J Vet Sci.  2014 Mar;15(1):81-89. 10.4142/jvs.2014.15.1.81.

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

Affiliations
  • 1Graduate Institute of Veterinary Pathobiology, College of Veterinary Medicine, National Chung Hsing University, Taichung 402, Taiwan, R.O.C. hsuan@nchu.edu.tw
  • 2Graduate Institute of Microbiology and Public Health, College of Veterinary Medicine, National Chung Hsing University, Taichung 402, Taiwan, R.O.C.
  • 3Division of Animal Medicine, Animal Technology Institute Taiwan, Miaoli, 350, Taiwan, R.O.C.

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.

Keyword

Actinobacillus pleuropneumoniae; ApxI; attenuation; cytotoxicity; mutant

MeSH Terms

Actinobacillus pleuropneumoniae/genetics/*pathogenicity/*physiology
Animals
*Apoptosis
Bacterial Proteins/genetics/metabolism
Blotting, Southern
Exotoxins/*genetics
Hemolysin Proteins/genetics/metabolism
*Hemolysis
Macrophages, Alveolar/metabolism/*microbiology
Polymerase Chain Reaction
Sequence Analysis, DNA
Swine
Virulence
Bacterial Proteins
Exotoxins
Hemolysin Proteins
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