Analysis of differential protein expression in Actinobacillus pleuropneumoniae by Surface Enhanced Laser Desorption Ionisation--ProteinChip (SELDI) technology.
Actinobacillus pleuropneumoniae (APP) is the aetiological agent of porcine pleuropneumonia. An increased understanding of its molecular basis of pathogenicity and vaccine development will be facilitated by the availability of sequence data from a complete genome which, by analogy to other bacteria, is predicted to encode many proteins in the molecular mass range 3-20kDa. However, conventional techniques to study bacterial protein expression, such as SDS-PAGE and 2-dimensional electrophoresis, typically focus on the 15-200kDa range. In this study we have evaluated Surface Enhanced Laser Desorption Ionisation-ProteinChip (SELDI) technology for the analysis of protein expression, in particular those of <20kDa, of APP grown under different environmental conditions. Cytoplasmic/periplasmic and outer membrane protein fractions were obtained from the APP wildtype serotype 1 strain 4074 grown in Brain Heart Infusion (BHI) broth (+different concentrations of NAD), BHI containing pig serum or defined medium. Optimum conditions for SELDI profiles included a sample size of 1 microg and the use of sinapinic acid as the energy absorbing matrix. In the <20kDa range, the SELDI profiles obtained from wild-type bacteria grown in rich medium plus 33-66% pig serum were most similar to those grown in defined medium. The SELDI profiles of extracts of the wild-type and of an rpoE mutant were similar although there were clear differences. The results suggest that SELDI is a useful complementary approach to conventional proteomic analytical methods with APP, and presumably other bacterial pathogens, being particularly suited for analysis of proteins in the <20kDa mass range.
Hodgetts
Andrea
A
Department of Paediatrics, Faculty of Medicine, Imperial College, St Mary's Campus, London W2 1PG, UK.
Bossé
Janine T
JT
Kroll
J Simon
JS
Langford
Paul R
PR
D016977:Actinobacillus pleuropneumoniae / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D003373:Coumaric Acids / Q000737:chemistry; D008970:Molecular Weight; D040081:Protein Array Analysis / Q000379:methods; D020543:Proteome / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
D001426:Bacterial Proteins; D003373:Coumaric Acids; D020543:Proteome; C073734:sinapinic acid