title: Differential gene expression profiling of Actinobacillus pleuropneumoniae during induction of primary alveolar macrophage apoptosis in piglets.
abstract: Actinobacillus pleuropneumoniae (A. pleuropneumoniae) is the causative agent of porcine pleuropneumonia, a disease that causes serious problems for the swine industry. Successful infection by this bacterium requires breaking the first line of defence in the lungs, the primary alveolar macrophages (PAMs). Therefore, exploring A. pleuropneumoniae-PAM interactions will provide vital groundwork for the scientific control of this infectious disease, which has been little studied up to now. In this work, PAMs were isolated from piglets and co-incubated with A. pleuropneumoniae serovar 5b strain L20 in vitro, and their interaction, PAM cell death, and differential gene expression of A. pleuropneumoniae in response to PAM cell death were observed and analysed using confocal microscopy, electron microscopy, RT-PCR, Western blot, flow cytometry and the use of a gene expression profile chip. A. pleuropneumoniae quickly adhered to and invaded PAMs, inducing apoptosis, which was confirmed using transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The highest percentage of apoptosis in cells was confirmed using flow cytometry when the cells were infected at a multiplicity of infection (MOI) of 10 and incubated for 5 h, with higher expression of activated caspase-3 as measured by Western blot. Using microarray gene chips with 2868 probes containing nearly all of the genomic sequence of A. pleuropneumoniae serotype 5b strain L20, a total of 185 bacterial genes were found to be differentially expressed (including 92 up-regulated and 93 down-regulated genes) and involved in the process of apoptosis, as compared with the expression of control bacteria cultured without PAMs in BHI medium (mean expression ratios >1.5-fold, p < 0.05). The up-regulated genes are involved in energy metabolism, gene transcription and translation, virulence related gene such as LPS, Trimeric Autotransporter Adhesin, RTX and similar genes. The down-regulated genes are involved in amino acid, cofactor, and vitamin metabolism, and also include ABC transporters. These data demonstrate that A. pleuropneumoniae induces apoptosis of PAMs and undergoes complex changes in gene transcription, including expression changes in known and potential virulence factors. Some potentially novel virulence targets have been identified, suggesting new strategies for the development of vaccines and medicines for both preventive and clinical use.
authors.affiliation: College of Veterinary Medicine, Jilin University, Changchun, PR China.
authors.forename: Lei
authors.initials: L
authors.lastname: Wang
authors.affiliation: College of Veterinary Medicine, Jilin University, Changchun, PR China.
authors.forename: Wanhai
authors.initials: W
authors.lastname: Qin
authors.affiliation: College of Veterinary Medicine, Jilin University, Changchun, PR China.
authors.forename: Zhai
authors.initials: Z
authors.lastname: Ruidong
authors.affiliation: College of Veterinary Medicine, Jilin University, Changchun, PR China.
authors.forename: Shiting
authors.initials: S
authors.lastname: Liu
authors.affiliation: College of Veterinary Medicine, Jilin University, Changchun, PR China.
authors.forename: Hu
authors.initials: H
authors.lastname: Zhang
authors.affiliation: College of Veterinary Medicine, Jilin University, Changchun, PR China.
authors.forename: Changjiang
authors.initials: C
authors.lastname: Sun
authors.affiliation: College of Veterinary Medicine, Jilin University, Changchun, PR China.
authors.forename: Xin
authors.initials: X
authors.lastname: Feng
authors.affiliation: College of Veterinary Medicine, Jilin University, Changchun, PR China.
authors.forename: Jingmin
authors.initials: J
authors.lastname: Gu
authors.affiliation: College of Veterinary Medicine, Jilin University, Changchun, PR China.
authors.forename: Chongtao
authors.initials: C
authors.lastname: Du
authors.affiliation: College of Veterinary Medicine, Jilin University, Changchun, PR China.
authors.forename: Wenyu
authors.initials: W
authors.lastname: Han
authors.affiliation: Section of Paediatrics, Imperial College London, London, UK.
authors.forename: P R
authors.initials: PR
authors.lastname: Langford
authors.affiliation: College of Veterinary Medicine, Jilin University, Changchun, PR China. Electronic address: leilc@jlu.edu.cn.
authors.forename: Liancheng
authors.initials: L
authors.lastname: Lei
mesh_terms: D000189:Actinobacillus Infections / Q000382:microbiology / Q000503:physiopathology / Q000662:veterinary*; D016977:Actinobacillus pleuropneumoniae / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D017209:Apoptosis; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial; D016676:Macrophages, Alveolar / Q000166:cytology* / Q000382:microbiology; D013552:Swine; D013553:Swine Diseases / Q000382:microbiology* / Q000503:physiopathology
chemical_list: D001426:Bacterial Proteins
keywords: Actinobacillus pleuropneumoniae; Apoptosis; Gene expression; Primary alveolar macrophages