Karin Di Padova

Bio: Karin Di Padova is an academic researcher from La Roche College. The author has contributed to research in topics: Gene & DNA repair. The author has an hindex of 3, co-authored 3 publications receiving 224 citations.

Gene Expression Changes Triggered by Exposure of Haemophilus influenzae to Novobiocin or Ciprofloxacin: Combined Transcription and Translation Analysis

Abstract: The responses of Haemophilus influenzae to DNA gyrase inhibitors were analyzed at the transcriptional and the translational level. High-density microarrays based on the genomic sequence were used to monitor the expression levels of >80% of the genes in this bacterium. In parallel the proteins were analyzed by two-dimensional electrophoresis. DNA gyrase inhibitors of two different functional classes were used. Novobiocin, as a representative of one class, inhibits the ATPase activity of the enzyme, thereby indirectly changing the degree of DNA supercoiling. Ciprofloxacin, a representative of the second class, obstructs supercoiling by inhibiting the DNA cleavage-resealing reaction. Our results clearly show that different responses can be observed. Treatment with the ATPase inhibitor Novobiocin changed the expression rates of many genes, reflecting the fact that the initiation of transcription for many genes is sensitive to DNA supercoiling. Ciprofloxacin mainly stimulated the expression of DNA repair systems as a response to the DNA damage caused by the stable ternary complexes. In addition, changed expression levels were also observed for some genes coding for proteins either annotated as “unknown function” or “hypothetical” or for proteins not directly involved in DNA topology or repair. [The sequence data described in this paper have been submitted to the EMBL data library under accession nos. {"type":"entrez-nucleotide","attrs":{"text":"AJ297131","term_id":"10880347","term_text":"AJ297131"}}AJ297131 and {"type":"entrez-nucleotide","attrs":{"text":"AL135960","term_id":"6635875","term_text":"AL135960"}}AL135960.]

Mechanism-related changes in the gene transcription and protein synthesis patterns of Haemophilus influenzae after treatment with transcriptional and translational inhibitors.

Abstract: High-resolution two-dimensional gel electrophoresis of pulse-labeled Haemophilus influenzae extracts allows for the separation and quantification of more than five hundred protein spots. We have determined the changes in the protein synthesis patterns triggered by treatment with inhibitors of transcription, Rifampicin (Rif) and translation, Chloramphenicol (Chl), Erythromycin (Ery), Fusidate (Fus), Puromycin (Pur), Kanamycin (Kan), Streptomycin (Str), and Tetracycline (Tet) relative to the total protein synthesis rate. More than 200 spots changed in intensity under at least one condition. With the exception of the aminoglycosides, Kan and Str, all inhibitors triggered a clear increase in the synthesis rates of ribosomal proteins and RNA polymerase subunits. Northern analysis of rpoA, rpoB, rpoC, and six ribosomal protein genes indicated induction of transcription as well as antitermination as part of the mechanism of the regulation of gene expression. Total RNA synthesis was increased after exposure to Chl, Ery, Fus, and Tet, whereas Str had no effect. Rif led to an almost complete shutdown of RNA synthesis. Exposure to Chl, Ery, Fus, Rif, and Tet resulted in a decrease in the concentration of the stringent factor, guanosine 5',3'-bis-diphosphate (ppGpp) whereas Str again had no effect. Thus, as in Escherichia coli, the response of H. influenzae to translational inhibitors appears to be mediated by the regulatory nucleotide ppGpp.

Strategies towards a better understanding of antibiotic action: Folate pathway inhibition in Haemophilus influenzae as an example

Abstract: Two-dimensional electrophoresis was applied to the global analysis of the cellular response of Haemophilus influenzae to sulfamethoxazole and trimethoprim, both inhibitors of tetrahydrofolate synthesis. Deregulation of the synthesis rate of 118 proteins, involved in different metabolic pathways, was observed. The regulation of the genes involved in the metabolism of the amino acids methionine, threonine, serine, glycine, and aspartate was investigated in detail by analysis of protein synthesis and Northern hybridization. The results suggested that the synthesis of methionine biosynthetic enzymes in H. influenzae is regulated in a similar fashion as in Escherichia coli. A good correlation between the results obtained by Northern hybridization and quantification of protein synthesis was observed. In contrast to trimethoprim, sulfamethoxazole triggered the increased synthesis of the heat shock proteins DnaK, GroEL, and GroES.

Proteomic Approach to Understanding Antibiotic Action

Abstract: We have used proteomic technology to elucidate the complex cellular responses of Bacillus subtilis to antimicrobial compounds belonging to classical and emerging antibiotic classes. We established on two-dimensional gels a comprehensive database of cytoplasmic proteins with pIs covering a range of 4 to 7 that were synthesized during treatment with antibiotics or agents known to cause generalized cell damage. Although each antibiotic showed an individual protein expression profile, overlaps in the expression of marker proteins reflected similarities in molecular drug mechanisms, suggesting that novel compounds with unknown mechanisms of action may be classified. Indeed, one such substance, a structurally novel protein synthesis inhibitor (BAY 50-2369), could be classified as a peptidyltransferase inhibitor. These results suggest that this technique gives new insights into the bacterial response toward classical antibiotics and hints at modes of action of novel compounds. Such a method should prove useful in the process of antibiotic drug discovery.

