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Dive into the research topics where Maria Rosalia Pasca is active.

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Featured researches published by Maria Rosalia Pasca.


Science | 2009

Benzothiazinones Kill Mycobacterium tuberculosis by Blocking Arabinan Synthesis

Vadim Makarov; Giulia Manina; Katarína Mikušová; Ute Möllmann; Olga Ryabova; Brigitte Saint-Joanis; Neeraj Dhar; Maria Rosalia Pasca; Silvia Buroni; Anna Paola Lucarelli; Anna Milano; Edda De Rossi; Martina Belanová; Adela Bobovská; Petronela Dianišková; Jana Korduláková; Claudia Sala; Elizabeth Fullam; Patricia Schneider; John D. McKinney; Priscille Brodin; Thierry Christophe; Simon J. Waddell; Philip D. Butcher; Jakob Albrethsen; Ida Rosenkrands; Roland Brosch; Vrinda Nandi; Sheshagiri Gaonkar; Radha Shandil

Ammunition for the TB Wars Tuberculosis is a major human disease of global importance resulting from infection with the air-borne pathogen Mycobacterium tuberculosis, which is becoming increasingly resistant to all available drugs. An antituberculosis benzothiazinone compound kills mycobacterium in infected cells and in mice. Makarov et al. (p. 801) have identified a sulfur atom and nitro residues important for benzothiazinones activity and used genetic methods and biochemical analysis to identify its target in blocking arabinogalactan biosynthesis during cell-wall synthesis. The compound affects the same pathway as ethambutol, and thus a benzothiazinone drug has the potential to become an important part of treatment of drug-resistant disease and, possibly, replace the less effective ethambutol in the primary treatment of tuberculosis. An isomerase required for cell-wall synthesis is a target for an alternative drug lead for tuberculosis treatment. New drugs are required to counter the tuberculosis (TB) pandemic. Here, we describe the synthesis and characterization of 1,3-benzothiazin-4-ones (BTZs), a new class of antimycobacterial agents that kill Mycobacterium tuberculosis in vitro, ex vivo, and in mouse models of TB. Using genetics and biochemistry, we identified the enzyme decaprenylphosphoryl-β-d-ribose 2′-epimerase as a major BTZ target. Inhibition of this enzymatic activity abolishes the formation of decaprenylphosphoryl arabinose, a key precursor that is required for the synthesis of the cell-wall arabinans, thus provoking cell lysis and bacterial death. The most advanced compound, BTZ043, is a candidate for inclusion in combination therapies for both drug-sensitive and extensively drug-resistant TB.


Journal of Bacteriology | 2007

Global Analysis of the Mycobacterium tuberculosis Zur (FurB) Regulon

Anna Maciąg; Elisa Dainese; G. Marcela Rodriguez; Anna Milano; Roberta Provvedi; Maria Rosalia Pasca; Issar Smith; Giorgio Palù; Giovanna Riccardi; Riccardo Manganelli

The proteins belonging to the Fur family are global regulators of gene expression involved in the response to several environmental stresses and to the maintenance of divalent cation homeostasis. The Mycobacterium tuberculosis genome encodes two Fur-like proteins, FurA and a protein formerly annotated FurB. Since in this paper we show that it represents a zinc uptake regulator, we refer to it as Zur. The gene encoding Zur is found in an operon together with the gene encoding a second transcriptional regulator (Rv2358). In a previous work we demonstrated that Rv2358 is responsible for the zinc-dependent repression of the Rv2358-zur operon, favoring the hypothesis that these genes represent key regulators of zinc homeostasis. In this study we generated a zur mutant in M. tuberculosis, examined its phenotype, and characterized the Zur regulon by DNA microarray analysis. Thirty-two genes, presumably organized in 16 operons, were found to be upregulated in the zur mutant. Twenty-four of them belonged to eight putative transcriptional units preceded by a conserved 26-bp palindrome. Electrophoretic mobility shift experiments demonstrated that Zur binds to this palindrome in a zinc-dependent manner, suggesting its direct regulation of these genes. The proteins encoded by Zur-regulated genes include a group of ribosomal proteins, three putative metal transporters, the proteins belonging to early secretory antigen target 6 (ESAT-6) cluster 3, and three additional proteins belonging to the ESAT-6/culture filtrate protein 10 (CFP-10) family known to contain immunodominant epitopes in the T-cell response to M. tuberculosis infection.


