Elucidating the role of ApxI in hemolysis and cellular damage by using a novel apxIA mutant of Actinobacillus pleuropneumoniae serotype 10

Abstract

Exotoxins produced by Actinobacillus (A.) pleuropneumoniae (Apx) play major roles in the pathogenesis of pleuropneumonia in swine. This study investigated the role of ApxI in hemolysis and cellular damage using a novel apxIA mutant, ApxIA336, which was developed from the parental strain A. pleuropneumoniae serotype 10 that produces only ApxI in vitro. The genotype of ApxIA336 was confirmed by PCR, Southern blotting, and gene sequencing. Exotoxin preparation derived from ApxIA336 was analyzed for its bioactivity towards porcine erythrocytes and alveolar macrophages. Analysis results indicated that ApxIA336 contained a kanamycin- resistant cassette inserted immediately after 1005 bp of the apxIA gene. Phenotype analysis of ApxIA336 revealed no difference in the growth rate as compared to the parental strain. Meanwhile, ApxI production was abolished in the bacterial culture supernatant, i.e. exotoxin preparation. The inability of ApxIA336 to produce ApxI corresponded to the loss of hemolytic and cytotoxic bioactivity in exotoxin preparation, as demonstrated by hemolysis, lactate dehydrogenase release, mitochondrial activity, and apoptosis assays. Additionally, the virulence of ApxIA336 appeared to be attenuated by 15-fold in BALB/c mice. Collectively, ApxI, but not other components in the exotoxin preparation of A. pleuropneumoniae serotype 10, was responsible for the hemolytic and cytotoxic effects on porcine erythrocytes and alveolar macrophages.

Keywords: Actinobacillus pleuropneumoniae; ApxI; attenuation; cytotoxicity; mutant.

Fig. 1

Schematic diagram of the apxIA mutant construction. A kanamycin-resistant cassette (kan^R) was inserted into the apxIA gene. Primer locations (arrows) and restriction enzyme cleavage sites are indicated.

Fig. 2

PCR results and Southern blot analysis of the apxIA insertion mutant. Genomic DNA of parental (P) or ApxIA336 mutant (M), or de-ionized water (W) was amplified by PCR with primers specific for the apxIA (A) or apxIVA (B) gene. Genomic DNA was also digested with the restriction enzyme PvuI and subjected to Southern blot analysis using DIG-labeled probe specific to apxIA (C) or kanamycin-resistant (D) gene.

Fig. 3

Growth curve analysis and stability of the kanamycin-resistant gene in the ApxIA336 mutant. (A) Growth rate of ApxIA336 or the parental strain was monitored for 7 h and the number of colony forming units (CFUs) was determined. Data are from three independent experiments. Error bars represent the standard error (SE). (B) Presence of the kanamycin-resistant gene in ApxIA336 10 serial passages was detected by PCR. De-ionized water (W) served as a negative control. The arrow indicates the amplified fragment corresponding to the kanamycin-resistant cassette (633 bp). NS: no significant difference between the two strains.

Fig. 4

ApxIA336 failed to produce ApxI. Cultural supernatant from the parental (P) or ApxIA336 mutant (M) strain was subjected to SDS-PAGE (A) and Western blot analysis (B). Arrows indicate the band corresponding to ApxI at 105 kDa.

Fig. 5

ApxIA336 cultural supernatant failed to induce hemolysis or PAM damage. Porcine RBCs were treated with different concentrations of the cultural supernatants derived from ApxIA336 or parental strain in hemolytic assay (A). Effects of ApxIA336 or parental cultural supernatant on PAMs were evaluated in LDH release (B), mitochondrial activity (C), and apoptosis (D) assays. Results are from three independent experiments of at least duplicate determinations. Error bars represent the SE. ^*p < 0.05, ^**p < 0.01, and ^***p < 0.001.