ABSTRACT
Pyroptosis, a form of programmed cell death, drives inflammation in the context of cerebral ischemia/reperfusion. The molecular mechanism of pyroptosis underlying ischemia/reperfusion, however, is not fully understood. The transient middle cerebral artery occlusion was applied to wild-type and caspase-1 knockout mice. 2,3,5-Triphenyltetrazolium chloride-staining and immunohistochemistry were used to identify the ischemic region, and western blot and immunofluorescence for the examination of neuronal pyroptosis. The expression of inflammatory factors and the behavioral function assessments were further conducted to examine the effects of caspase-1 knockout on protection against ischemia/reperfusion injury. Ischemia/reperfusion injury increased pyroptosis-related signals represented by the overexpression of pyroptosis-related proteins including caspase-1 and gasdermin D (GSDMD). Meanwhile, the number of GSDMD positive neurons increased in penumbra by immunofluorescence staining. Compared with wild-type mice, those with caspase-1 knockout exhibited decreased levels of pyroptosis-related proteins following ischemia/reperfusion. Furthermore, ischemia/reperfusion attack-induced brain infarction, cerebral edema, inflammatory factors, and neurological outcomes were partially improved in caspase-1 knockout mice. The data indicate that pyroptosis participates in ischemia/reperfusion induced-damage, and the caspase-1 might be involved, it provides some new insights into the molecular mechanism of ischemia.
Subject(s)
Caspase 1 , Infarction, Middle Cerebral Artery , Pyroptosis , Reperfusion Injury , Animals , Male , Mice , Brain Ischemia/metabolism , Brain Ischemia/pathology , Caspase 1/metabolism , Disease Models, Animal , Infarction, Middle Cerebral Artery/metabolism , Infarction, Middle Cerebral Artery/pathology , Mice, Inbred C57BL , Mice, Knockout , Neurons/metabolism , Neurons/pathology , Pyroptosis/physiology , Reperfusion Injury/metabolism , Reperfusion Injury/pathologyABSTRACT
The type III secretion system (T3SS), encoded by hrp (hypersensitive response and pathogenicity) genes in Gram-negative phytopathogenic bacteria, delivers repertoires of T3SS effectors (T3SEs) into plant cells to trigger the hypersensitive response (HR) in nonhost or resistant-host plants and promote pathogenicity in susceptible plants. The expression of hrp genes in Xanthomonas is regulated by two key regulatory proteins, HrpG and HrpX. However, the interactions between hrp gene products in directing T3SE secretion are largely unknown. Here we demonstrated that HrcT of X. oryzae pv. oryzicola functions as a T3SS component and positively regulates the expression of hrpX. Transcription of hrcT occurs via two distinct promoters; one (T1) is with the hrpB operon and the second (T3) within hrpB7 Via either promoter T1 or T3, the defect in Hrp phenotype by hrcT deletion was corrected in the presence of hrcT only from Xanthomonas species but not from other phytopathogenic bacteria. An N-terminally truncated HrcT was able to bind the hrpX promoter and activate the expression of hrpX, supporting that HrcT is a positive regulator of hrpX. A revised model showing the regulatory interactions between HrcT, HrpX, and HrpG is proposed.
Subject(s)
Bacterial Proteins/biosynthesis , Bacterial Secretion Systems , Gene Expression Regulation, Bacterial , Trans-Activators/metabolism , Transcription Factors/biosynthesis , Xanthomonas/genetics , Xanthomonas/metabolism , Bacterial Proteins/genetics , DNA, Bacterial/metabolism , Gene Deletion , Gene Expression Profiling , Genetic Complementation Test , Promoter Regions, Genetic , Protein Binding , Trans-Activators/genetics , Transcription Factors/genetics , Transcription, GeneticABSTRACT
The function of some hypothetical proteins, possibly regulated by key hrp regulators, in the pathogenicity of phytopathogenic bacteria remains largely unknown. In the present study, in silicon microarray data demonstrated that the expression of 17 HrpX-regulated protein (Xrp) genes of X. oryzae pv. oryzicola (Xoc), which causes bacterial leaf streak in rice, were either positively or negatively regulated by HrpX or/and HrpG. Bioinformatics analysis demonstrated that five Xrps possess a putative type III secretion (T3S) signal in the first 50 N-terminal amino acids, six xrp genes contain a PIP-box-like sequence (TTCGB-NX-TTCGB, 9 ≤ X ≤ 25) in the promoter regions, and two Xrps have both motifs. Twelve Xrps are widely conserved in Xanthomonas spp., whereas four are specific for X. oryzae (Xrp6) or Xoc (Xrp8, Xrp14 and Xrp17). In addition to the regulation by HrpG/HrpX, some of the 17 genes were also modulated by another hrp regulator HrpD6. Mutagenesis of these 17 genes indicated that five Xrps (Xrp1, Xrp2, Xrp5, Xrp8 and Xrp14) were required for full virulence and bacterial growth in planta. Immunoblotting assays and fusion with N-terminally truncated AvrXa10 indicated that Xrp3 and Xrp5 were secreted and translocated into rice cells through the type-III secretion system (T3S), suggesting they are novel T3S effectors. Our results suggest that Xoc exploits an orchestra of proteins that are regulated by HrpG, HrpX and HrpD6, and these proteins facilitate both infection and metabolism.
