Analysis of the Actinobacillus pleuropneumoniae HlyX (FNR) regulon and identification of iron-regulated protein B as an essential virulence factor

• Published in: Proteomics (Weinheim) 2009-05, Vol.9 (9), p.2383-2398
• Main Authors: Buettner, Falk F.R, Bendalla, Ibrahim M, Bossé, Janine T, Meens, Jochen, Nash, John H.E, Härtig, Elisabeth, Langford, Paul R, Gerlach, Gerald-F
• Format: Article
• Language: English
• Subjects: Actinobacillus pleuropneumoniae; Actinobacillus pleuropneumoniae - genetics; Actinobacillus pleuropneumoniae - growth & development; Actinobacillus pleuropneumoniae - pathogenicity; Actinobacillus pleuropneumoniae - physiology; Analytical, structural and metabolic biochemistry; Animals; Bacteria; Bacterial Outer Membrane Proteins - genetics; Bacterial Outer Membrane Proteins - physiology; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Bacterial Proteins - physiology; Bacteriology; Biological and medical sciences; cDNA microarray; Computational Biology; Computer Simulation; DNA-Binding Proteins - genetics; DNA-Binding Proteins - physiology; Electrophoresis, Gel, Two-Dimensional; Electrospray ionization‐quadrupole time of flight tandem mass spectrometry; Fluorescence two‐dimensional difference gel electrophoresis; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation, Bacterial; Male; Matrix‐assisted laser desorption/ionization time of flight mass spectrometry; Microbiology; Miscellaneous; Oligonucleotide Array Sequence Analysis; Proteins; Regulon; Reverse Transcriptase Polymerase Chain Reaction; Sequence Deletion; Swine; Transcription Factors - genetics; Transcription Factors - physiology; Virulence Factors - genetics; Virulence Factors - physiology
• ISSN: 1615-9853; 1615-9861
• DOI: 10.1002/pmic.200800439

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 (greater-than-or-equal 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 ΔfrpB did not cause any disease and could not be re-isolated from experimentally infected pigs, thereby identifying FrpB as a previously unknown virulence factor.