RESUMEN
The heterogeneous environment of the lung of the cystic fibrosis (CF) patient gives rise to Pseudomonas aeruginosa small colony variants (SCVs) with increased antibiotic resistance, autoaggregative growth behavior, and an enhanced ability to form biofilms. In this study, oligonucleotide DNA microarrays were used to perform a genome-wide expression study of autoaggregative and highly adherent P. aeruginosa SCV 20265 isolated from a CF patient's lung in comparison with its clonal wild type and a revertant generated in vitro from the SCV population. Most strikingly, SCV 20265 showed a pronounced upregulation of the type III protein secretion system (TTSS) and the respective effector proteins. This differential expression was shown to be biologically meaningful, as SCV 20265 and other hyperpiliated and autoaggregative SCVs with increased TTSS expression were significantly more cytotoxic for macrophages in vitro and were more virulent in a mouse model of respiratory tract infection than the wild type. The observed cytotoxicity and virulence of SCV 20265 required exsA, an important transcriptional activator of the TTSS. Thus, the prevailing assumption that P. aeruginosa is subject to selection towards reduced cytotoxicity and attenuated virulence during chronic CF lung infection might not apply to all clonal variants.
Asunto(s)
Adhesión Bacteriana , Proteínas Bacterianas/metabolismo , Fibrosis Quística/microbiología , Regulación Bacteriana de la Expresión Génica , Pulmón/microbiología , Proteoma , Pseudomonas aeruginosa/patogenicidad , Animales , Proteínas Bacterianas/genética , Biopelículas , Línea Celular , Genoma Bacteriano , Humanos , Macrófagos/microbiología , Ratones , Ratones Endogámicos BALB C , Análisis de Secuencia por Matrices de Oligonucleótidos , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/clasificación , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/fisiología , Transcripción GenéticaRESUMEN
Gene expression patterns associated with resistance and susceptibility to tuberculosis (TB) were investigated at the macrophage level in the well-defined mouse model of infection. Oligonucleotide microarrays were used to analyse the regulation of gene expression in murine bone marrow-derived macrophages infected with Mycobacterium tuberculosis. Four mouse strains, known to differ in terms of growth permissiveness for M. tuberculosis in infected tissues, in the development of pulmonary pathology, and in the rate of premature death due to tuberculosis, were compared: C57BL/6 and BALB/c representing resistant, DBA/2 and CBA/J representing susceptible mouse strains. Genes (55) were regulated more than two-fold in macrophages of all strains investigated following M. tuberculosis infection. Importantly, 18 genes were commonly regulated only in macrophages of the two resistant strains upon infection, and 102 genes were commonly regulated exclusively in macrophages of the two susceptible strains. Using this approach, we have therefore identified more than 100 genes potentially associated with resistance and susceptibility, respectively, to TB at the macrophage level. A tentative interpretation of our microarray data suggests that macrophages from susceptible mice predominantly stimulate the recruitment of cells that contribute disproportionately to tissue damage rather than to microbial elimination. In conclusion, microarray gene chips are useful tools for generating new hypotheses about resistance and susceptibility to TB, and the mouse model can now be used to subject candidate genes identified by this approach to further functional analyses.
Asunto(s)
Predisposición Genética a la Enfermedad , Macrófagos/microbiología , Tuberculosis/genética , Animales , Células Cultivadas , Quimiocina CXCL5 , Quimiocinas CXC/biosíntesis , Modelos Animales de Enfermedad , Femenino , Regulación Bacteriana de la Expresión Génica , Interleucina-10/biosíntesis , Activación de Macrófagos , Macrófagos/inmunología , Ratones , Ratones Endogámicos , Análisis de Secuencia por Matrices de Oligonucleótidos , Especificidad de la Especie , Tuberculosis/inmunología , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
Pathogenesis of Pseudomonas aeruginosa is controlled to a major extent by the two quorum-sensing systems las and rhl. The previously uncharacterized gene PA2591 was identified as a major virulence regulator, vqsR, in the quorum-sensing hierarchy. vqsR is a member of the LuxR family and possesses a las box in its upstream region. Transposon inactivation of vqsR abrogated the production of N-acylhomoserine lactones and the secretion of exoproducts and diminished bacterial virulence for Caenorhabditis elegans. Cytotoxicity towards macrophages was not affected. vqsR mRNA was expressed more strongly in the presence of human serum and oxidative stress than under standard growth conditions. High-density oligonucleotide microarrays were used to compare the global expression profile of a wild-type strain and a vqsR mutant. One-hundred-and-fifty-one and 113 genes were significantly differentially expressed in the presence of H(2)O(2) and human serum, respectively. The disruption of vqsR repressed the expression of genes that are known to be promoted by quorum sensing and activated the expression of genes that are known to be repressed by quorum sensing. Moreover, the vqsR mutant harboured less mRNA transcript for the production of siderophores and membrane-bound elements of antibiotic resistance. The protein encoded by PA2591 regulates several traits of pathogenicity; hence, the name vqsR ('virulence and quorum-sensing regulator') was assigned to PA2591.
