Adherent-invasive E. coli (AIEC) is closely related to inflammatory bowel disease (IBD). However, its pathogenic mechanism has not yet been fully elucidated. Using a BLASTP search, we discovered that the amino acid sequence of a putative protein (UFP37798.1) in the AIEC LF82 strain is highly homologous to some regulators in the SlyA family. We named it EruA. We displayed the secondary structures of EruA using bioinformatics, overexpressed the His6-tagged EruA protein using SDS-PAGE, and dissected the genetic organization of the eruA chromosomal region using 5′RACE. We constructed an eruA deletion mutant (ΔeruA) and a complementary strain (CΔeruA) of the LF82 strain. The transcriptomes of wild-type (WT) and ΔeruA bacteria were compared using RNA sequencing and qRT-PCR, thereby identifying 32 differentially expressed genes (DEGs). Based on YASARA software and EMSA analysis, EruA directly binds to the consensus sequences (PfimA and PtnaB) in the promoter region of the fimA and tnaB genes from these DEGs. By using a super-resolution confocal microscope (SCM), counting CFUs of colonies on plates, indole quantification, and crystal violet staining of biofilms adhered to tubes or 96-well plates, we found that EruA activates the fimA to promote bacterial adhesion to intestinal epithelial cells and activates the tnaB to enhance bacterial indole production and biofilm formation. Moreover, EruA helps AIEC resist environmental stress and enhances bacterial survival within macrophages as well as loading in mouse tissues. Notably, EruA promotes AIEC colonization in the colons of mice and exacerbates intestinal inflammation caused by bacterial infection in mice with DSS-induced inflammatory colitis, manifested by weight loss, colon length shortening, and pathological changes in colon tissues. Therefore, EruA plays a key role in the pathogenicity of AIEC.

Inflammatory bowel disease (IBD) involves the complex interplay among the mucosal barrier, microbiota, immunity and genetic factors. There are currently no satisfactory treatments for IBD. Administration of the probiotic Bacillus licheniformis (Bl) can improves colitis by regulating the gut microbiota. The phytohormone abscisic acid (ABA) treatment has favorable effects on immunity, as well as on inflammatory diseases like colitis. We hypothesized that the expression of an additional cyp gene by the Bl to increase its ABA production would enhance its effects. In this study, we found that a Bl-cyp strain overexpressing the cyp gene secreted more ABA into its supernatant than either the parental Bl stain or a Bl-pET82a strain expressing only a vector pET82a when these bacteria were grown in Nfb medium for 48 h. The prophylactic administration of the Bl-cyp strain culture more effectively attenuated dextran sodium sulfate (DSS)-induced colitis in mice compared to the Bl and Bl-pET28a strains. These findings were associated with significantly reduced epithelial barrier damage, as well as increased number of goblet cells and expression levels of occludin gene in the colonic epithelial layer, and decreased serum LPS levels in the Bl-cyp group. In addition, the administration of Bl-cyp strain effectively regulated the disordered gut microbiota by improving their diversity, richness and compositions more than the Bl or Bl-pET82a strain, including the ratio of Bacteroidota: Bacillota. It also inhibited the excessive growth of opportunistic pathogen Escherichia just like the Bl or Bl-pET82a strain. Moreover, the preventive administration of the Bl-cyp strain to mice following DSS-induced colitis enhanced the proportion of Treg cells and suppressed the proportion of Th17 cells in mesenteric lymph nodes (MLNs), decreased the levels of pro-inflammatory cytokines TNF-α, IL-6, and IL-22, and increased the level of anti-inflammatory IL-10 in colon tissues, similar to treatment with a high concentration of the ABA standard (ABA-H). Notably, the treatment with the Bl-cyp strain more effectively regulated the disordered microbiota than the ABA-H. The administration of the Bl-cyp strain may provide a novel preventive approach for IBD, and may exert its effects by modulating the gut microbiota and host’s immune status.