Antibiotics as Signal Molecules

Abstract: Antibiotics have been extensively used in the treatment of infectious diseases The lethality of these compounds has been exploited in clinical and laboratory approaches and their specific targets in bacterial physiology elucidated However, along with this development of drugs has come the problem of increasing resistance in microbes, resulting in dramatically reduced therapeutic effectiveness 1 Pathogenic microbes have rapidly evolved efficient mechanisms of resistance, including increased efflux, enzymatic inactivation, target modification, or biofilm formation 2 The concentrations of these molecules required to achieve an antimicrobial effect are likely extremely high compared to the concentrations in which these molecules can be found in natural environments While we know their effect at lethal concentrations, the activities of these molecules at concentrations below the inhibitory limit needs deeper investigation 3 The findings of the nineteenth-century pharmacologist Hugo Schulz, who noted that certain disinfectants could have stimulatory effect on yeast growth at low concentrations, could be considered the first evidence that the action of an antimicrobial can cause a differential response depending on the concentration His observation was the first example of what it would be later called hormesis This term was coined by Chester Southam and John Ehrlich in the mid-1920s and it is used to refer to the ability of certain molecules to induce diverse responses depending on the concentration used 3 The vast amount of information related to the lethal concentration of antibiotics, targets, or side effects, contrasts dramatically with the relatively few studies focused on their effect at concentrations below the MIC (minimal inhibitory concentration) As pointed by Davies, only a small fraction of natural products that have antimicrobial activity have been extensively studied, and their role in natural settings is poorly understood Thus it is possible that many of these molecules formerly considered antibiotics might have a different function in nature It is now believed that many of these compounds might act as signaling molecules that modulate gene expression in microbial populations, or physiological functions such as motility, pigmentation, and production of metabolites, and thus facilitate inter- and intra-species communication 4 This fundamental lack of understanding may be rooted in our conception of the microbial world as single and separated species, as they are usually studied under laboratory conditions However, in nature, each niche is complex, and to different extents, variable in microbial community composition 5,6–8 Therefore it is conceivable that molecules fluctuate in concentration and diversity, thus facilitating communication among species 9 Many interesting lines of research are currently focused on understanding how some antibiotics may affect, either positively or negatively, cell-cell communication systems, and the physiological responses that are affected as a result In some cases, natural products can influence the ability of bacteria to transition from a planktonic state to complex multicellular aggregates attached to surfaces known as biofilms Cells in bioflms are encased in an extracellular matrix which can serve as a barrier for antibiotics 10 One example is the biofilm produced by the gram-negative pathogen, Pseudomonas aeruginosa, which is resistant to antibiotics produced by gram-positive competitors 11 Studies on many model microorganisms from the genera Bacillus, Streptomyces, and Pseudomonas are shedding new light on the fascinating world of cell signaling and communication in microbial world 5,12,13 We will discuss in this review several examples of the dual functions of some well-known antibiotics, including those that when used at sub-inhibitory concentrations (SIC) promote an interesting response in bacterial populations and the ecological implications of such varied responses Also, the role of other naturally synthesized antibiotics will be discussed in the context of cell communication in natural environments, including one of the most exploited environmental niches for antibiotic discovery, the soil

Fluoroquinolones: action and resistance

Abstract: Fluoroquinolones trap gyrase and topoisomerase IV on DNA as ternary complexes that block the movement of replication forks and transcription complexes. Studies with resistant mutants indicate that during complex formation quinolones bind to a surface alpha-helix of the GyrA and ParC proteins. Lethal action is a distinct event that is proposed to arise from release of DNA breaks from the ternary complexes. Many bacterial pathogens are exhibiting resistance due to alterations in drug permeability, drug efflux, gyrase-protecting proteins, and target topoisomerases. When selection of resistant mutants is described in terms of fluoroquinolone concentration, a threshold (mutant prevention concentration, MPC) can be defined for restricting the development of resistance. MPC varies among fluoroquinolones and pathogens; when combined with pharmacokinetics, MPC can be used to identify compounds least likely to enrich mutant subpopulations. Use of suboptimal doses and compounds erodes the efficacy of the class as a whole because resistance to one quinolone reduces susceptibility to others and/or increases the frequency at which resistance develops. When using fluoroquinolones in combination therapy, the development of resistance may be minimized by optimizing regimens for pharmacokinetic overlap.

Perspectives for mass spectrometry and functional proteomics.

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