Antimicrobial Agents and Chemotherapy | 2012

MmpL3 is the cellular target of the antitubercular pyrrole derivative BM212

Valentina La Rosa; Giovanna Poce; Julio Ortiz Canseco; Silvia Buroni; Maria Rosalia Pasca; Mariangela Biava; Ravikiran M. Raju; Salvatore Alfonso; Claudio Battilocchio; Babak Javid; Flavia Sorrentino; Thomas R. Ioerger; James C. Sacchettini; Fabrizio Manetti; Maurizio Botta; Alessandro De Logu; Eric J. Rubin; Edda De Rossi

ABSTRACT The 1,5-diarylpyrrole derivative BM212 was previously shown to be active against multidrug-resistant clinical isolates and Mycobacterium tuberculosis residing within macrophages as well as against Mycobacterium avium and other atypical mycobacteria. To determine its mechanism of action, we identified the cellular target. Spontaneous Mycobacterium smegmatis, Mycobacterium bovis BCG, and M. tuberculosis H37Rv mutants that were resistant to BM212 were isolated. By the screening of genomic libraries and by whole-genome sequencing, we found that all the characterized mutants showed mutations in the mmpL3 gene, allowing us to conclude that resistance to BM212 maps to the MmpL3 protein, a member of the MmpL (mycobacterial membrane protein, large) family. Susceptibility was unaffected by the efflux pump inhibitors reserpine, carbonylcyanide m-chlorophenylhydrazone, and verapamil. Uptake/efflux experiments with [14C]BM212 demonstrated that resistance is not driven by the efflux of BM212. Together, these data strongly suggest that the MmpL3 protein is the cellular target of BM212.


Antimicrobial Agents and Chemotherapy | 2004

Rv2686c-Rv2687c-Rv2688c, an ABC Fluoroquinolone Efflux Pump in Mycobacterium tuberculosis

Maria Rosalia Pasca; Paola Guglierame; Fabio Arcesi; Marco Bellinzoni; Edda De Rossi; Giovanna Riccardi

ABSTRACT The Mycobacterium tuberculosis Rv2686c-Rv2687c-Rv2688c operon, encoding an ABC transporter, conferred resistance to ciprofloxacin and, to a lesser extent, norfloxacin, moxifloxacin, and sparfloxacin to Mycobacterium smegmatis. The resistance level decreased in the presence of the efflux pump inhibitors reserpine, carbonyl cyanide m-chlorophenylhydrazone, and verapamil. Energy-dependent efflux of ciprofloxacin from M. smegmatis cells containing the Rv2686c-Rv2687c-Rv2688c operon was observed.


Science Translational Medicine | 2012

Structural Basis for Benzothiazinone-Mediated Killing of Mycobacterium tuberculosis

João Neres; Florence Pojer; Elisabetta Molteni; Laurent R. Chiarelli; Neeraj Dhar; Stefanie Boy-Röttger; Silvia Buroni; Elizabeth Fullam; Giulia Degiacomi; Anna Paola Lucarelli; Randy J. Read; Giuseppe Zanoni; Dale E. Edmondson; Edda De Rossi; Maria Rosalia Pasca; John D. McKinney; Paul J. Dyson; Giovanna Riccardi; Andrea Mattevi; Stewart T. Cole; Claudia Binda

