Regulation Mechanism Of Edwardsiella Piscicida Virulence By Phosphoenolpyruvate:Carbohydrate Phosphotransferase System

Bacterial pathogens rely on sophisticated protein regulatory networks to adapt to nutritionally restricted host environments.The pho sphoeno lpyruvate:carbohydrate phosphotransferase system(PTS)is a conserved carbohydrate transportation and phosphorylation system in bacteria that combine PTS-sugar transportation with phosphorylation to monitor the availability of host carbohydrates,thus globally control the bacterial intracellular energy metabolism and other important physiological and biochemical processes.PTS exists in bacteria and archaea,but has not been found in eukaryotes so far.PTS usually consists of three proteins or protein complexes,i.e.,the cytosolically soluble Enzyme I(EI),the thermally stable,histidine-containing protein HPr,and the membrane-bound protease Ⅱ(EⅡ).They are phosphorylated at histidine or cysteine residues and play important roles in phosphorylation cascades to potentiate the use of different sugars and then transferred to the cytoplasm.The phosphorylation levels and phosphorylation sites of PTS components vary depending on the phosphorylated state of carbohydrates and the metabolic state of the cell.In addition to helping microbes intake of sugars and participation in the metabolism of carbon source,PTS also regulates nitrogen source utilization and bacterial virulence,including biofilm formation,chemotaxis,virulence gene expression,and is thus a potential antimicrobial target.As an important marine pathogen,Edwardsiella piscicida is highly pathogenic and infects some maricultured fish species leading to edwardsiellosis,doing huge harm to the healthy development of aquaculture industries.Due to the lack of many specific PTS system components,the saccharides that could be used by the bacterium are very limited and slowly fermented.Thus,the bacterium was initially named after "tarda"(slow).In the process of host infection and colonization by E.piscicida,type Ⅲ and Ⅵ secretion systems(T3/T6SS)are the deadlist weapons for bacterial resisting and invading host macrophages and other cells against the defense of host,but the mechanism underlying their expression regulation during infection is not very clear.Illumination of the regulatory network of T3/T6SS will enrich our understanding of pathgonesis of the bacterium and other enteric pathogens such as Salmonella causing systemic infections.The previous investigations in our laboratory revealed that mannose-6-phosphate(Man-6-P)binds to regulation factor EvrA and allosterically controls its activity to regulate the transcription of T3/T6SS genes.This study systematically characterized the roles of E.piscicida PTS system components in assisting in the transportation of various specific PTS-sugars.Meanwhile,PTS system was unexpectedly found to show a significantly regulatory effect on T3/T6SS gene expression,participating in the virulence program and in vivo infection of the bacterium.Furthermore,the regulatory roles of PTS component HPr on T3/T6SS were investigated.HPr was found to be involved in EvrA-dependent regulation of virulence by modulating mannose-PTS system.The deletions of each ptsH and manZ encoding respectively HPr and mannose-PTS enzyme ⅡD resulted in markedly decreases of the some T3/T6SS-involved protein produciton,the bacterial growth rate,and the colonization ability in turbot.In addition,the HPr paralog FPr that supports utilization of fructose while not mannose,was not found to be indispensable for T3/T6SS expression.These data collectively demonstrated that mannose-PTS transportation and its derivative metabolite Man-6-P consists vital regulatory circuits for T3/T6SS,and that the mannose from host represents an important metabolic cue specifically sensed and recognized by the pathogen to integrate into the known virulence programs.Meanewhile,the interaction between AdhE and HPr protein was firstly identified with HPr-based pull-down technology.AdhE is a bifocal enzyme with the N-terminus of acetaldehyde-CoA dehydrogenase,responsible for converting acetaldehyde-CoA to acetaldehyde,and the C-terminus of alcohol dehydrogenase,responsible for converting acetaldehyde to alcohol.We further validated their interaction by co-immunoprecipitation assays,and it was found that adhE-deleted strain showed poor growth probably due to their incapacities to utilize all PTS-sugars,as well as intracellular accumulation of acidic metabolites and cytoplasmic acidification.It was further demonstrated with metabolomics analysis that the metabolome profillings in adhE-and ptsH-deleted strains significantly changed as compared to the wild type parental strain.AdhE modulates a large quantity of metabolic pathways,including amino acids,tricarboxylic acid cycle intermediates,sugar and fatty acids.More importantly,the adhE-deleted strain also showed significantly decreaed expressions of many T3/T6SS proteins,and weakened its colonization ability in turbot.These findings confirmed the interaction between HPr and AdhE participated in PTS-sugar transportation and mediated various physiological processes and virulence programs.To summarize,this study investigated the relationship between carbon resources and virulence regulation in pathogen E.piscicida.By referring to the distinct functions and localization of HPr,ManZ and AdhE in cells,a virulence regulation model of HPr,ManZ and AdhE was proposed.AdhE is located in the cytoplasmic spaces and acts as a "parking lot" to recruit HPr and other PTS components in the cytoplasm due to the AdhE oligomerization and assembly into supermacromolecule form of spriosome.Spatial proximity enables HPr to perform its phosphorylation function to PTS and PTS-sugars including mannose.Among all PTS components of E.piscicida,mannose-PTS has been shown to be involved in T3/T6SS activation and virulence regulation dependent on EvrA.At the current stage it remains unclear that whether or not the metabolites underlying AdhE-HPr interaction are involved in the recruitment of AdhE to HPr and other proteins.Nevertheless,we could conclude that the interaction between AdhE and HPr is definitely essential to support the function of PTS-sugar utilization.Results of this study showed that PTS system controls not only the metabolism of the bacterium,but also the expression of virulence factors,which promotes the understanding of the relationship between metabolism and virulence in the bacterium,and may facilitate development of novel therapeutics against the bacterial infection.