Beatrice Cameron

Analysis of gyrA and grlA mutations in stepwise-selected ciprofloxacin-resistant mutants of Staphylococcus aureus.

Fluoroquinolone-resistant mutants were obtained in vitro from Staphylococcus aureus RN4220 by stepwise selection on increasing concentrations of ciprofloxacin. Results from sequence analysis of the quinolone resistance-determining region of GyrA and of the corresponding region of GrlA, the DNA topoisomerase IV subunit, showed an alteration of Ser-80 to Tyr (corresponding to Ser-83 of Escherichia coli GyrA) or Glu-84 to Lys in GrlA of both low- and high-level quinolone-resistant mutants. Second-step mutants were found to have, in addition to a mutation in grlA, reduced accumulation of norfloxacin or an alteration in GyrA at Ser-84 to Leu or Glu-88 to Lys. Third-step mutants derived from second-step mutants with reduced accumulation were found to have a mutation in gyrA. The results from this study demonstrated that mutations in gyrA or mutations leading to reduced drug accumulation occur after alteration of GrlA, supporting the previous findings (L. Ferrero, B. Cameron, B. Manse, D. Lagneaux, J. Crouzet, A. Famechon, and F. Blanche, Mol. Microbiol. 13:641-653, 1994) that DNA topoisomerase IV is a primary target of fluoroquinolones in S. aureus.

A new DNA vehicle for nonviral gene delivery: supercoiled minicircle.

Plasmids currently used for nonviral gene transfer have the disadvantage of carrying a bacterial origin of replication and an antibiotic resistance gene. There is, therefore, a risk of uncontrolled dissemination of the therapeutic gene and the antibiotic resistance gene. Minicircles are new DNA delivery vehicles which do not have such elements and are consequently safer as they exhibit a high level of biological containment. They are obtained in E. coli by att site-specific recombination mediated by the phage λ integrase. The desired eukaryotic expression cassette bounded by the λ attP and attB sites was cloned on a recombinant plasmid. The expression cassette was excised in vivo after thermoinduction of the integrase gene leading to the formation of two supercoiled molecules: the minicircle and the starting plasmid lacking the expression cassette. In various cell lines, purified minicircles exhibited a two- to 10-fold higher luciferase reporter gene activity than the unrecombined plasmid. This could be due to either the removal of unnecessary plasmid sequences, which could affect gene expression, or the smaller size of minicircle which may confer better extracellular and intracellular bioavailability and result in improved gene delivery properties.

Differential behaviors of Staphylococcus aureus and Escherichia coli type II DNA topoisomerases.

Staphylococcus aureus gyrA and gyrB genes encoding DNA gyrase subunits were cloned and coexpressed in Escherichia coli under the control of the T7 promoter-T7 RNA polymerase system, leading to soluble gyrase which was purified to homogeneity. Purified gyrase was catalytically indistinguishable from the gyrase purified from S. aureus and did not contain detectable amounts of topoisomerases from the E. coli host. Topoisomerase IV subunits GrlA and GrlB from S. aureus were also expressed in E. coli and were separately purified to apparent homogeneity. Topoisomerase IV, which was reconstituted by mixing equimolar amounts of GrlA and GrlB, had both ATP-dependent decatenation and DNA relaxation activities in vitro. This enzyme was more sensitive than gyrase to inhibition by typical fluoroquinolone antimicrobial agents such as ciprofloxacin or sparfloxacin, adding strong support to genetic studies which indicate that topoisomerase IV is the primary target of fluoroquinolones in S. aureus. The results obtained with ofloxacin suggest that this fluoroquinolone could also primarily target gyrase. No cleavable complex could be detected with S. aureus gyrase upon incubation with ciprofloxacin or sparfloxacin at concentrations which fully inhibit DNA supercoiling. This suggests that these drugs do not stabilize the open DNA-gyrase complex, at least under standard in vitro incubation conditions, but are more likely to interfere primarily with the DNA breakage step, contrary to what has been reported with E. coli gyrase. Both S. aureus gyrase-catalyzed DNA supercoiling and S. aureus topoisomerase IV-catalyzed decatenation were dramatically stimulated by potassium glutamate or aspartate (500- and 50-fold by 700 and 350 mM glutamate, respectively), whereas topoisomerase IV-dependent DNA relaxation was inhibited 3-fold by 350 mM glutamate. The relevance of the effect of dicarboxylic amino acids on the activities of type II topoisomerases is discussed with regard to the intracellular osmolite composition of S. aureus.

