RESUMO
The Gram-negative rod Actinobacillus pleuropneumoniae is a facultative anaerobic pathogen of the porcine respiratory tract, and HlyX, the A. pleuropneumoniae homologue of fumarate and nitrate reduction regulator (FNR), has been shown to be important for persistence. An A. pleuropneumoniae hlyX deletion mutant has a decreased generation time but highly prolonged survival in comparison to its wild type parent strain when grown anaerobically in glucose-supplemented medium. Applying a combination of proteomic and transcriptomic approaches as well as in silico analyses, we identified 23 different proteins and 418 genes to be modulated by HlyX (> or = twofold up- or down-regulated). A putative HlyX-box was identified upstream of 54 of these genes implying direct control by HlyX. Consistent with its role as a strong positive regulator, HlyX induced the expression of genes for anaerobic metabolism encoding alternative terminal reductases and hydrogenases. In addition, expression of virulence-associated genes encoding iron uptake systems, a putative DNA adenine modification system, and an autotransporter serine protease were induced by HlyX under anaerobic growth conditions. With respect to virulence-associated genes, we focused on the iron-regulated protein B (FrpB) as it is the outer membrane protein most strongly up-regulated by HlyX. An frpB deletion mutant of A. pleuropneumoniae had the same growth characteristics as wild type grown aerobically and anaerobically. In contrast, A. pleuropneumoniae DeltafrpB did not cause any disease and could not be re-isolated from experimentally infected pigs, thereby identifying FrpB as a previously unknown virulence factor.
Assuntos
Actinobacillus pleuropneumoniae/fisiologia , Proteínas da Membrana Bacteriana Externa/fisiologia , Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/genética , Regulon , Fatores de Transcrição/genética , Actinobacillus pleuropneumoniae/genética , Actinobacillus pleuropneumoniae/crescimento & desenvolvimento , Actinobacillus pleuropneumoniae/patogenicidade , Animais , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/fisiologia , Biologia Computacional , Simulação por Computador , Proteínas de Ligação a DNA/fisiologia , Eletroforese em Gel Bidimensional , Regulação Bacteriana da Expressão Gênica , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Deleção de Sequência , Suínos , Fatores de Transcrição/fisiologia , Fatores de Virulência/genética , Fatores de Virulência/fisiologiaRESUMO
A ferrichrome receptor, FhuA, was identified in Actinobacillus pleuropneumoniae serotype 7. An isogenic mutant with a deletion in the ferrichrome uptake receptor gene (fhuA) was constructed and examined in an aerosol infection model. The disease caused by the mutant was indistinguishable from disease induced by A. pleuropneumoniae serotype 7 wild-type; an isogenic mutant lacking expression of the exbB gene that is required for the uptake of transferrin-bound iron retained the ability to utilize ferrichrome, thereby indicating that an energy-coupling mechanism involved in ferrichrome transport remains to be identified.