The Investigation Of The Influence Of S. Typhi Plasmid PR_ST98and Spv Genes On Macrophage Autophagy And Apoptosis And The Molecular Mechanisms

Objective:To investigate the influence of S. typhi plasmid pR_ST98and Salmonella plasmidvirulence gene spv on macrophage autophagy and apoptosis and the molecularmechanisms by cell model.Methods:One. Investigation of the influence of S. typhi plasmid pR_ST98on macrophageautophagy and apoptosis and the molecular mechanisms.Wild-type S. typhi strain carrying pR_ST98ST8, pR_ST98-deletion S. typhi strainST8-ΔpR_ST98and pR_ST98-complemented S. typhi strain ST8-c-pR_ST98constructed byconjugative transfer were used to infect human-derived macrophage-like cell lineTHP-1to build infection model. Some groups were pretreated with autophagy inducerrapamycin（RAPA） and nitric oxide synthase inhibitor nitro-L-arginine methylester（L-NAME）. Cells and bacteria were co-incubated for30min in culture plates, thenthe medium was removed. This time was regarded as0point. Medium containing100mg amikacin per ml was added to culture plates to kill the remaining extracellularbacteria. After2h of further incubation the medium in culture plates was replaced withmedium containing10mg amikacin per ml to prevent intracellular growth of bacteriareleased from infected cells and incubation was continued. Cells and supernatant wereharvested at0point,2h and6h post-infection. The expression of autophagy proteinmicrotubule-associated protein1light chain3（LC3）-II and Sub-G1apoptosis peakwere determined by flow cytometry （FCM）; ultrastructure of THP-1cells was observed by transmission electron microscope（TEM）; the expression of the autophagy proteinBeclin-1and anti-apoptotic protein Bcl-2were detected by Western blot; Caspase-3activity was assayed by colorimetry; NO production was assayed by Griess method;TNF-α production was assayed by ELISA; and the intracellular bacterial survival wasdetected by plate count method.Two. Investigation of the influence of spv genes on macrophage apoptosis andautophagy and the molecular mechanisms.S. typhi is highly adapted to humans and no ideal animal model is suitable for S.typhi, for further investigation spv function on pR_ST98in vivo, this section S.typhimurium was selected. Wild-type S. typhimurium strain harboring spv UF009andspv-deletion S. typhimurium strain UF110were used to infect mouse-derivedmacrophage-like cell line J774A.1to build infection model. Some groups werepretreated with p38inhibitor SB203580. Cells and bacteria were co-incubated for1h inculture plates, then the medium was removed. This time was regarded as0point. Theuse of amikacin was the same as section one. Cells and supernatant were harvested at0point,2h,6h and24h post-infection. The expression of autophagy protein LC3-II andSub-G1apoptosis peak were determined by FCM; the autophagy protein Beclin-1andanti-apoptotic protein Bcl-2were detected by Western blot; and NO production wasassayed by Griess method.Results:One. Investigation of the influence of S. typhi plasmid pR_ST98on macrophageautophagy and apoptosis and the molecular mechanisms.1. FCM analysis and Western blot detection showed that the expression ofautophagy protein LC3-II（P <0.01） and Beclin-1of ST8and ST8-c-pR_ST98infectedgroups was significantly lower than ST8-ΔpR_ST98infected group at0point; theexpression of autophagy protein LC3-II（P>0.05） and Beclin-1of every infected groupwas no significant difference at2h post-infection.2. FCM analysis showed that apoptosis rate of every infected group was nosignificant difference at2h post-infection（P>0.05）; apoptosis rate of ST8andST8-c-pR_ST98infected groups was significantly higher than ST8-ΔpR_ST98infected groupat6h post-infection （P <0.01）. The result of observation of ultrastructure of THP-1cells is consistent with the result of FCM analysis, cells exhibited morphological features of apoptosis, such as marginated nuclear chromatin at2h post-infection, butthere was no significant difference between these three groups in amount of apoptoticcells at this point; more cells showed morphological features of apoptosis in ST8andST8-c-pR_ST98infected groups than ST8-ΔpR_ST98infected group at6h post-infection.Western blot detection showed that the expression of anti-apoptotic protein Bcl-2ofST8and ST8-c-pR_ST98infected