Dominique Aubel

Characterization of a prolidase from Lactobacillus delbrueckii subsp. bulgaricus CNRZ 397 with an unusual regulation of biosynthesis.

Lactobacillus delbrueckii subsp. bulgaricus CNRZ 397 (Lb. bulgaricus) is characterized by a high level of peptidase activities specific to proline-containing peptides. A prolidase (PepQ, EC 3.4.13.9) was purified to homogeneity and characterized as a strict dipeptidase active on X-Pro dipeptides, except Gly-Pro and Pro-Pro. The values for Km and Vmax were, respectively, 2.2 mM and 0.33 mmol min(-1) mg(-1), with Leu-Pro as the substrate. The enzyme exhibited optimal activity at 50 degrees C and pH 6.0, and required the presence of Zn2+. Size exclusion chromatographies and SDS-PAGE analysis led to the conclusion that this prolidase was a homodimer. Antibodies raised against the purified protein allowed the detection of PepQ among several Lactobacillus species but not lactococci. The pepQ gene and the upstream region were isolated and sequenced. The deduced peptide sequence showed that PepQ belongs to the M24 family of metallopeptidases. The pepR1 gene is located immediately upstream of pepQ and its product is homologous to the transcription factor CcpA, which is involved in catabolite repression of catabolic operons from Gram-positive bacteria. The pepR1-pepQ intergenic region contains a consensus catabolite-responsive element (CRE) which could be a target for PepR1 protein. Moreover, in contrast to other proline-specific enzymes from Lb. bulgaricus, PepQ biosynthesis was shown to be dependent on the composition of the culture medium, but not on the peptide concentration. A possible regulation mechanism is discussed.

Corynebacterium freneyi sp. nov., alpha-glucosidase-positive strains related to Corynebacterium xerosis.

Three coryneform strains from clinical specimens were studied. They belonged to the genus Corynebacterium, since they had type IV cell walls containing corynemycolic acids. They had phenotypic characteristics that included alpha-glucosidase, pyrazinamidase and alkaline phosphatase activities and fermentation of glucose, ribose, maltose and sucrose. These are the characteristics of Corynebacterium xerosis. Since this species is very rare in human pathology, the strains were studied in more detail by comparing the 16S-23S intergenic spacers, rDNA sequences and levels of DNA similarity of these three strains and those of the reference strains C. xerosis ATCC 373T and Corynebacterium amycolatum CIP 103452T. According to DNA-DNA hybridization data, the three novel strains are members of the same species (level of DNA similarity >72%). Phylogenetic analysis revealed that these strains are closely related to C. xerosis and C. amycolatum, but DNA-relatedness experiments showed clearly that they constitute a distinct new species, with levels of DNA relatedness of less than 23% to C. xerosis ATCC 373T and less than 5% to C. amycolatum CIP 103452T. Two other alpha-glucosidase-positive strains presenting the same biochemical characteristics were included in the study and proved to be C. amycolatum. This new species can be differentiated from C. xerosis and C. amycolatum strains by carbon source utilization, intergenic spacer region length profiles and some biochemical characteristics such as glucose fermentation at 42 degrees C and growth at 20 degrees C. The name Corynebacterium freneyi sp. nov. is proposed with the type strain ISPB 6695110T (= CIP 106767T = DSM 44506T).

Novel theranostic agents for next-generation personalized medicine: small molecules, nanoparticles, and engineered mammalian cells

Modern medicine is currently undergoing a paradigm shift from conventional disease treatments based on the diagnosis of a generalized disease state to a more personalized, customized treatment model based on molecular-level diagnosis. This uses novel biosensors that can precisely extract disease-related information from complex biological systems. Moreover, with the recent progress in chemical biology, materials science, and synthetic biology, it has become possible to simultaneously conduct diagnosis and targeted therapy (theranostics/theragnosis) by directly connecting the readout of a biosensor to a therapeutic output. These advances pave the way for more advanced and better personalized treatment for intractable diseases with fewer side effects. In this review, we describe recent advances in the development of cutting-edge theranostic agents that contain both diagnostic and therapeutic functions in a single integrated system. By comparing the advantages and disadvantages of each modality, we discuss the future challenges and prospects of developing ideal theranostic agents for the next generation of personalized medicine.

