RESUMEN

Polymyxin B-sensitive mutants in Burkholderia vietnamiensis (Burkholderia cepacia genomovar V) were generated with a mini-Tn5 encoding tetracycline resistance. One of the transposon mutants had an insertion in the norM gene encoding a multi-drug efflux protein. Expression of B. vietnamiensis norM in an Escherichia coli acrAB deletion mutant complemented its norfloxacin hypersensitivity, indicating that the protein functions in drug efflux. However, no effect on antibiotic sensitivity other than sensitivity to polymyxin B was observed in the B. vietnamiensis norM mutant. We demonstrate that increased polymyxin sensitivity in B. vietnamiensis was associated with the presence of tetracycline in the growth medium, a phenotype that was partially suppressed by expression of the norM gene.

Asunto(s)

Antiportadores/genética , Antiportadores/metabolismo , Burkholderia/efectos de los fármacos , Clonación Molecular , Farmacorresistencia Bacteriana/genética , Proteínas de Escherichia coli , Antibacterianos/metabolismo , Antibacterianos/farmacología , Antiinfecciosos/metabolismo , Antiinfecciosos/farmacología , Antiportadores/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Burkholderia/genética , Burkholderia/crecimiento & desarrollo , Burkholderia/metabolismo , Datos de Secuencia Molecular , Norfloxacino/metabolismo , Norfloxacino/farmacología , Polimixinas/metabolismo , Polimixinas/farmacología , Análisis de Secuencia de ADN , Tetraciclina/metabolismo , Tetraciclina/farmacología

RESUMEN

Members of the Burkholderia cepacia complex can secrete proteases, lipases, and hemolysins. We report in this study the identification of a general secretory pathway present in a B. vietnamiensis (formerly genomovar V) clinical isolate, which is required for the efficient secretion of phospholipase C and hemolysin activities. Southern blot hybridization experiments revealed that this general secretion pathway is highly conserved among the different genomovars of the B. cepacia complex and is homologous to a similar system described in B. pseudomallei. We also show that this pathway appears not to be necessary for intracellular survival of B. vietnamiensis within Acanthamoeba polyphaga.

Asunto(s)

Burkholderia cepacia/fisiología , Proteínas Hemolisinas/fisiología , Fosfolipasas de Tipo C/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Southern Blotting , Infecciones por Burkholderia/microbiología , Burkholderia cepacia/genética , Burkholderia cepacia/metabolismo , Elementos Transponibles de ADN/genética , ADN Bacteriano/genética , Proteínas Hemolisinas/metabolismo , Humanos , Datos de Secuencia Molecular , Mutación , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido

RESUMEN

In the present study the involvement of VLA-6 (α6ß1) integrin, a laminin receptor, was characterized during the course of mouse bone marrow-derived mast cell (BMMC) development. Flow cytometry and immunoprecipitation revealed increases in α6 integrin expression during the first 3 weeks, followed by a decline, such that α6ß1 was no longer detectable by week 13. Using RT-PCR, transcripts for α6A but not the α6B isoform were detected. Results from immunoprecipitation and costaining with ß1-or ß4-specific mAb showed the expression of VLA-6 (α6ß1) and not α6ß4 heterodimers. Moreover, the ability of BMMC to interact with laminin correlated with the time period of VLA-6 expression. However, only 40% of adhesion to laminin was inhibited by blocking mAb for α6, indicating the involvement of additional laminin receptor(s). This is supported by the immunoprecipitation of VLA-2 integrin, also known to have laminin binding properties. Heterogeneity of VLA-6 expression was also found in connective tissue-type mast cells; thus, VLA-6hi and VLA-6lo subpopulations of peritoneal mast cells were observed. The heterogeneity of VLA-6 integrin expression in BMMC and CTMC may be relevant to the concept of mast cell heterogeneity as well as to the ability of mast cell precursors to migrate and complete their course of maturation within tissues.