groups was also significantly lower than ST8-ΔpR_ST98infected group at6h post-infection.3. Colorimetry assay showed that the Caspase-3activity of every infected groupwas no significant difference at2h post-infection（P>0.05）; Caspase-3activity of ST8and ST8-c-pR_ST98infected groups was higher than ST8-ΔpR_ST98infected group at6hpost-infection（P <0.05）.4. Assaying the content of NO and TNF-α in the supernatant showed that NO（P <0.01） and TNF-α（2h, P <0.01;6h, P <0.05） production of ST8and ST8-c-pR_ST98infected groups were lower than ST8-ΔpR_ST98infected group at2h and6h post-infection. But plate counting showed that ST8and ST8-c-pR_ST98multiplied faster ininfected macrophages than ST8-ΔpR_ST98（2h, P <0.05;6h, P <0.01）.5. Pretreated with autophagy inducer RAPA reduced apoptosis（P <0.01） andintracellular bacterial survival（2h, P <0.05;6h, P <0.01） of ST8and ST8-c-pR_ST98infected groups; but had no significant influence on ST8-ΔpR_ST98infected group（P>0.05）.6. Pretreated with L-NAME suppressed the expression of autophagy proteinLC3-II（P <0.01） and enhanced apoptosis（P <0.01） and Caspase-3activity（P <0.05） ofST8-ΔpR_ST98infected group; but had no significant influence on ST8and ST8-c-pR_ST98infected groups （P>0.05）.Two. Investigation of the influence of spv genes on macrophage apoptosis andautophagy and the molecular mechanisms.1. FCM analysis and Western blot detection showed that the expression ofautophagy protein LC3-II（P <0.01） and Beclin-1of UF009infected group wassignificantly lower than UF110infected group at0point; the expression of autophagyprotein LC3-II（P>0.05） and Beclin-1of two infected groups was no significantdifference at2h post-infection.2. Pretreated with p38inhibitor SB203580suppressed the expression of autophagyprotein LC3-II（P <0.01） and Beclin-1of UF110infected group, but had no significant influence on the expression of autophagy protein LC3-II（P>0.05） and Beclin-1ofUF009infected group at0point.3. FCM analysis showed that apoptosis rate of two infected groups was nosignificant difference at2h post-infection（P>0.05）; apoptosis rate of UF009infectedgroups was significantly higher than UF110infected group at6h and24h post-infection（P <0.01）. Western blot detection showed that the expression of anti-apoptoticprotein Bcl-2of two infected groups was no significant difference at2h post-infection;the expression of anti-apoptotic protein Bcl-2of UF009infected groups was slightlylower than UF110infected group at6h post-infection, but the difference was notsignificant; the expression of anti-apoptotic protein Bcl-2of UF009infected groups wassignificantly lower than UF110infected group at24h post-infection.4. Pretreated with p38inhibitor SB203580enhanced apoptosis rate of UF110infected group（P <0.05）, but had no significant influence on UF009infected group at6h and24h post-infection （P>0.05）. Pretreated with p38inhibitor SB203580alsosuppressed the expression of anti-apoptotic protein Bcl-2of UF110infected group, butbut had no significant influence on UF009infected group at24h post-infection.5. Assaying the content of NO in the supernatant showed that NO production ofUF009infected group was significantly lower than UF110infected group at2h,6h and24h post-infection（P <0.01）.6. Pretreated with p38inhibitor SB203580suppressed NO production of UF110infected group（P <0.01）, but had no significant influence on UF009infected group atall three time point（P>0.05）.Conclusions:The above results demonstrated that pR_ST98suppressed macrophage autophagy toincrease the intracellular bacteria survival at early phase after infection and inducedmacrophage apoptosis at later phase after infection. Pretreated with autophagy inducerRAPA to activate autophagy can alleviate macrophage apoptosis induced by S. typhicarrying pR_ST98. The influence of plasmid pR_ST98on macrophage autophagy andapoptosis is related to suppresion of NO and TNF-α production by this plasmid. Spv notonly suppressed autophagy of infected macrophages, but also enhanced Salmonella-induced macrophage apoptosis. Pretreated with p38inhibitor SB203580could suppressmacrophage autophagy and enhance macrophage apoptosis induced by Salmonellawithout spv demonstrated that the influence of spv on macrophage autophagy and apoptosis is closely related to p38MAP kinase signaling.