A Multifunction ABC Transporter (Opt) Contributes to Diversity of Peptide Uptake Specificity within the Genus Lactococcus

Growth of Lactococcus lactis in milk depends on the utilization of extracellular peptides. Up to now, oligopeptide uptake was thought to be due only to the ABC transporter Opp. Nevertheless, analysis of several Opp-deficient L. lactis strains revealed the implication of a second oligopeptide ABC transporter, the so-called Opt system. Both transporters are expressed in wild-type strains such as L. lactis SK11 and Wg2, whereas the plasmid-free strains MG1363 and IL-1403 synthesize only Opp and Opt, respectively. The Opt system displays significant differences from the lactococcal Opp system, which made Opt much more closely related to the oligopeptide transporters of streptococci than to the lactococcal Opp system: (i) genetic organization, (ii) peptide uptake specificity, and (iii) presence of two oligopeptide-binding proteins, OptS and OptA. The fact that only OptA is required for nutrition calls into question the function of the second oligopeptide binding protein (Opts). Sequence analysis of oligopeptide-binding proteins from different bacteria prompted us to propose a classification of these proteins in three distinct groups, differentiated by the presence (or not) of precisely located extensions.

Genomic Diversity of Several Corynebacterium Species Identified by Amplification of the 16s-23s rRNA Gene Spacer Regions

In order to investigate whether 16S–23S ribosomal DNA (rDNA) spacer region length polymorphisms are suitable identification of Corynebacterium strains at the species level, the 16S–23S rDNA intergenic spacer region strains belonging to 11 Corynebacterium species were studied by a PCR-based method. The lengths 16S–23S rDNA spacer regions varied from 394 to 585 bp, fragment lengths which are similar to those described for other genera. A single PCR profile was obtained for each of the following species: Corynebacterium renale, Corynebacterium urealyticum, Corynebacterium diphtheriae, Corynebacterium ulcerans, Corynebacterium pseudodiphtheriticum, and Corynebacterium kutscheri. In contrast, two and three PCR patterns were detected for Corynebacterium minutissimum, Corynebacterium striatum, Corynebacterium amycolatum, and Corynebacterium jeikeium, suggesting that genomic heterogeneity occurs in these four species. The 16S–23S rDNA spacer region length polymorphisms allowed us to discriminate among C. minutissimum, C. striatum, and C. amycolatum, three species that are frequently isolated and misidentified in clinical laboratories. Type strain Corynebacterium xerosis ATCC 373, which exhibited a PCR pattern similar to that of C. amycolatum strains classified in PCR group I, could nevertheless be discriminated from PCR group II (C. amycolatum) strains, as Well as minutissimum and C. striatum strains. Type strain C. xerosis ATCC 373 and C. amycolatum strains classified PCR group I could not be distinguished from strains belonging to C. diphtheriae, C. ulcerans, and C. pseudodiphtheriticum. The lipophilic species C. urealyticum and C. jeikeium, which are frequently encountered in clinical specimens, could be clearly distinguished from each other by this method. The use of 16S–23S spacer region length data determined by PCR-mediated amplification is suitable for identification of several Corynebacterium species. This rapid and easy method may be a useful identification tool for clinical microbiologists.

Isolation of the patC gene encoding the cystathionine beta-lyase of Lactobacillus delbrueckii subsp. bulgaricus and molecular analysis of inter-strain variability in enzyme biosynthesis.

