The Identification And Preliminary Functional Study Of Putative T3SS Effector CPSIT₀959 In Chlamydia Psittaci

Objective:Chlamydia psittaci（C.psittaci）is an obligate intracellular human pathogen.It is auxotrophic for a variety of essential metabolites and obtains amino acids from their eukaryotic host cells to proliferation.Moreover,a pathway secreted effectors can be delivered is the type III secretion system（T3SS）.The T3SS is universal to several gram-negative bacteria and is closely associated with their pathogenesis.This experiment has identified the characteristics,subcellular localization and time-course expression of the putative cysteine desulfurase protein CPSIT₀959 in C.psittaci,and its effect both in Chlamydia and host cells,which has directive significance on the understanding of chlamydial replication and the research of drug target for Chlamydia.Methods:The phylogenetic tree was constructed by MEGA 5.0,and the cysteine desulfurase and PLP-depending activity was detected by using sodium borohydride（NaBH₄）and D-4-amino-3isoxazolidone（DCS）via ultraviolet spectrophotometer and circular dichroism（CD）.We calculated the inclusion-forming units（IFU）in infected HeLa cells after the treatment of free-endotoxin protein,DCS and both,respectively.Cultivate HeLa cells,and monolayer cells were culture C.psittaci 6BC standard strain for 6,12,18,24,36,48 and 60h.The subcellular localization of CPSIT₀959 and its secretion were detected by indirect immunofluorescence assay（IFA）and determined by confocal laser scanning microscope（CLSM）.CPSIT₀959 mRNA and its protein expression in HeLa cells were detected by Real-time quantitative PCR（RT-qPCR）and western blot（wb）after chlamydial infection with different hours.Monolayer HeLa cells were culture C.psittaci 6BC standard strain for 12h.The T3SS inhibitor INP0007 was added at 12 hpi,subsequently collected samples at 18,24,36,48 and 60h for IFA and wb to test its secretary pathway.T3SS inhibitor was added in 12,18 or 24hpi,and then the samples were collected at 36hpi for IFA to detect the effect of INP0007.We estimate the expression of Fas,Fas-L,Caspase 3,tBid,Bim and Caspase 8 in host cells proteins by wb,and detected mitochondrial membrane potential（MMP）by IFA.Results:（1）The phylogenetic tree showed that CPSIT₀959 was a PLP-dependent enzyme and the addition of PLP inhibitors NaBH₄ and DCS results in the shift of UV-VIS spectrum;（2）IFU of infected-cells was increased after the treatment of free-endotoxin recombinant protein,which can invert the decrease of DCS-treatment;（3）The results of RT-qPCR and wb shown that CPSIT₀959 was transcript at 6 hours post-infection（hpi）,and peaking at 12 hpi,the protein was peaking at 24hpi;（4）CPSIT₀959 was secreted into host cellular cytoplasm during36-60 hpi;（5）Size of inclusion was significantly decrease after T3SS inhibitor treatment,and CPSIT₀959 was located in inclusion but not in cytoplasm;CPSIT₀959 was secreted from inclusions into cytoplasm at12-18 hpi;（6）Free-endotoxin CPSIT₀959 treatment has no effect on Fas/Fas-L,slightly increased the expression of Caspase 8,significantly increased the expression of tBid and Bim in time-dependent manner.The depressed tBid and Bim declined the MMP.Conclusion:1.CPSIT₀959 was highly homology in pathogenic chlamydia and possess the cysteine desulfurase activity and PLP-dependent activity,which can invert the decrease of IFU after DCS-treatment..2.CPSIT₀959 was secreted into cytoplasm of host cell by T3SS in the mid of development cycle,and up-regulated the expression of tBid and Bim,subsequently declined the MMP,which induce the apoptosis of host cells and improve the quantity of offsprings.