An improved anion-exchange HPLC method for the detection and purification of adenoviral particles

we have developed an anion-exchange high-performance liquid chromatography (hplc) method using q sepharose xl (amersham pharmacia biotech) as adsorbent to analyze samples containing adenovirus. this method has several major advantages over the hplc method previously described for quantitating particles, namely (1) a >10-fold improvement in the detection limit of adenovirus in crude preparations; (2) absence of interferences originating from nucleic acids and proteins which usually contaminate crude samples; (3) unprecedented sharpness and symmetry of adenovirus peak, rendering the identification of the viral peak unambiguous, even in extremely crude and dilute preparations; and (4) no enzymatic treatment required even for crude samples. This assay was used to quantitate particles in samples taken at the transfection and amplification stages of production of various recombinant adenovirus, and in cultures of wild-type adenovirus of different serotypes. A modification of this analytical method was also developed for the purification of infectious adenovirus particles, including fiber-modified and third-generation recombinant viruses, giving highly purified preparations from low-titer crude lysates with an excellent overall recovery (50–74%).

Application of lipids and plasmid design for gene delivery to mammalian cells

Cationic lipids are widely used for in vitro gene transfer due to their efficiency. The major challenges for the improvement of in vivo cationic lipid-mediated gene delivery reside in the design of more biocompatible lipoplexes mimicking viral-mediated gene delivery and in understanding the fate of the lipoplexes within the cells.

Production of a new DNA vehicle for gene transfer using site-specific recombination

Abstract Supercoiled DNA molecules, minicircles, were produced by in vivo site-specific recombination. They contained exclusively the desired excisable fragment. Recombination was driven by bacteriophage λ integrase from a plasmid substrate containing the attP and attB recombination sites in the same orientation. Conditions for minicircle production within the lysogen Escherichia coli D1210HP were optimised. Up to 1.5 mg minicircles could be produced per litre bacterial culture, and the remaining, unrecombined plasmid comprised less than about 15% of the minicircle produced. However minicircle multimers were also produced, and comprised up to 30% of all minicircles synthesised. The parABCDE′ locus from plasmid RK2 was introduced into the minicircle fragment, resulting in minicircle dimers being reduced to less than 2% of all minicircles. The parA gene encodes a resolvase that catalyses recombination at the multimer resolution site in the parABCDE′ locus. Minicircle multimers were also resolved when parA was introduced downstream from the integrase gene of the λpL transcript in D1210HP together with a multimer resolution site carried by the minicircle fragment.

Topoisomerase I of Helicobacter pylori: juxtaposition with a flagellin gene (flaB) and functional requirement of a fourth zinc finger motif

Cloning and nucleotide sequence analysis showed that in Helicobacter pylori the gene encoding topoisomerase I (topA) lies about 170 nucleotides upstream from flaB, a gene encoding one of the two flagellin proteins that is required for virulence. The topA and flaB genes are divergently transcribed. The orientation and spatial relationship between flaB and topA are remarkably conserved among strains of a bacterium in which genomic rearrangements are common. The deduced amino acid sequence of topoisomerase I revealed four zinc finger motifs, one more than has been reported previously for the Escherichia coli homologue. The additional motif, which is near the C-terminus of the protein, appears to be essential for function since mutations in that region are lethal. These data show that TopA proteins can be divided into several classes on the basis of zinc finger motifs and raise the interesting possibility that the H. pylori enzyme has local topological effects focussed on a flagellin gene.

Construction of a broad-host-range non-mobilizable stable vector carrying RP4 par-region

Plasmid pXL1635 was constructed from the already segregationally stable incP-derived pRK290. Plasmid pXL1635 should be suitable for industrial and environmental uses in Gram- bacteria since (i) it contains the par fragment from RP4 which increases its stability in Pseudomonas denitrificans, a cobalamin-producing and industrially used bacterium, and (ii) the RK2 oriT has been deleted, leading to a non-mobilizable plasmid.

