Growth phase mediated regulation of the Actinobacillus pleuropneumoniae ApxI and ApxII toxins

Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia, a highly contagious and often fatal respiratory tract disease of pigs. The Apx toxins are primary virulence factors of this pathogen, with ApxI and ApxII being produced by all highly virulent strains in North America. Further characterization of these hemolytic toxins is needed to fully understand their role in disease and elucidate the environmental signals and genes that affect their production during infection. Many bacteria regulate genes in response to growth phase, and in this report we examined the effect of growth phase on ApxI and ApxII gene expression. Batch cultures of ApxI- and ApxII-producing strains were grown in heart infusion broth supplemented with β-NAD, and samples were prepared throughout the growth curve. Maximal gene expression occurred in late exponential or early stationary phase, as indicated by a peak in apx mRNA concentration in Northern blot analysis. The amount of accumulated Apx protein and Apx hemolytic activity confirmed this increase in gene expression. These findings suggest a novel transcriptional regulatory mechanism that enhances Apx gene expression under in vitro conditions of high cell density and/or slow growth rate.