The crystal structure of the mycobacterial DprE1 reveals how the TB drug benzothiazinone BTZ043 blocks this microbial enzyme target. New TB Drug Snapped in Action Tuberculosis (TB) is a major global health problem that claimed 1.4 million lives in 2010. TB is becoming incurable with existing antibiotics, as infections with multidrug-resistant strains of the causative pathogen Mycobacterium tuberculosis continue to climb. To make matters worse, many patients with TB also suffer from HIV/AIDS, making both diseases even more difficult to treat. It has been more than 40 years since a new drug for TB was approved for clinical use. In 2009, a study published in Science described a promising new drug candidate, a synthetic organic molecule known as BTZ043, which is active in the low nanomolar range against mycobacteria. BTZ043 inhibits a bacterial epimerase enzyme that produces the sugar d-arabinose, the sole precursor for the synthesis of a polysaccharide that is an essential component of the bacterial cell wall. In a key follow-up study, Neres et al. use x-ray crystallography to obtain a picture of the epimerase at the atomic level. They demonstrate that the drug serves as a suicide substrate that is converted by the epimerase into a highly reactive species, and they present a snapshot that shows covalent binding of this species to the active site of the enzyme. Together with biochemical work, the three-dimensional structure explains why BTZ043 inactivates its target so effectively, thus killing the bacteria. By attaching a fluorescent probe to one side of the drug, the authors discovered that the epimerase enzyme becomes localized to the poles of live bacteria, thus pinpointing the site of action. The availability of the epimerase structure and a deeper understanding of its catalytic properties open a host of avenues for rational drug discovery that hopefully will result in new medicines for fighting TB. The benzothiazinone BTZ043 is a tuberculosis drug candidate with nanomolar whole-cell activity. BTZ043 targets the DprE1 catalytic component of the essential enzyme decaprenylphosphoryl-β-d-ribofuranose-2′-epimerase, thus blocking biosynthesis of arabinans, vital components of mycobacterial cell walls. Crystal structures of DprE1, in its native form and in a complex with BTZ043, reveal formation of a semimercaptal adduct between the drug and an active-site cysteine, as well as contacts to a neighboring catalytic lysine residue. Kinetic studies confirm that BTZ043 is a mechanism-based, covalent inhibitor. This explains the exquisite potency of BTZ043, which, when fluorescently labeled, localizes DprE1 at the poles of growing bacteria. Menaquinone can reoxidize the flavin adenine dinucleotide cofactor in DprE1 and may be the natural electron acceptor for this reaction in the mycobacterium. Our structural and kinetic analysis provides both insight into a critical epimerization reaction and a platform for structure-based design of improved inhibitors.


Antimicrobial Agents and Chemotherapy | 2005

mmpL7 Gene of Mycobacterium tuberculosis Is Responsible for Isoniazid Efflux in Mycobacterium smegmatis

Maria Rosalia Pasca; Paola Guglierame; Edda De Rossi; Francesca Zara; Giovanna Riccardi

ABSTRACT The Mycobacterium tuberculosis mmpL7 gene, encoding a hypothetical resistance nodulation division transporter, confers a high resistance level to isoniazid when overexpressed in Mycobacterium smegmatis. The resistance level decreased in the presence of the efflux pump inhibitors reserpine and CCCP (carbonyl cyanide m-chlorophenylhydrazone). Energy-dependent efflux of isoniazid from M. smegmatis cells expressing the mmpL7 gene was observed.


Tuberculosis | 2009

Azole resistance in Mycobacterium tuberculosis is mediated by the MmpS5–MmpL5 efflux system

Anna Milano; Maria Rosalia Pasca; Roberta Provvedi; Anna Paola Lucarelli; Giulia Manina; Ana Luisa de Jesus Lopes Ribeiro; Riccardo Manganelli; Giovanna Riccardi

