Analysis Of Signaling Pathway Of Stress Hormones Sensing By Two-components System In Actinobacillus Pleuropneumoniae

Actinobacillus pleuropneumonia (APP) is a Gram-nagetive pathogen which can cause porcine contagious pleuropneumonia (PCP). APP can be spread with high virulence, strong infectivity and a wide range. It has become a major threat to pig industry in China and even throughout the world which causes dramatic economic loss every year. Stress has been one of the most important factors causing APP infection.It is reported in E.coli and Salmonella that bacterial two-component systems (TCSs) such as QseCB, QseEF may be responsible for host stress hormones sensing. Especially, there is direct evidence showing that stress hormone such as epinephrine (Epi) and norepinephrine (NE) could bind to QseCB in Enterohemorrhagic Escherichia coli (EHEC). TCSs widely exist in bacterial, archaea, fungi and some plant species which are a very common and significant regulatory system responding to changes in extracellular environment. Host stress hormones sensed by the pathogen two-component systems could enhance its virulence so as to make the pathogen more infective. There involves a pair of two-component systems ygiYX in APP genome which is homologous to qseCB in E.coli. But it remains to be examined whether ygiYX possesses similar function of sensing host stress hormones with qseCB.Furthermore, there are more than 30 pairs of two-component systems in E.coli while only five are found in APP genome. Recent research shows cross regulations between different two-component systems in which different two-component systems could sense and respond to same signals. Yet, this phenomenon so far has not been verified in APP.Therefore, my study is attempting to find sensors in APP for host hormones based on two-component systems. Meanwhile, it is demonstrated in my study through cross phosphorylation and bacterial two-hybrid system that cross interactions exist between different histidine kinases and response regulators in APP.1. Study of stress hormones sensing by histidine kinase of APP.There involves five pairs of two-component systems in APP among which histidine kinase YgiY shares high identity with QseC in E.coli. In my study, YgiY protein was successfully purified in native conditions. Using γ-32P labeled ATP, the phosphorylation level was measured after YgiY was exposed to epinephrine, norepinephrine and 5-hydroxytryptamine to demonstrate whether they could be sensed by YgiY. The results showed that the phosphorylation level was enhanced when YgiY was exposed to epinephrine and norepinephrine while no significant change was observed when exposed to 5-hydroxytryptamine. The results showed that YgiY could be a possible sensor for stress hormones such as epinephrine and norepinephrine in APP.2. Cross phosphorylation between two-components systems of APP.The signal transduction between two-components system is accomplished through auto-phosphorylation of histidine kinase and the transfering of phosphate from histidine kinase to response regulator. As the signal molecues sensed by two-components systems of APP all remain unkown, we purified cytoplasmic region of histidine kinases YgiY-C, ArcB-C, PhoR-C, CpxA-C, which are truncated histidine kinases containing all catalytic domains. And all five full-length response regulators YgiX, ArcA, PhoB, CpxR, NarP were purified. Using γ-32P labeled ATP, auto-phosphorylation of truncated histidine kinases and phosphorylation between all histidine kinases and response regulators were measured. It was demonstrated in the results that phosphorylation not only occurred between cognate two-components systems such as YgiY-YgiX, ArcB-ArcA, PhoR-PhoB, CpxA-CpxR but between non-cognate two-components systems such as YgiY-PhoB, YgiY-NarP, ArcB-YgiX, ArcB-PhoB, ArcB-CpxR, ArcB-NarP, PhoR-ArcA, CpxA-PhoB. The results showed that the phenomenon of cross-phosphorylation between two-components systems exist in APP.3. Protein-protein interactions between two-components systems of APP.Protein-protein interactions between histidine kinases and response regulators were investigated by bacterial two-hybird system. Complicated interactions were observed among two-components system proteins of APP.Through the above study, we identified a epinephrine and norepinephrine sensor YgiY and characterized a regulatory network among two-components systems. We have preliminary elucidated stress hormones sensing and cross-signaling pathway by two-components systems of APP which provides a theoretical basis for the enhanced susceptibility to APP when hosts are under stress.