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1030694963

银虫 (小有名气)

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专业: 波谱分析与成像分析

[求助] Francisella tularensis Harvests Nutrients Derived Autophagy

Autophagy derived amino acids are transferred from host proteins to F. tularensis
To confirm that F. tularensis imports amino acids derived from host proteins, we monitored transfer of radiolabelled amino acids from host proteins into bacterial proteins. MEFs were first metabolically labeled with 35S-labeled methionine and cysteine for 18 hours to fully label all host proteins. Then the radiolabel was removed and the cells were incubated in unlabeled media for two hours prior to infection with F. tularensis to remove 35S that was not incorporated into protein. At 16 hours post infection (18 hours after the radiolabel was removed) we lysed the MEFs and purified F. tularensis by mixing cell lysate from either uninfected or infected cells with magnetic beads linked to an anti- F. tularensis lipopolysaccharide (LPS) antibody. We then determined if F. tularensis proteins contained radiolabeled amino acids by examining the trichloroacetic acid (TCA) insoluble fraction of
purified F. tularensis. There was a significant increase of radiolabel in the TCA insoluble, F. tularensis bead purified fraction from infected MEFs as compared to uninfected control samples (Figure 2B). Indeed, 6.22%+/24.15% (average +/2 SEM, n = 5 samples) of the TCA insoluble radiolabel present prior to infection transferred to the bacteria during the 16 hour infection. To control for possible direct transfer of labeled amino acids that were not incorporated into host proteins we analyzed infected MEFs that were treated with cycloheximide during 35S labeling prior to infection. There were negligible amounts of radiolabel present in the bead purified fraction of cycloheximide treated cells (Figure 2B). F. tularensis survived and replicated within cycloheximide pre-treated cells and F. tularensis was present in the bacterial purified fraction (data not shown). Thus, host cell lysis due to the
cycloheximide treatment was not solely responsible for the lack of radiolabel in the bacterial fraction. 35S radiolabel was primarily incorporated into host proteins, rather than as free 35S labeled amino acids. Taken together, these data demonstrate that F. tularensis synthesized proteins using amino acids derived from host cell proteins.
Treating the radiolabeled cells with either Baf or 3-MA resulted in significantly decreased incorporation of the radiolabel by F. tularensis (Figure 2C). Since F. tularensis proliferation is reduced in 3MA and Baf treated MEFs, several fold fewer bacteria were present in the bacteria purified fraction of the treated MEFs (data not shown). Nevertheless, the median 35S counts per bacteria were significantly lower in the 3MA or Baf treated samples compared to untreated samples (untreated: 0.016 CPM/bacteria, 3MA: 0.000 CPM/bacteria, Baf: 0.000 CPM/bacteria [n = 3 experiments done in duplicate]). Therefore, transfer of radiolabeled amino acids to bacterial proteins was reduced by both 3MA and Baf treatment, indicating that under normal culture conditions, amino acids derived by the degradation of host cell proteins via autophagy were used by F. tularensis.

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