Tuberculosis (TB) remains the leading cause of mortality due to a bacterial pathogen, Mycobacterium tuberculosis. Moreover, the recent isolation of M. tuberculosis strains resistant to both first- and second-line antitubercular drugs (XDR-TB) threatens to make the treatment of this disease extremely difficult and becoming a threat to public health worldwide. Recently, it has been shown that azoles are potent inhibitors of mycobacterial cell growth and have antitubercular activity in mice, thus favoring the hypothesis that these drugs may constitute a novel strategy against tuberculosis disease. To investigate the mechanisms of resistance to azoles in mycobacteria, we isolated and characterized several spontaneous azoles resistant mutants from M. tuberculosis and Mycobacterium bovis BCG. All the analyzed resistant mutants exhibited both increased econazole efflux and increased transcription of mmpS5-mmpL5 genes, encoding a hypothetical efflux system belonging to the resistance-nodulation-division (RND) family of transporters. We found that the up-regulation of mmpS5-mmpL5 genes was linked to mutations either in the Rv0678 gene, hypothesized to be involved in the transcriptional regulation of this efflux system, or in its putative promoter/operator region.


PLOS ONE | 2011

Deciphering the role of RND efflux transporters in Burkholderia cenocepacia

Silvia Bazzini; Claudia Udine; Andrea Sass; Maria Rosalia Pasca; Francesca Longo; Giovanni Emiliani; Marco Fondi; Elena Perrin; Francesca Decorosi; Carlo Viti; Luciana Giovannetti; Livia Leoni; Renato Fani; Giovanna Riccardi; Eshwar Mahenthiralingam; Silvia Buroni

Burkholderia cenocepacia J2315 is representative of a highly problematic group of cystic fibrosis (CF) pathogens. Eradication of B. cenocepacia is very difficult with the antimicrobial therapy being ineffective due to its high resistance to clinically relevant antimicrobial agents and disinfectants. RND (Resistance-Nodulation-Cell Division) efflux pumps are known to be among the mediators of multidrug resistance in Gram-negative bacteria. Since the significance of the 16 RND efflux systems present in B. cenocepacia (named RND-1 to -16) has been only partially determined, the aim of this work was to analyze mutants of B. cenocepacia strain J2315 impaired in RND-4 and RND-9 efflux systems, and assess their role in the efflux of toxic compounds. The transcriptomes of mutants deleted individually in RND-4 and RND-9 (named D4 and D9), and a double-mutant in both efflux pumps (named D4-D9), were compared to that of the wild-type B. cenocepacia using microarray analysis. Microarray data were confirmed by qRT-PCR, phenotypic experiments, and by Phenotype MicroArray analysis. The data revealed that RND-4 made a significant contribution to the antibiotic resistance of B. cenocepacia, whereas RND-9 was only marginally involved in this process. Moreover, the double mutant D4-D9 showed a phenotype and an expression profile similar to D4. The microarray data showed that motility and chemotaxis-related genes appeared to be up-regulated in both D4 and D4–D9 strains. In contrast, these gene sets were down-regulated or expressed at levels similar to J2315 in the D9 mutant. Biofilm production was enhanced in all mutants. Overall, these results indicate that in B. cenocepacia RND pumps play a wider role than just in drug resistance, influencing additional phenotypic traits important for pathogenesis.


BMC Microbiology | 2006

Efflux pump genes of the resistance-nodulation-division family in Burkholderia cenocepacia genome

Paola Guglierame; Maria Rosalia Pasca; Edda De Rossi; Silvia Buroni; Patrizio Arrigo; Giulia Manina; Giovanna Riccardi