Asunto(s)
Proteínas Bacterianas/metabolismo , Caenorhabditis elegans/microbiología , Regulación Bacteriana de la Expresión Génica , Pseudomonas aeruginosa/crecimiento & desarrollo , Pseudomonas aeruginosa/patogenicidad , Transactivadores/metabolismo , 4-Butirolactona/análogos & derivados , 4-Butirolactona/metabolismo , Animales , Proteínas Bacterianas/genética , Humanos , Monocitos , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteoma , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Transducción de Señal , Transactivadores/genética , Transcripción Genética , VirulenciaRESUMEN
Regulatory T cells (Tregs) fulfill a central role in immune regulation. We reported previously that the integrin alphaEbeta7 discriminates distinct subsets of murine CD4+ regulatory T cells. Use of this marker has now helped to unravel a fundamental dichotomy among regulatory T cells. alphaE-CD25+ cells expressed L-selectin and CCR7, enabling recirculation through lymphoid tissues. In contrast, alphaE -positive subsets (CD25+ and CD25-) displayed an effector/memory phenotype expressing high levels of E/P-selectin-binding ligands, multiple adhesion molecules as well as receptors for inflammatory chemokines, allowing efficient migration into inflamed sites. Accordingly, alphaE -expressing cells were found to be the most potent suppressors of inflammatory processes in disease models such as antigen-induced arthritis.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Memoria Inmunológica/inmunología , Animales , Artritis Experimental/inmunología , Antígenos CD4/inmunología , Inmunofenotipificación , Inflamación/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Análisis de Secuencia por Matrices de Oligonucleótidos , Receptores de Interleucina-2/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Subgrupos de Linfocitos T/inmunologíaRESUMEN
PURPOSE: During the last decade numerous different reverse transcriptase-polymerase chain reaction (RT-PCR) techniques have been described. However, the lack of highly sensitive, quantitative and reliable methodology has been responsible for its limited use in modern urology. Early semiquantitative RT-PCR techniques often proved not to produce consistent results and have a high failure rate due to complicated working models. In this article we provide a comprehensive and intelligible description of real-time PCR technology, which is a novel quantitative methodology to analyze gene expression. In addition, we report the first preclinical and clinical applications in molecular urology. MATERIALS AND METHODS: The current literature was reviewed in regard to different current real-time RT-PCR protocols and their use in modern urological oncology. RESULTS: Real-time RT-PCR is a reliable, rapid and relatively inexpensive technique that can be easily adapted for standardized preclinical and clinical applications at different centers. Its sensitivity equals at least that of conventional RT-PCR and the option of exact quantification of gene expressions allows proper differentiation among high, low and illegitimate RNA transcription. It eliminates post-PCR processing of PCR products, thereby, increasing throughput and decreasing the chance of carryover contamination. CONCLUSIONS: Although the application of real-time RT-PCR has gained wide acceptance in urological research, its routine clinical use is still in its infancy. However, due to its high sensitivity and exact quantitation real-time RT-PCR may be the method of choice for modern preclinical and clinical studies in the future.
Asunto(s)
Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Neoplasias Urológicas/genética , Humanos , Factores de TiempoRESUMEN
Enterohemorrhagic Escherichia coli (EHEC) is the major cause of hemolyticuremic syndrome (HUS) characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure. EHEC produces one or more Shiga toxins (Stx1 and Stx2), and it was assumed that Stx's only relevant biologic activity was cell destruction through inhibition of protein synthesis. However, recent data indicate that in vivo the cytokine milieu may determine whether endothelial cells survive or undergo apoptosis/necrosis when exposed to Stxs. In this study, we analyzed the genome-wide expression patterns of human endothelial cells stimulated with subinhibitory concentrations of Stxs in order to characterize the genomic expression program involved in the vascular pathology of HUS. We found that Stxs elicited few, but reproducible, changes in gene expression. The majority of genes reported in this study encodes for chemokines and cytokines, which might contribute to the multifaceted inflammatory response of host endothelial cells observed in patients suffering from EHEC disease. In addition, our data provide for the first time molecular insights into the epidemiologically well-established higher pathogenicity of Stx2 over Stx1.