Salmonella enterica serovar typhi plasmid pR ST98 enhances intracellular bacterial growth and S. typhi-induced macrophage cell death by suppressing autophagy

OBJECTIVES: Plasmid pR ST98 is a hybrid resistance-virulence plasmid isolated from Salmonella enterica serovar Typhi (S. typhi). Previous studies demonstrated that pR ST98 could enhance the virulence of its host bacteria. However, the mechanism of pR ST98-increased bacterial virulence is still not fully elucidated. This study was designed to gain further insight into the roles of pR ST98 in host responses. METHODS: Human-derived macrophage-like cell line THP-1 was infected with wild-type (ST8), pR ST98-deletion (ST8-ΔpR ST98), and complemented (ST8-c-pR ST98) S. typhi strains. Macrophage autophagy was performed by extracting the membrane-unbound LC3-I protein from cells, followed by flow cytometric detection of the membrane-associated fraction of LC3-II. Intracellular bacterial growth was determined by colony-forming units (cfu) assay. Macrophage cell death was measured by flow cytometry after propidium iodide (PI) staining. Autophagy activator rapamycin (RAPA) was added to the medium 2 h before infection to investigate the effect of autophagy on intracellular bacterial growth and macrophage cell death after S. typhi infection. RESULTS: Plasmid pR ST98 suppressed autophagy in infected macrophages and enhanced intracellular bacterial growth and S. typhi-induced macrophage cell death. Pretreatment with RAPA effectively restricted intracellular bacterial growth of ST8 and ST8-c-pR ST98, and alleviated ST8 and ST8-c-pR ST98-induced macrophage cell death, but had no significant effect on ST8-ΔpR ST98. CONCLUSIONS: Plasmid pR ST98 enhances intracellular bacterial growth and S. typhi-induced macrophage cell death by suppressing autophagy.

Macrophage apoptosis associated with Salmonella enterica serovar Typhi plasmid.

The present study was undertaken to investigate the relationship between plasmid isolated from S. enterica serovar Typhi (pRST98) and macrophage apoptosis. pRST98 was transferred into an attenuated S. enterica serovar Typhimurium strain RIA to create a transconjugant pRST98/RIA. Standard S. enterica serovar Typhimurium virulence strain SR-11 was used as a positive control, and RIA as a negative one. Murine macrophage-like cell line (J774A.1) was used as an infectious cell model in vitro. In order to determine the inhibition and bactericidal effect of amikacin (AMK) to extracellular bacteria and the best optimization co-culture ratio between Salmonella and J774A.1, the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of AMK to strains SR-11, pRST98/RIA and RIA and multiplicity of infection (MOI) were detected first, and then J774A.1 was infected by the above three serovar Typhimurium strains. Apoptosis of J774A.1 was examined with electron microscopy and flow cytometry after annexin-V/propidium iodide labeling at 0, 1, 3, 6, 12 and 24 h. Mitochondrial membrane potential was detected by JC-1 staining method. It was demonstrated that MIC of AMK to the three strains was 10µg/ml, MBC was 80µg/ml, and optimal MOI was 100:1. pRST98/RIA resulted in a higher apoptosis of J774A.1 than RIA, apoptotic features such as chromatin margination could be observed after 3 h, and death of J774A.1 cells was associated with the loss of mitochondrial membrane potential. These results indicated that pRST98 could enhance the virulence of its host bacteria, evidenced by increased macrophage apoptosis.

Rapamycin induces autophagy and exacerbates metabolism associated complications in a mouse model of type 1 diabetes.

Type 1 diabetes mellitus (T1DM) is characterized by lack of insulin production as a consequence of massive beta cells destruction. The contributions of autophagy to loss of beta cell mass were not clearly elucidated. Rapamycin is a specific and potent inhibitor of mammalian target of rapamycin (mTOR) and is used as the central immunosuppressant in T1DM patients especially for those who received islet transplantation. In the present study, effects of rapamycin on autophagy of T1DM were investigated in a mouse model treated with multiple low doses of streptozotocin. Rapamycin treatment led to hyperglycemia, weight loss, increased intake of food and drinking water, and islet inflammation in T1DM mice. Pathological changes including autophagy and apoptosis in pancreas, kidney, spleen and thymus, accompanied with an accumulation of LC3-II, Beclin1 and Caspase-3 protein were observed. The results indicate that rapamycin may exacerbate metabolism associated complications by activating autophagy and apoptosis in T1DM.