Study Of Catecholamines Regulation On Actinobacillus Pleuropneumoniae Growth

Actinobacillus pleuropneumoniae, a member of Pasteurella Branch, Actinobacillus, is the etiologic agent of porcine contagious pleuropneumonia (PCP) that causes great economic losses worldwide. Stress is one of the most important factor caused PCP.Recently, reports show that bacteria can use mammalian hormones released under stress as an environmental signal to modulate pathogenic processes that play essential roles in disease development. Our previous work found out that catecholamine can regulate gene expression of APP, including the important virulence, metabolism, and regulation genes which confirmed that APP can sense catecholamine. In different expressed genes influenced by stress hormones, there are many iron metabolism-related genes. In this research, we investigated the effect of catecholamine on APP’s growth when iron is deficient. Through the research on iron metabolism mutants of APP and the microarray of APP, we studied the regulation of catecholamines on Actinobacillus pleuropneumoniae growth. The main results are as follows:1. The effect of catecholamines on the growth of APP under iron deficient conditionsThe growth of APP was inhibited in CDM after adding iron chelating agent bipyridine. After adding serum, the growth of APP was also inhibited. The inhibition was only relived by adding Fe3+ while adding Mg2+, Ca2+ or Fe3+. Meanwhile, the effect of non-iron ATF was as the same as serum but iron saturated HTF was not. These phenomena showed that serum or ATF caused an iron deficient environment. The growth curves of APP were detected under these conditions and we found that Epi, NE and DA could promote the growth of APP. Further testing investigated that mammalian hormone receptor inhibitors didn’t inhibit the growth of APP promoted by hormones. The growth of the mutants ΔqseB, ΔqseC and ΔqseBC, two-component system of APP, were not significantly different with parent strains. It revealed that hormones could regulate the growth of APP, not needing the similar receptors of mammalian and known hormones of bacteria. The growth of mutants ΔtonBl, Δfur and ΔtonB2, associated with iron metabolism were decreasing under iron deficient environment. However, the growth of these mutants strain could be increased after adding hormones in serum medium. The growth of ΔtonBl, Δfur were promoted by hormones in ATF medium while ΔtonB2 was no longer changing. It showed that tonB2 might play an important role in the growth of APP promoted by hormones.2. Regulation of catecholamine on gene expression of APP under iron deficient environmentIn order to explore the molecular mechanism of APP growth with catecholamine, microarray was used to identify the differentially expressed genes of APP under CDM, CDM+S (serum), CDM+S+Epi, CDM+S+NE, CDM+S+DA respectively. CDM+S compared to CDM and CDM+S+Epi/NE/DA compared to CDM+S, there were 363 genes expressed differentially by serum regulation, including 172 up-regulated genes and 191 down-regulated genes. There were 255 genes expressed differentially by Epi regulation, including 139 up-regulated genes and 116 down-regulated genes.723 genes, regulated by NE, were expressed differentially, including 336 up-regulated genes and 387 down-regulated genes. And there were 716 genes expressed differentially by DA regulation, including 339 up-regulated genes and 377 down-regulated genes. According to functions of these genes, they were divided into 21 categories, including energy metabolism, material transport, signal transduction, transcription and translation.21 iron metabolism-related genes were regulated by serum, Epi, NE and DA,20 of these genes were up-regulated in serum medium while they were down-regulated significantly after adding Epi, NE or DA. There were two important iron uptake systems of APP in these different expressed proteins, TonB1-ExbB-ExbD and TonB2-ExbB2-ExbD2. In addition, chelated iron transport protein YfeD, the hemoglobin transport protein HgBa, transferrin transport protein TbpA1-TbpB2 were even belong to these differentially expressed gene. It showed that these three hormones could affect APP iron metabolism to regulate its growth through these complex iron uptake system. It might also be that the single mutant of iron uptake-related gene could not make hormones to promote APP growth.