RESUMO
LasB elastase is a broad-spectrum exoprotease and a key virulence factor of Pseudomonas aeruginosa, a major pathogen causing lung damage and inflammation in acute and chronic respiratory infections. Here, we describe the chemical optimization of specific LasB inhibitors with druglike properties and investigate their impact in cellular and animal models of P. aeruginosa infection. Competitive inhibition of LasB was demonstrated through structural and kinetic studies. In vitro LasB inhibition was confirmed with respect to several host target proteins, namely, elastin, IgG, and pro-IL-1ß. Furthermore, inhibition of LasB-mediated IL-1ß activation was demonstrated in macrophage and mouse lung infection models. In mice, intravenous administration of inhibitors also resulted in reduced bacterial numbers at 24 h. These highly potent, selective, and soluble LasB inhibitors constitute valuable tools to study the proinflammatory impact of LasB in P. aeruginosa infections and, most importantly, show clear potential for the clinical development of a novel therapy for life-threatening respiratory infections caused by this opportunistic pathogen.
Assuntos
Pseudomonas aeruginosa , Fatores de Virulência , Animais , Camundongos , Cinética , Modelos Animais , Elastase PancreáticaRESUMO
Why is P. aeruginosa LasB elastase an attractive target for antivirulence therapy and what is the state-of-the art in LasB inhibitor design and development?
Assuntos
Antibacterianos/uso terapêutico , Proteínas de Bactérias/antagonistas & inibidores , Metaloendopeptidases/antagonistas & inibidores , Elastase Pancreática/antagonistas & inibidores , Infecções por Pseudomonas/tratamento farmacológico , Animais , Proteínas de Bactérias/metabolismo , Desenvolvimento de Medicamentos , Humanos , Metaloendopeptidases/metabolismo , Elastase Pancreática/metabolismo , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/patogenicidade , VirulênciaRESUMO
The cytotoxic macrolide kendomycin was identified as a ligand of Bcl-xl, an anti-apoptotic member of the Bcl-2 protein family. Hydrolysis-stable and protonable semi-synthetic analogues have been obtained that retain cytotoxicity and Bcl-xl binding.
Assuntos
Apoptose/efeitos dos fármacos , Rifabutina/análogos & derivados , Proteína bcl-X/química , Linhagem Celular Tumoral , Humanos , Rifabutina/química , Rifabutina/farmacologiaRESUMO
Alternative sigma factors are key global regulators that coordinate bacterial responses to environmental changes necessary for adaptation and survival. In turn these sigma factors are controlled by regulators such as anti-sigma and anti-anti-sigma factors. In this report, using a cDNA-total RNA subtractive hybridisation strategy that we have developed previously, we identified increased transcription of a potential sigma factor regulatory gene, Rv1364c, in Mycobacterium bovis BCG upon phagocytosis by macrophages and this was confirmed by Northern blot analysis. Primer extension analysis revealed the use of alternative promotors, P1 and P2, and that the increased expression inside macrophages coincided with promoter switching from P2 to P1. Rv1364c (653 amino acids), originally annotated as RsbU, contains structural domains homologous to the PAS redox sensor, the protein phosphatases anti-anti-sigma factor RsbU/SpoIIE, the protein kinase anti-sigma factor RsbW/SpoIIAB and the anti-anti-sigma factor RsbV/SpoIIAA found in other bacteria. These findings have important implications for understanding coordination of the expression of sigma factors under intra-macrophage conditions. Other potentially differentially expressed genes, including genes for fatty acid metabolism, membrane transportors, heat shock proteins, potential sigma factors and energy metabolic pathways are also listed and their biological significance discussed.
Assuntos
Macrófagos/microbiologia , Infecções por Mycobacterium/microbiologia , Mycobacterium bovis/genética , Mycobacterium bovis/metabolismo , Fator sigma/genética , Sequência de Bases , Regulação Bacteriana da Expressão Gênica , Humanos , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Regiões Promotoras Genéticas/fisiologia , Transcrição Gênica/fisiologiaRESUMO
A ciprofloxacin-resistant, nalidixic acid-susceptible mutant of Staphylococcus aureus (F145) contained no mutations within gyrA, gyrB, grlA, and grlB or within norA or its promoter region. MICs and accumulation studies suggest the role of a novel multidrug efflux pump.
