Transcriptional Responses Of Actinobacillus Pleuropneumoniae To LuxS/AI-2 And Catecholamines

The communication between bacteria is achieved by quorum sensing （QS）. QS is the phenomenon that bacteria can regulate their own gene expression according to cell densities through some autoinducers （AIs） within a bacterial community. In Gram-positive and -negative bacteria, different and species-specific signals （AIs） are used to regulate many bacterial behaviors. But the LuxS/AI-2 system is wide spread in both Gram-positive and -negative bacteria, playing multiple roles in virulence and metabolism of various bacterial species. In recent years, many studies revealed that the communication is not only limited in bacteria community, but also present between bacteria and host. During stress and disease, bacteria can actively respond to host hormones to regulate their growth ability and virulence traits. This communication plays critical roles in stress, disease and immunity. In this study, we investigated the genes and features regulated by LuxS/AI-2 and host hormones （catecholamines） in Actinobacillus pleuropneumoniae, an important swine respiratory pathogen.1. Transcriptional response of Actinobacillus pleuropneumoniae to LuxS/AI-2LuxS is an enzyme involved in the activated methyl cycle in methionine metabolism and the by-product autoinducer 2 （AI-2） was a quorum sensing signal in some species. LuxS/AI-2 plays multiple roles in virulence and metabolism of different bacteria. AI-2 was discovered to be present in the supernatant of A. pleuropneumoniae and luxS is an essential gene in the process of A. pleuropneumoniae infection. In our previous study, the functional LuxS in AI-2 production was identified in A.pleuropneumoniae. Enhanced biofilm formation and reduced virulence were observed in the luxS mutant. To comprehensively understand the LuxS/AI-2 function, in this study, the transcriptional profiles were compared between the A. pleuropneumoniae luxS mutant and its parental strain in four different growth phases using microarray.138,113,351 and 58 genes were found to be differentially expressed in early, middle, late exponenetial and stationary phase respectively. Real-time RT-PCR was used to validate the microarry results.Many genes associated with infection were differentially expressed, which agrees well with the previous observations that the luxS mutant showed reduced virulence. The biofilm formation genes pgaABC in the luxS mutant were up-regulated in early exponential phase, while 9 genes associated with adhesion were down-regulated in late exponential phase. A group of genes involved in iron acquisition and metabolism were regulated in four growth phases. Phenotypic investigations using luxS mutant and both genetic and chemical （AI-2） complementation on these virulence traits were performed. The results demonstrated that the luxS mutant showed enhanced biofilm formation and reduced adhesion ability and these effects were not due to lack of AI-2. But AI-2 could increase biofilm formation and adhesion of A. pleuropneumoniae independent of LuxS. Growth under iron restricted condition could be controlled by LuxS through AI-2 production. These results revealed pleiotropic roles of LuxS and AI-2 on A. pleuropneumoniae virulence traits.Besides the infection associated genes, a large group of genes belonging to carbohydrate metabolism and energy metabolism were differentially expressed. The related metabolic pathways were analyzed. The maximal change of carbohydrate metabolism happened in late exponential phase during which AI-2 activity delined rapidly. Hence, the regulation on carbohydrate metabolism could be resulted from AI-2 uptake or transport. Using medium added with different carbon sources, the growth and extracellular AI-2 level of A pleuropneumoniae were investigated. The results suggested AI-2 uptake or transport was affected by carbon source selection and availability. Genes encoding ribose transporter were identified to be differentially expressed in late exponential phase. Further investigations indicated that RbsB2 could bind extra-cellular AI-2 from both A. pleuropneumoniae andâ…¤. harveyi, suggesting the ribose transporter could be one of the AI-2 transporter in A. pleuropneumoniae. Many regulators were also differentially expressed in late exponential phase. These regulators could be important in LuxS/AI-2 regulation. On the other hand, several genes involved in methionie, cystein and sulfur metabolism were affected in luxS mutant, indicated that LuxS was also functional in the AMC in methione metabolism in A. pleuropneumoniae. But under the condition used in this study, the influence of LuxS mutation on these metabolism pathways was limited.2. Transcriptional response of Actinobacillus pleuropneumoniae to catecholaminesBacteria can use mammalian stress hormones to modulate their pathogenic processes, which plays essential role in disease development. Stresses were found to contribute to the outcome of A. pleuropneumoniae infection. To test whether A. pleuropneumoniae could respond to the stress hormone catecholamines, the gene expression profiles after epinephrine （Epi） or norepinephrine （NE） treatment were compared with the untreated bacteria using microarray. Real-time RT-PCR was used to validate the microarry results. 158 and 105 genes belonging to various function categories were differentially expressed by Epi and NE respectively. Few genes involved in central metabolism were affected, whereas many virulence or infection associated genes were differentially expressed. These results revealed that A pleuropneumoniae could respond to catecholamines.Only 18 common genes were regulated by the two hormones, these genes included Apxl structure gene apxIA, biofilm formation gene pgaB, APL₀443 encoding an autotransporter adhesin and potential hormone responsors tyrP2, ygiY-ygiX （qseC-qseB） and narQ-narP. The genes regulated by Epi and NE involved in infection and regulations were analyzed respectively. The different regulations of the two hormones on A. pleuropneumoniae suggested that this pathogen may have multiple responsive systems for different type of catecholamines.Further investigations demonstrated that cytotoxic activity was enhanced by Epi but repressed by NE in accordance with the regulations on apxIA. Bothα-adrenergic receptor antagonist phentolamine （PE） and theβ-adrenergic receptor antagonistpropranolol （PO） could block these effects. Biofilm formation was not affected by the two hormones despite that pgaB was affected. Adhesion to host cells was induced by NE but not by Epi when the two hormones were added into the bacterial medium. Both PE and PO could block the enhanced adhesion by NE.When the two hormones were added after bacteria-cell contact, both Epi and NE could increase the adhesion of A. pleuropneumoniae. PO but not PE could block the enhanced adhesion. The effects of Epi and NE on adhesion may involve other putative adhesins affected by the hormones in addition to APL₀443.