Biochemical and Genetic Studies on Vitamin B12 Synthesis in Pseudomonas dentrificans

Vitamin B12 is produced industrially by Pseudomonas denitrificans or by bacteria belonging to Propionibacterium species (i. e. Propionibacterium shermanii or Propionibacterium freudenreichii). Industrial strains were obtained after long lasting and intense programs aimed at improving the fermentation caracteristics and the productivity of natural isolates (Florent, 1986). These programs consist of numerous mutagenesis steps, each of them introducing a property resulting in improved characteristics of the strain for industrial purpose (i. e. higher productivity, higher growth rate in a defined medium, growth to a higher biomass, etc). At each mutagenesis step the clone of interest is selected randomly either (i) for its property to produce higher level of vitamin B12 or (ii) for its resistance to an antibiotic for instance or (iii) for a phenotype related to an improved production of vitamin B12 (Florent, 1986). These selection programs have allowed various companies to improve the production of the starting bacteria. For instance the Pseudomonas denitrificans wild type isolate, strain MB580, from which the industrial strain used by Rhone-Poulenc Sante derives produces 2.5 mg/1 of cobalamin. In the same conditions, the industrial strain presently used produces much more (Florent, 1986).

Abstract 1197: Outstanding preclinical efficacy of a novel maytansinoid-antibody-drug conjugate targeting LAMP1 in patient-derived xenograft solid tumors

Lysosome-associated membrane protein 1 (LAMP1) is involved in the maintenance of lysosome membrane integrity and phagolysosome formation. LAMP1 shows very limited expression at the cell surface in normal tissues while, a moderate to high expression was found in a number of solid tumors. We generated SAR428926, an antibody drug conjugate (ADC) comprising an anti-LAMP1 humanized monoclonal antibody conjugated via a cleavable SPDB linker, to the maytansinoid derivative DM4, a tubulin interfering molecule. Here we report the annotation of LAMP1 expression in a large panel of human tumors and correlate it to the in vivo efficacy of SAR428926 in a panel of over 50 human patient-derived xenograft (PDX) solid tumors. Indications of interest were identified by immunohistochemistry (IHC) conducted on a large panel of frozen tumor samples using the murine version of the therapeutic anti-human LAMP1 antibody. The prevalence of tumor samples with positive membrane staining was 49% in breast invasive lobular or ductal carcinoma, and in particular in a TNBC subset (87%), 25% in gastric adenocarcinoma, 52% in colon/rectum adenocarcinoma, 22% in lung adenocarcinoma, 20% in lung squamous cell carcinoma, 25% in prostate adenocarcinoma and 31% in ovary adenocarcinoma. In vivo efficacy of SAR428926 was evaluated in PDX models engrafted subcutaneously into immunocompromised SCID mice. PDX models retain the molecular diversity, cellular heterogeneity, and histology typically seen in patient tumors and offer a distinct advantage over cell line models. The PDX models used in the study, which include colon, breast, lung, prostate, gastric and ovarian cancer, were selected by IHC based on LAMP1 expression levels distribution and the frequency of LAMP1 membrane positive cells, which varied between 5% and 100% of the tumor cells, allowing coverage of a wide variety of cases. Outstanding efficacy with complete regressions was observed at 5 mg/kg iv single administration, a dose that gives an exposure tolerated in toxicology species. Efficacy was also observed at lower doses of 2.5 mg/kg and 1.25 mg/kg after single administration. Our results show that while the presence of the antigen is required as there is no efficacy in LAMP1-negative PDX, there is no linear correlation between the level of antigen expression and the antitumor activity across the panel of PDX tested. In addition to membrane LAMP1 expression, a key driver for efficacy was the intrinsic sensitivity of the PDX models to DM4. These results highlight the potential of ADCs that target human cancers expressing membrane LAMP1 at the surface of tumor cells. These encouraging preclinical data have prompted us to initiate IND-enabling studies with the goal to progress anti LAMP1-ADC to the clinic in patients with solid tumors expressing LAMP1 at the cell surface. The phase I study started in October 2015. Citation Format: Loreley Calvet, Anne-Marie Lefebvre, Celine Nicolazzi, Lydia Blot, Corinne Thomas, Yves Baudat, Beatrice Cameron, Carlos Garcia-Echeverria, Jean-Francois Mayaux, Veronique Blanc, Cecile Combeau, Souad Naimi, Sukhvinder Sidhu. Outstanding preclinical efficacy of a novel maytansinoid-antibody-drug conjugate targeting LAMP1 in patient-derived xenograft solid tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1197.