Escherichia coli ghosts promote innate immune responses in human keratinocytes

Bacterial ghosts (BGs) as non-living bacterial envelopes devoid of cytoplasmic content with preserved and intact inner and outer membrane structures of their living counterparts have been used to study the ability of their surface components for the induction of antimicrobial peptides and pro-inflammatory cytokines in human primary keratinocytes (KCs). Quantitative real-time PCR analysis revealed that incubation of KCs with BGs generated from wild-type Escherichia coli induced the mRNA expression of antimicrobial psoriasin (S100A7c) in a BGs particle concentration-dependent manner. Using immunoblot analysis we showed that BGs generated from the flagellin-deficient (ΔFliC) E. coli strain NK9375 were as effective as its isogenic wild-type (wt) E. coli strain NK9373 to induce psoriasin expression when normalized to BG particles being taken up by KCs. However, results obtained from endocytic activity of KCs reflect that internalization of BGs is greatly dependent on the presence of flagellin on the surface of BGs. Moreover, BGs derived from wt E. coli NK9373 strongly induced the release of the pro-inflammatory cytokines IL-6 and IL-8, compared to ΔFliC E. coli NK9375 BGs. Taken together, obtained data demonstrate that non-living BGs possessing all bacterial bio-adhesive surface properties in their original state while not posing any infectious threat have the capacity to induce the expression of innate immune modulators and that these responses are partially dependent on the presence of flagellin.     

铁皮石斛DoWRKY6转录因子基因的克隆与分析

目的:植物生长发育由多种转录因子调控,探索WRKY转录因子调控铁皮石斛组织生长发育机理。方法:根据铁皮石斛原球茎转录组数据中的WRKY6序列设计引物,并利用RACE技术克隆该基因;用生信分析软件预测其蛋白结构,并进行系统发育分析;利用实时荧光定量PCR(q RT-PCR)技术研究该基因在铁皮石斛幼苗根、茎、叶等组织中的表达。结果:通过RACE技术得到1个1,238bp的Ⅱ类WRKY转录因子基因,命名为DoWRKY6,其ORF长1,017 bp,编码339个氨基酸残基,与梅[PrmuWRKY27(XM_008236601.1)]及大豆[GlmaWRKY27(XM_003555347.3)]有较近的亲缘关系,且DoWRKY6在茎中的相对表达量最大。结论:我们预测在铁皮石斛的生长发育过程中DoWRKY6基因对根、叶、茎的调控作用依次增强,表明DoWRKY6与已发表的铁皮石斛WRKY基因在组织的差异性表达上有较大差别,这对丰富WRKY家族对铁皮石斛的生长发育调控机制提供更多依据。     

Molecular cloning and characterization of an Hsp90/70 organizing protein gene from Frankliniella occidentalis (Insecta: Thysanoptera,Thripidae)

The heat shock 90/70 organizing protein (Hop), also known as Sti-1 (stress-induced protein-1), is a co-chaperone that usually mediates the interaction of Hsp90 and Hsp70 and has been extensively characterized in mammals and plants. However, its role in insects remains unknown. In the present study, we isolated and characterized a Hop homologue gene from Frankliniella occidentalis (Fohop). The Fohop contains a 1659 bp ORF encoding a protein of 552 amino acids with a caculated molecular mass of approximately 62.25 kDa, which displays a reasonable degree of identity with the known Hops and shares several canonical motifs, including three tetratricopeptide repeated motif domains (TPR1, TPR2A and TPR2B) and two aspartic acid–proline (DP) repeat motifs (DP1 and DP2). As in other hops, Fohop contains introns, but the number and the position are quite variable. The mRNA expression patterns indicated that Fohop was constitutively expressed throughout the developmental stages, but was obviously upregulated by heat stress both in larvae and adults. Our studies imply that Hop, as in other Hsps, may play an important role in heat shock response of F. occidentalis.