Actinobacillus pleuropneumoniae, the etiological agent of porcine pleuropneumonia, is able to persist on respiratory epithelia, in tonsils, and in the anaerobic environment of encapsulated lung sequesters. We have demonstrated previously that putative HlyX-regulated genes, coding for dimethyl sulfoxide (DMSO) reductase and aspartate ammonia lyase, are upregulated during infection and that deletions in these genes result in attenuation of the organism. The study presented here investigates the role of HlyX, the fumarate nitrate reductase regulator (FNR) homologue of A. pleuropneumoniae. By constructing an isogenic A. pleuropneumoniae hlyX mutant, the HlyX protein is shown to be responsible for upregulated expression of both DMSO reductase and aspartate ammonia lyase (AspA) under anaerobic conditions. In a challenge experiment the A. pleuropneumoniae hlyX mutant is shown to be highly attenuated, unable to persist in healthy lung epithelium and tonsils, and impaired in survival inside sequestered lung tissue. Further, using an A. pleuropneumoniae strain carrying the luxAB genes as transcriptional fusion to aspA on the chromosome, the airway antioxidant glutathione was identified as one factor potentially responsible for inducing HlyX-dependent gene expression of A. pleuropneumoniae in epithelial lining fluid.Actinobacillus pleuropneumoniae, the etiological agent of porcine pleuropneumonia (10), is able to persist in host tissues, such as tonsillar crypts and sequestered necrotic lung, where the oxygen supply is scarce. The resulting carrier animals are the major source of infection for previously A. pleuropneumoniae-free herds (10) and, therefore, unraveling the mechanisms of persistence is highly relevant to effective vaccination and control of the infection.In Escherichia coli a number of genes expressed under anaerobic conditions are regulated by the global regulator FNR (for fumarate nitrate reductase regulator) (24). An A. pleuropneumoniae FNR homologue, HlyX, has been found to induce hemolytic activity in E. coli under anoxic conditions and to be able to complement E. coli fnr deletions (18, 26). Like FNR, HlyX contains four iron-sulfur clusters responsible for the DNA-binding ability of the protein (12).In A. pleuropneumoniae, anaerobically regulated genes appear to play a role in virulence and persistence. We have shown previously that A. pleuropneumoniae genes upregulated under anaerobic conditions in culture are not only upregulated in sequestered necrotic lung tissue where anoxic conditions are to be expected (1) but also upon the supplementation of culture medium with bronchoalveolar lavage fluid from A. pleuropneumoniae-infected pigs, which mimics conditions as they occur on respiratory epithelium (1-3, 15). Further, we have shown that isogenic mutants lacking aspartate ammonia lyase (aspartase) activity or both dimethyl sulfoxide (DMSO) reductase and aspartase activity were reduced in virulence and in their ability to persist on unaltered respiratory epithelium (2, 15).Since both the DMSO reductase (d...