Assuntos
Anti-Infecciosos/farmacologia , Ciprofloxacina/farmacologia , Ácido Nalidíxico/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Testes de Sensibilidade Microbiana , Mutação , Staphylococcus aureus/genéticaRESUMO
Consecutive isolates of quinolone-resistant campylobacter isolated over a 5 year period (1990-1995) from the faeces of patients with enteritis in Plymouth, UK, were examined for the epidemiology of mutations in gyrA (n = 127). In addition, clinical isolates and poultry isolates from Germany, The Netherlands and other regions of the UK collected before 1995 were examined for mutations in the quinolone resistance-determining region of gyrA by single-stranded conformational polymorphism analysis and direct sequencing of a 270 bp fragment of PCR-generated DNA. The majority of isolates (173/208) carried a mutation at codon 86 in gyrA resulting in substitution of Ile for Thr; all of these were resistant to ciprofloxacin (MIC > or = 2 mg/L). One isolate of Campylobacter jejuni had a mutation at Asp-90, and another had a double mutation at Thr-86 and Pro-104. Only two resistant isolates showed no mutation in gyrA. A novel gyrA sequence was amplified from two Campylobacter lari and one C. jejuni, which exhibited a valine at codon 86. Only 8/192 isolates had changes in gyrB; all were shown to relate to silent mutations in gyrB and presumably reflect natural polymorphisms in the gene.
Assuntos
Anti-Infecciosos/farmacologia , Campylobacter/enzimologia , Campylobacter/genética , DNA Girase/genética , Farmacorresistência Bacteriana/genética , Genes Bacterianos/genética , Mutação , Animais , Anti-Infecciosos/uso terapêutico , Sequência de Bases , Campylobacter/efeitos dos fármacos , Fluoroquinolonas , Humanos , Testes de Sensibilidade Microbiana/estatística & dados numéricos , Dados de Sequência Molecular , Polimorfismo Conformacional de Fita Simples , Aves Domésticas/microbiologia , Homologia de Sequência do Ácido NucleicoRESUMO
Identifying genes that are differentially expressed by Mycobacterium bovis BCG after phagocytosis by macrophages will facilitate the understanding of the molecular mechanisms of host cell-intracellular pathogen interactions. To identify such genes a cDNA-total RNA subtractive hybridization strategy has been used that circumvents the problems both of limited availability of bacterial RNA from models of infection and the high rRNA backgrounds in total bacterial RNA. The subtraction products were used to screen a high-density gridded Mycobacterium tuberculosis genomic library. Sequence data were obtained from 19 differential clones, five of which contained overlapping sequences for the gene encoding mycocerosic acid synthase (mas). Mas is an enzyme involved in the synthesis of multi-methylated long-chain fatty acids that are part of phthiocerol dimycocerosate, a major component of the complex mycobacterial cell wall. Northern blotting and primer extension data confirmed up-regulation of mas in intracellular mycobacteria and also revealed a putative extended -10 promoter structure and a long untranslated upstream region 5' of the mas transcripts, containing predicted double-stranded structures. Furthermore, clones containing overlapping sequences for furB, groEL-2, rplE and fadD28 were identified and the up-regulation of these genes was confirmed by Northern blot analysis. The cDNA-RNA subtractive hybridization enrichment and high density gridded library screening, combined with selective extraction of bacterial mRNA represents a valuable approach to the identification of genes expressed during intra-macrophage residence for bacteria such as M. bovis BCG and the pathogenic mycobacterium, M. tuberculosis.
Assuntos
Aciltransferases/metabolismo , Regulação Bacteriana da Expressão Gênica , Macrófagos/microbiologia , Mycobacterium bovis/enzimologia , Mycobacterium bovis/crescimento & desenvolvimento , Hibridização de Ácido Nucleico/métodos , Aciltransferases/química , Aciltransferases/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Primers do DNA , DNA Complementar , Biblioteca Genômica , Humanos , Dados de Sequência Molecular , Mycobacterium bovis/genética , Mycobacterium tuberculosis/genética , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Mensageiro/metabolismo , Transcrição Gênica , Células Tumorais CultivadasRESUMO
A library of Mycobacterium tuberculosis insertional mutants was generated with the transposon Tn5370. The junction sequence between the transposon and the mycobacterial chromosome was determined, revealing the positions of 1329 unique insertions, 1189 of which were located in 351 different ORFs. Transposition was not completely random and examination of the most susceptible genome regions revealed a lower-than-average G+C content ranging from 54 to 62 mol%. Mutants were obtained in all of the recognized M. tuberculosis functional protein-coding gene classes. About 30% of the disrupted ORFs had matches elsewhere in the genome that suggested redundancy of function. The effect of gene disruption on the virulence of a selected set of defined mutants was investigated in a severe combined immune deficiency (SCID) mouse model. A range of phenotypes was observed in these mutants, the most notable being the severe attenuation in virulence of a strain disrupted in the Rv1290c gene, which encodes a protein of unknown function. The library described in this study provides a resource of defined mutant strains for use in functional analyses aimed at investigating the role of particular M. tuberculosis genes in virulence and defining their potential as targets for new anti-mycobacterial drugs or as candidates for deletion in a rationally attenuated live vaccine.