Drastic changes in the tissue-specific expression of secreted phospholipases A2 in chicken pulmonary disease

Abstract

Infectious bronchitis is one of the most important diseases in poultry and it causes major economic losses. Infectious bronchitis is an acute, highly contagious, viral disease of chickens, characterized by rales, coughing, and sneezing. Because secreted phospholipases A2 (sPLA2) are involved in inflammatory processes, the gene expressions of sPLA2s were investigated in both healthy chickens and chickens with infectious bronchitis and lung inflammation. The draft chicken genome was first scanned using human sPLA2 sequences to identify chicken sPLA2s (ChPLA2), chicken total mRNA were isolated and RT-PCR experiments were performed to amplify and then sequence orthologous cDNAs. Full-length cDNA sequences of ChPLA2-IB, -IIA, -IIE, -V and -X were cloned. The high degree of sequence identity of 50-70% between the avian and mammalian (human and mouse) sPLA2 orthologs suggests a conservation of important enzymatic functions for these phospholipases. Quantitation by qPCR of the transcript levels of ChPLA2-IB, -IIA, -IIE, -V and -X in several tissues from healthy chicken indicated that the expression patterns and mRNA levels diverged among the phospholipases tested. In chicken with infectious bronchitis, an over expression of ChPLA2-V was observed in lungs and spleen in comparison with healthy chicken. These findings suggest that ChPLA2-V could be a potential biomarker for lung inflammation. Conversely, a down regulation of ChPLA2-IB, -IIA and -X was observed in lungs and spleen in case of infectious bronchitis. A significant increase in the expression level of ChPLA2-X and ChPLA2-IB was also noticed in pancreas. No or minor changes have been detected in the expression of ChPLA2-IIE in lungs and small intestine, but it shows a significant increase in several infected tissues.

Graphical abstract

Fig. 1

Nucleotide sequences of the cDNA of ChPLA2s and deduced amino acid sequences. Sequencing was performed in triplicate with three independent PCRs. A: ChPLA2-IB, B: ChPLA2-IIA, C: ChPLA2-IIE, D: ChPLA2-V and E: ChPLA2-X.

Fig. 1

Nucleotide sequences of the cDNA of ChPLA2s and deduced amino acid sequences. Sequencing was performed in triplicate with three independent PCRs. A: ChPLA2-IB, B: ChPLA2-IIA, C: ChPLA2-IIE, D: ChPLA2-V and E: ChPLA2-X.

Fig. 2

Comparison of the conserved chicken and human phospholipase A2 core modules. The GenBank accession numbers for the chicken, mouse and human sequences are provided in Table 1. The identities between full-length PLA2 sequences of the human and chicken proteins are for sPLA2-IB, 63%; sPLA2-IIA, 45%; sPLA2-IIE, 42%; sPLA2-V, 54% and sPLA2-X, 45%. Overall, the sequences show little conservation. The active-site motifs, however, are well conserved. The phospholipase consensus motif with the catalytic HD catalytic diad, CCXXHDX, is fully conserved. In addition, the calcium loop consensus sequences YGCXCGXGG is conserved in several sPLA2 sequence. We note also a conserved arrangement of Disulfides Bridge. Identical amino acids in all 5 proteins are marked with an asterisk (∗), conservative substitutions with a colon (:), and semi-conservative substitutions with a period (.).

Fig. 3

Comparison of mRNA expression profiles of PLA2s in normal and infection bronchitis tissues. a: ChPLA2-IB, b: ChPLA2-IIA, c: ChPLA2-IIE, d: ChPLA2-V and e: ChPLA2-X. Gene-specific primers were used to determine the expression profiles of the avian PLA2. Each chicken gene was analyzed twice independently using the Light Cycler 480 system. Error bars represent the observed standard deviation. The relative expression values were scaled to a range of 0–100. The values shown were calculated using β-actin as the housekeeping gene. Results are expressed as the mean +/− SEM percentage of the control value (n _ 3).∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.