FAT10 deficiency or overexpression does not change bacterial

Although the percentage of FAT10 decorated SHF2 is less than for ubiquitin, it was investigated whether this might have an impact on intracellular replication of SHF2. In HUVECs, FAT10 decoration was observable with highest percentages. Hence, in a first approach, FAT10 was knocked down in cytokine treated HUVECs and bacterial replication was quantified (Figure 27 A). Intracellular replication can be determined by means of gentamicin protection assays. Therefore gentamicin is added to the cells after invasion is completed. This kills extracellular bacteria but does not harm intracellular bacterial replication. In HUVECs rapid replication of SHF2 was observable in uninduced control cells within six hours of infection. In contrast, cells that were primed with IFN-γ and TNF-α for FAT10 induction could control bacterial replication, whether they were depleted of FAT10 expression or not (Figure 27 A). The efficiency of the FAT10 siRNA targeting during infection was controlled by monitoring FAT10 protein levels at the time point of infection (Figure 27 B). Additionally FAT10 mRNA levels were analyzed by quantitative real-time RT-PCR (Figure 27 C).

When the same gentamicin protection assays were performed with wild type and FAT10 knockout murine embryonic fibroblasts (MEFs) the same outcome was observable (Figure 28 A). Also for MEFs the induction of FAT10 expression was controlled by real-time RT-PCR at the time point of infection (Figure 28 B).

Figure 27: FAT10 deficiency in HUVECs does not change bacterial replication in vitro. (A) siRNA and SHF2 infected HUVECs were incubated with gentamicin and subsequently lysed at indicated time points. For cfu enumeration dilutions of lysates were plated on agar plates. The average +/- SEM is shown for at least two independent experiments and triplicate colony counts. (B) Immunoblot of total lysate of IFN-γ and TNF-α stimulated and untreated HUVECs treated with FAT10 specific or control siRNA for the indicated time period. GAPDH served as a loading control. (C) Quantitative real time RT-PCR of IFN-γ and TNF-α stimulated and control HUVECs treated with FAT10 or control siRNA for the indicated time period. FAT10 expression was normalized to GAPDH.

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Primary fibroblasts and epithelial cell lines are commonly used for in vitro infection assays, although they represent tissues with less physiological relevance. Macrophages are considered to be the principal cell type involved in the activation of the immune system by S. Typhimurium, which can invade these cells and survive within them (Schwan et al., 2000;

Vazquez-Torres et al., 2000). Schwan et al. observed that bacterial survival inside tissue culture cells can offer some useful information on pathogenesis but may not always reflect what occurs in host primary macrophage cells. Therefore, the expression of FAT10 and its potential impact on bacterial replication in peritoneal macrophages was likewise investigated.

In comparison to MEFs and HUVECs, bacterial replication was rapidly blocked and macrophages reduced the bacterial load over time (Figure 29 A and B). Minor differences between cytokine primed and untreated peritoneal macrophages were detectable but less severe (Figure 29 A and B). But more important, no significant difference in bacterial cfu could be observed in FAT10 knockout compared to wild type macrophages in Figure 29 A and B. The induced expression of FAT10 in cytokine treated macrophages in comparison to untreated cells was controlled by real time RT-PCR (Figure 29 C). Additionally, specific markers for the macrophage linage were used in FACS analysis to confirm the enrichment of macrophages from peritoneal lavage (Figure 29 D). Both preparations from C57BL/6 wild type and FAT10-deficient mice revealed about 70% F4/80 and CD11b double positive cells, therefor representing cells of the macrophage linage.

Figure 28: FAT10 deficiency in MEFs does not change bacterial replication in vitro. (A) SHF2 infected MEFs were incubated with gentamicin and subsequently lysed at indicated time points. For cfu enumeration dilutions of lysates were plated on agar plates. The average +/- SEM is shown for at least three independent experiments and triplicate colony counts. (B) Quantitative real-time RT-PCR of IFN-γ and TNF-α stimulated and untreated wild type and FAT10 KO MEFs. FAT10 expression was

To address the same question whether FAT10 influences intracellular bacterial replication without the need of cytokines, stable Flag-FAT10 overexpressing HEK293 cells were infected (Figure 30 A). Since these cells are under geneticin selection pressure and prokaryotes like SHF2 are much more sensitive to this agent than eukaryotic cells, geneticin was withdrawn three day prior to infection. Additionally, to have suitable control cells for the gentamicin protection assay we generated stable geneticin resistant HEK293 clones. Gentamicin protection assays with two Flag-FAT10 expressing (A2 and D2) and two control (B12 and F12) clones revealed no significant result but a tendency for reduced bacterial replication in the FAT10 overexpressing clones A2 and D2 compared to control clones (Figure 30 A). The

Figure 29: FAT10 deficiency in macrophages does not change bacterial replication in vitro.

SHF2 infected murine peritoneal macrophages treated with IFN-γ and TNF-α (A) or left untreated (B) were incubated with gentamicin and subsequently lysed at indicated time points. For cfu enumeration, dilutions of lysates were plated on agar plates. The average +/- SEM is shown for at least three independent experiments and triplicate colony counts. (C) Quantitative real-time RT-PCR of IFN-γ and TNF-α stimulated and untreated wild type and FAT10-deficient peritoneal macrophages. FAT10 expression was normalized to HPRT. (D) Cells obtained by peritoneal lavage were stained for CD11b and F4/80 and analyzed by flow cytometry.

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Flag-FAT10 expression levels were monitored by immunoblot to confirm persistent FAT10 expression after three days without selection pressure (Figure 30 B). In summary, primary cells of different origin with FAT10 knock down or knockout phenotypes as well as FAT10 overexpressing cells were analyzed. But no significant effect of FAT10 expression by enumeration of bacterial replication could be shown.

3.2.7 FAT10 deficiency in NRAMP1^r/r mice reveals a higher susceptibility to