BackgroundBurkholderia cenocepacia is recognized as opportunistic pathogen that can cause lung infections in cystic fibrosis patients. A hallmark of B. cenocepacia infections is the inability to eradicate the organism because of multiple intrinsic antibiotic resistance. As Resistance-Nodulation-Division (RND) efflux systems are responsible for much of the intrinsic multidrug resistance in Gram-negative bacteria, this study aims to identify RND genes in the B. cenocepacia genome and start to investigate their involvement into antimicrobial resistance.ResultsGenome analysis and homology searches revealed 14 open reading frames encoding putative drug efflux pumps belonging to RND family in B. cenocepacia J2315 strain.By reverse transcription (RT)-PCR analysis, it was found that orf3, orf9, orf11, and orf13 were expressed at detectable levels, while orf10 appeared to be weakly expressed in B. cenocepacia. Futhermore, orf3 was strongly induced by chloramphenicol. The orf2 conferred resistance to fluoroquinolones, tetraphenylphosphonium, streptomycin, and ethidium bromide when cloned and expressed in Escherichia coli KAM3, a strain lacking the multidrug efflux pump AcrAB. The orf2-overexpressing E. coli also accumulate low concentrations of ethidium bromide, which was restored to wild type level in the presence of CCCP, an energy uncoupler altering the energy of the drug efflux pump.ConclusionThe 14 RND pumps gene we have identified in the genome of B. cenocepacia suggest that active efflux could be a major mechanism underlying antimicrobial resistance in this microorganism. We have characterized the ORF2 pump, one of these 14 potential RND efflux systems. Its overexpression in E. coli conferred resistance to several antibiotics and to ethidium bromide but it remains to be determined if this pump play a significant role in the antimicrobial intrinsic resistance of B. cenocepacia. The characterization of antibiotic efflux pumps in B. cenocepacia is an obligatory step prior to the design of specific, potent bacterial inhibitors for the improved control of infectious diseases. Consequently, the topic deserves to be further investigated and future studies will involve systematic investigation on the function and expression of each of the RND efflux pump homologs.


BMC Microbiology | 2009

Assessment of three Resistance-Nodulation-Cell Division drug efflux transporters of Burkholderia cenocepacia in intrinsic antibiotic resistance

Silvia Buroni; Maria Rosalia Pasca; Ronald S. Flannagan; Silvia Bazzini; Anna Milano; Iris Bertani; Vittorio Venturi; Miguel A. Valvano; Giovanna Riccardi

BackgroundBurkholderia cenocepacia are opportunistic Gram-negative bacteria that can cause chronic pulmonary infections in patients with cystic fibrosis. These bacteria demonstrate a high-level of intrinsic antibiotic resistance to most clinically useful antibiotics complicating treatment. We previously identified 14 genes encoding putative Resistance-Nodulation-Cell Division (RND) efflux pumps in the genome of B. cenocepacia J2315, but the contribution of these pumps to the intrinsic drug resistance of this bacterium remains unclear.ResultsTo investigate the contribution of efflux pumps to intrinsic drug resistance of B. cenocepacia J2315, we deleted 3 operons encoding the putative RND transporters RND-1, RND-3, and RND-4 containing the genes BCAS0591-BCAS0593, BCAL1674-BCAL1676, and BCAL2822-BCAL2820. Each deletion included the genes encoding the RND transporter itself and those encoding predicted periplasmic proteins and outer membrane pores. In addition, the deletion of rnd-3 also included BCAL1672, encoding a putative TetR regulator. The B. cenocepacia rnd-3 and rnd-4 mutants demonstrated increased sensitivity to inhibitory compounds, suggesting an involvement of these proteins in drug resistance. Moreover, the rnd-3 and rnd-4 mutants demonstrated reduced accumulation of N-acyl homoserine lactones in the growth medium. In contrast, deletion of the rnd-1 operon had no detectable phenotypes under the conditions assayed.ConclusionTwo of the three inactivated RND efflux pumps in B. cenocepacia J2315 contribute to the high level of intrinsic resistance of this strain to some antibiotics and other inhibitory compounds. Furthermore, these efflux systems also mediate accumulation in the growth medium of quorum sensing molecules that have been shown to contribute to infection. A systematic study of RND efflux systems in B. cenocepacia is required to provide a full picture of intrinsic antibiotic resistance in this opportunistic bacterium.

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Michel Baltas

Centre national de la recherche scientifique

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Vadim Makarov

Russian Academy of Sciences

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