The patC gene encoding the cystathionine beta-lyase (CBL) of Lactobacillus delbrueckii subsp. bulgaricus NCDO 1489 was cloned and expressed in Escherichia coli. Overexpression of CBL complemented the methionine auxotrophy of an E. coli metC mutant, demonstrating in vivo that this enzyme functions as a CBL. However, PatC is distinguishable from the MetC CBLs by a low identity in amino acid sequence, a sensitivity to iodoacetic acid, greater thermostability and a lower substrate affinity. Homologues of patC were detected in the 13 Lb. delbrueckii strains studied, but only seven of them showed CBL activity. In constrast to CBL(+) strains, all CBL-deficient strains analysed were auxotrophic for methionine. This supports the hypothesis that CBLs from lactobacilli are probably involved in methionine biosynthesis. Moreover, the results of this study suggest that post-transcriptional mechanisms account for the differences in CBL activities observed between strains of Lb. delbrueckii.

Toward a world of theranostic medication: Programming biological sentinel systems for therapeutic intervention☆

Theranostic systems support diagnostic and therapeutic functions in a single integrated entity and enable precise spatiotemporal control of the generation of therapeutic molecules according to the individual patients disease state, thereby maximizing the therapeutic outcome and minimizing side effects. These systems can also incorporate reporter systems equipped with a disease-sensing module that can be used to estimate the efficacy of treatment in vivo. Among these reporter systems, biological sentinel systems, such as viruses, bacteria, and mammalian cells, have great potential for use in the development of novel theranostic systems because of their ability to sense a variety of disease markers and secrete various therapeutic molecules. Furthermore, recent advances in biotechnology and synthetic biology have made it possible to treat these biological systems as true programmable entities capable of conducting complex operations, to accurately identify each individual patients disease state. In this review, we introduce the basic design principles of these rapidly expanding classes of biological sentinel system-based theranostic agents, with a focus on recent advances, and we also discuss potential enabling technologies that can further improve these systems and provide more sophisticated therapeutic interventions in the near future. In addition, we consider the possibility of synergistic use of theranostic agents that use different modalities and discuss the prospects for next-generation theranostic agents.

Prosthetic gene networks as an alternative to standard pharmacotherapies for metabolic disorders

Synthetic biology makes inroads into clinical therapy with the debut of closed-loop prosthetic gene networks specifically designed to treat human diseases. Prosthetic networks are synthetic sensor/effector devices that could functionally integrate and interface with host metabolism to monitor disease states and coordinate appropriate therapeutic responses in a self-sufficient, timely and automatic manner. Prosthetic networks hold particular promise for the current global epidemic of closely interrelated metabolic disorders encompassing obesity, type 2 diabetes, hypertension and hyperlipidaemia, which arise from the unhealthy lifestyle and dietary factors in the modern urbanised world. This review will critically examine the various attempts at constructing prosthetic gene networks for the treatment of these metabolic disorders, as well as provide insight into future developments in the field.

Synthetic gene circuits for the detection, elimination and prevention of disease

In living organisms, naturally evolved sensors that constantly monitor and process environmental cues trigger corrective actions that enable the organisms to cope with changing conditions. Such natural processes have inspired biologists to construct synthetic living sensors and signalling pathways, by repurposing naturally occurring proteins and by designing molecular building blocks de novo, for customized diagnostics and therapeutics. In particular, designer cells that employ user-defined synthetic gene circuits to survey disease biomarkers and to autonomously re-adjust unbalanced pathological states can coordinate the production of therapeutics, with controlled timing and dosage. Furthermore, tailored genetic networks operating in bacterial or human cells have led to cancer remission in experimental animal models, owing to the network’s unprecedented specificity. Other applications of designer cells in infectious, metabolic and autoimmune diseases are also being explored. In this Review, we describe the biomedical applications of synthetic gene circuits in major disease areas, and discuss how the first genetically engineered devices developed on the basis of synthetic-biology principles made the leap from the laboratory to the clinic.This Review discusses the main diagnostic and therapeutic applications of synthetic gene circuits, and their translational prospects.