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关于transposon mutant library,文献中的方法求指导

soulfeather实名认证 发表于 2016-6-16 09:40 |查看: 33765|回复: 16|显示全部楼层 |阅读模式
Genome-wide screening for factors involved in IL-1β secretion
reveals a role for H. pylori LPS and urease. Known activators of the
NLRP3 inflammasome include both foreign and endogenous
compounds, with the best-understood being urate crystals, asbestos, ATP, and bacterial pore-forming toxins (15). We were able
to exclude a role for the H. pylori immunomodulator γ-glutamyltranspeptidase GGT and the Cag pathogenicity island in CASP1
activation and IL-1β secretion (Supplemental Figure 3, A and B).
To search for H. pylori factors involved in inflammasome activation in a genome-wide manner, we took advantage of a previously
described tn mutant library (16). As IL-1β secretion by H. pylori–exposed DCs is dependent on CASP1 (10), we opted for IL-1β ELISA
as a screening readout (Supplemental Figure 3C). The insertion
sites of 64 mutants with defects in inducing IL-1β secretion (<25%
of the corresponding WT infection) were sequenced and mapped
to 32 different loci (Supplemental Table 1). Loci belonging to two
mutant categories were identified repeatedly; these harbored tn
insertions in genes involved in LPS biosynthesis and in the urease
gene cluster (Figure 2, A and B). The LPS synthesis gene hit most
often was LPS-1,2-glycosyltransferase (HPG27_146). LPS from
H. pylori deficient for this gene lack the O-side chain and therefore Lewis antigens (Supplemental Figure 4A). A gene-specific
deletion mutant (Δ146) in strain G27 recapitulated the tn mutant
phenotype, as it failed to induce IL-1β secretion in BMDCs (Figure
2C). The phenotype of this mutant was attributable to its failure
to induce pro–IL-1β expression at the transcriptional level, rather
than to a defect in CASP1 activation, and could be rescued by
E. coli or H. pylori LPS (Figure 2, C–E, and Supplemental Figure 4,B and C). IL-1β expression upon stimulation with both types of LPS
was MyD88- and TLR4-dependent (Supplemental Figure 4D).
Remarkably, of the 8 tn insertions mapping to the urease
gene cluster, all affected 2 genes encoding the structural urease
subunits, ureA or ureB (Supplemental Table 1). A gene-specific
deletion mutant lacking both UreA and UreB proteins (G27Δure,
Supplemental Figure 5A) phenocopied the effect of the tn insertion mutants, which could be attributed to its failure to activate
CASP1 (Figure 2, C and D). In contrast, Il1b transcription was normal (Figure 2E). Coculturing of murine splenic CD11c+ DCs and
human blood-derived DCs confirmed the defect of the H. pylori
Δure and Δ146 mutants with respect to IL-1β secretion; in contrast,
the secretion of IL-18, which does not require transcriptional activation, was almost at WT levels in the case of the Δ146 mutant
(Figure 2, F–H). In summary, our screen identified H. pylori factors
regulating CASP1-dependent cytokine secretion at two distinct
levels, one transcriptional and one posttranslational.
这一段的描述看不明白
 楼主| soulfeather实名认证 发表于 2016-6-16 09:57 显示全部楼层
求大神指点
若绾君 发表于 2016-9-29 02:30 显示全部楼层
论坛有你更精彩!
1084268272 发表于 2016-9-29 03:47 显示全部楼层
论坛有你更精彩!
doudou 发表于 2016-9-29 06:42 显示全部楼层
大家都不容易!
radieh 发表于 2016-9-29 09:30 显示全部楼层
大家都不容易!
欢乐谷 发表于 2016-9-29 13:29 显示全部楼层
论坛有你更精彩!
gxrshoulderknee 发表于 2016-9-29 15:45 显示全部楼层
以后多分享一些这样的有价值的帖子啊
昆虫记小木虫公众人物认证 发表于 2016-9-29 21:27 显示全部楼层
大家都不容易!
lj5353553 发表于 2016-9-29 23:39 显示全部楼层
论坛有你更精彩!
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