Analysis of theActinobacillus pleuropneumoniaeHlyX (FNR) regulon and identification of iron‐regulated protein B as an essential virulence factor

Abstract

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.

A computational strategy for the search of regulatory small RNAs in Actinobacillus pleuropneumoniae

“…We used aerobiosis as it is the typical atmosphere used in experiments involving A. pleuropneumoniae and anaerobiosis as, based on mutant Jacobsen et al 2005;Buettner et al 2009) or transcriptome (Deslandes et al 2010;Klitgaard et al 2012) studies, it is representative of the growth conditions found in necrotic lungs of pigs. As shown in Figure 4 and Table 1, some trans-acting sRNA candidates, like Arrc05 and Arrc08, are located between genes within operons, which could allow them to be mistaken for subproducts of polycistronic mRNA maturation.…”

Glycoprotein analysis of porcine bronchoalveolar lavage fluid reveals potential biomarkers corresponding to resistance toActinobacillus pleuropneumoniaeinfection

“…After evaluation of the glycoproteome expression patterns, prominent protein spots were excised from preparative gels, trypsinized according to the method of Wilm and Mann [43] and identified by mass spectrometric methods as previously described [6]. Briefly, matrix-assisted laser desorption ionizationtime of flight (MALDI-TOF) MS was carried out by using a VoyagerDE Pro (Applied Biosystems, Foster City, CA, USA); 1 lL of the peptide solution was mixed with 1 lL of matrix (alpha-cyano-4-hydroxycinnamic acid (Bruker Daltonics, Billerica, MA, USA), 5 mg/mL, in 50% acetonitrile with 0.1% trifluoroacetic acid) and then spotted on the target plate.…”

Regulation of pga Operon Expression and Biofilm Formation in Actinobacillus pleuropneumoniae by σ E and H-NS

“…After the second DNase I treatment, the RNA was again purified, according to the RNeasy minikit cleanup protocol, and finally stored at Ϫ80°C. cDNA synthesis and labeling were done as previously described (12,13). Labeled samples were combined and hybridized to the A. pleuropneumoniae 5b strain L20 microarray AppChip2 (31).…”

“…Slides were scanned using a GenePix 4000B scanner (Axon Instruments) and analyzed with GenePix Pro6.0 to generate the data format compatible for further statistical analysis using GeneSpring GX7.3 (Agilent). Microarray analysis was carried out as described previously (12,13,34,45).…”