Deletion of the anaerobic regulator HlyX causes reduced colonization and persistence of Actinobacillus pleuropneumoniae in the porcine respiratory tract

Abstract

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.

FIG. 1.

Map of the A. pleuropneumoniae ΔhlyX locus with primers and restriction sites used in the present study. Primer binding sites are indicated by arrowheads; restriction sites used are indicated by upward vertical lines.

FIG. 2.

Expression of the A. pleuropneumoniae DmsA protein as assessed by Western blot analysis. Lanes: 1, A. pleuropneumoniae AP76; 2, A. pleuropneumoniae ΔhlyX; 3, A. pleuropneumoniae ΔhlyX transformed with pHLYX1300; 4, A. pleuropneumoniae ΔhlyX transformed with pHLYX1301.

FIG. 3.

Virulence studies of A. pleuropneumoniae ΔhlyX in an aerosol infection model. Symbols: •, A. pleuropneumoniae AP76 wild-type strain (AP76); ▴, A. pleuropneumoniae ΔhlyX (ΔdmsA). The central symbol within the hourglass shape represents the geometric mean, the hinges present the values in the middle of each half of data, and the top and bottom symbols mark the maximum and minimum value. Asterisks denote statistical significance (P < 0.05) in the Wilcoxon signed-rank test. (A) Body temperatures of pigs over the course of 6 days, with day 0 marking the day of infection; (B) lung lesion scores assessed according to the method of Hannan et al. (14). Statistical significance (P < 0.05) in the Mann-Whitney Test is denoted by an asterisk.

FIG. 4.

Influence of GSH on HlyX-induced activation of aspA transcription in a luciferase assay using A. pleuropneumoniae ΔaspA::luxAB. Lanes: 1, NaCl control; 2, 1.2 μM GSH; 3, 12 μM GSH; 4, 120 μM GSH; 5, 600 μM GSH; 6, 1,200 μM GSH; 7, 12 mM GSH.