining the specificity of these three methodologies is of great interest in developing an alternative to conventional time-consuming culture-based technologies for the detection of viable bacteria for industrial applications. First, optimization experiments with phage T4 spiked in complex matrices (without a phage amplification event) were performed and demonstrated specific, sensitive, and reproducible phage capture and detection in complex matrices including Luria-Bertani broth, orange juice, and skimmed milk. The method developed was then applied to the detection of E. coli spiked in foodstuffs (with a phage amplification event). After having evaluated the impact of infection duration on assay sensitivity, we showed that our assay specifically detects viable E. coli in milk at an initial count of ≥1 colony-forming unit (cfu)/mL after an 8-h infection. This excellent detection limit makes our new approach an alternative to PCR-based assays for rapid bacterial detection.

Mutants with higher stability and specific activity from a single thermosensitive variant of T7 RNA polymerase.

A single strategy to select RNA polymerase from bacteriophage T7 (T7 RNAP) mutants in Escherichia coli with enhanced thermostability or enzymatic activity is described. T7 RNAP has the ability to specifically transcribe genes under control of T7 phage promoter. By using random mutagenesis of the T7 RNAP gene in combination with an appropriate screening at 25 and 42°C, we have generated and selected E.coli clones with temperature-sensitive phenotype in the presence of chloramphenicol. The resistance to chloramphenicol used to select these clones results from expression control of the chloramphenicol acetyl transferase gene by the T7 promoter. In a second phase, and using the thermosensitive T7 RNAP variants as template, a new round of random mutagenesis was performed. Combined to an appropriate screening strategy, 11 mutations (second-site T7 RNAP revertants) that restore the initial resistance to chloramphenicol at 42°C were identified. Nine of these mutations increase the thermal resistance of the wild-type T7 RNA. They include the five mutations previously described using different approaches and four novel mutations. One improves T7 RNA catalytic activity and one has no positive effect on the natural enzyme but increases the activity of some combined mutants. Additive effects of mutations amount to an increase of as much as 10°C in T1/2 compared with the wild-type enzyme and up to a 2-fold activity enhancement.

Metabolomic Investigation of Staphylococcus aureus Antibiotic Susceptibility by Liquid Chromatography Coupled to High-Resolution Mass Spectrometry

Staphylococcus aureus is a major human pathogen that can readily acquire antibiotic resistance. For instance, methicillin-resistant S. aureus represents a major cause of hospital- and community-acquired bacterial infections. In this chapter, we first provide a detailed protocol for obtaining unbiased and reproducible S. aureus metabolic profiles. The resulting intracellular metabolome is then analyzed in an untargeted manner by using both hydrophilic interaction liquid chromatography and pentafluorophenyl-propyl columns coupled to high-resolution mass spectrometry. Such analyses are done in conjunction with our in-house spectral database to identify with high confidence as many meaningful S. aureus metabolites as possible. Under these conditions, we can routinely monitor more than 200 annotated S. aureus metabolites. We also indicate how this protocol can be used to investigate the metabolic differences between methicillin-resistant and susceptible strains.