目的:探讨aroC基因在迟缓爱德华氏菌(Edwardsiella tarda,Et)致病过程中的作用.方法:采用自杀质粒同源重组方法制备Et的aroC基因缺失株(ΔaroC)和aroC的互补株(CΔaroC);通过生物膜形成、运动性、细胞黏附、斑马鱼致病性、组织细菌载量等实验,分析aroC对Et致病性相关的生物学功能的影响.结果:aroC基因的缺失不影响Et生长速度;与野生株和CΔaroC相比,ΔaroC的生物膜形成能力、运动能力、黏附及胞外吲哚生成能力均显著降低(P<0.05 或<0.01);qRT-PCR结果显示,相比于野生株,ΔaroC鞭毛合成基因fliA和fliC的转录水平显著下降(P<0.01),但flgK、flgL和fliS基因的转录水平无明显变化(P>0.05);与野生株相比,感染ΔaroC的斑马鱼症状明显减轻、存活率高,且ΔaroC在小鼠肠道和肝脏中的定植数目显著减少(P<0.05 或<0.01).结论:aroC基因影响Et的运动性、生物膜形成、黏附性等,进而调控Et对宿主细胞的致病力.

目的 评估成人接种严重急性呼吸综合征冠状病毒2(SARS-CoV-2)疫苗后SARS-CoV-2 IgM、IgG和中和抗体的形成情况.方法 选取接受SARS-CoV-2疫苗接种的成人80名(疫苗接种组),检测疫苗接种完成后7 d内和第14天的血清SARS-CoV-2 IgM抗体、IgG抗体及中和抗体水平.以未接种SARS-CoV-2疫苗的健康成人80名作为对照组.按年龄和性别分别分组分析.结果 疫苗接种组接种结束后第14天,血清SARS-CoV-2 IgM抗体、IgG抗体及中和抗体阳性率分别为37.5％、98.8％和95.0％,IgM或IgG抗体的假阳性率为3.8％,中和抗体假阳性率为8.8％.男性SARS-CoV-2中和抗体阳性率高于女性(P<0.05).疫苗接种组中,20～39岁受试者与40～60岁受试者之间3种抗体阳性率差异均无统计学意义(P>0.05).接种第14天的血清SARS-CoV-2 IgG抗体水平显著高于接种7 d内(P=0.007),血清SARS-CoV-2 IgM抗体和中和抗体水平2个时间点之间差异均无统计学意义(P>0.05).与接种第14天的血清SARS-CoV-2 IgG抗体、中和抗体阳性率比较,接种7 d内均较低(P<0.01),血清SARS-CoV-2 IgM抗体2个时间点之间差异无统计学意义(P>0.05).结论 成年人完成SARS-CoV-2疫苗接种2周后,SARS-CoV-2 IgG抗体的阳性率可达95.0％,中和抗体阳性率可达86.2％.

Developing an integrated multimodal diagnosis and therapeutics nanoplatform is of great importance to enhance the outcome of cancer therapy. Herein, we report a highly efficient and biocompatible nanoplatform based on the assembly of a graphene oxide (GO) and metal-organic framework (MOF) Fe-porphyrin, which was coated with a folate-functionalized erythrocyte membrane (FA-EM@GO-MOF). The nanoplatform could be targeted to cancer cells precisely, and could avoid immune elimination and had prolonged blood circulation due to the presence of FA-EM on its surface. The presence of GO and paramagnetic Fe ions endowed the nanoplatform with robust fluorescence imaging and T-2-weighted magnetic resonance imaging capacities. The porous structure and large surface area of GO-MOF make it desirable for drug delivery in chemotherapy. More importantly, with one operation, under the same laser (808 nm) irradiation, both photothermal therapy and photodynamic therapy could be triggered for efficient synergistic treatment by using MOF as a photosensitizer. This synergistic anticancer therapy promoted the generation of tumor-associated antigens and evoked an antitumor immune response. In vitro and in vivo therapy studies highlighted that the as-fabricated biomimetic nanoplatform for dual imaging-guided synergistic cancer therapy was highly effective yet straightforward, paving a new avenue for cancer diagnosis and therapy.

Biofilms contribute to the resistance of Edwardsiella tarda to antibiotics and host immunity. AroC in the shikimate pathway produces chorismate to synthesize crucial intermediates such as indole. In this study, the differences between biofilms produced by aroC mutants (△aroC), wild-type (WT) strains, and △aroC complementary strains (C△aroC) were detected both in vitro with 96-well plates, tubes, or coverslips and in vivo using a mouse model of subcutaneous implants. When examining potential mechanisms, we found that the diameters of the movement rings in soft agar plates and the flagellar sizes and numbers determined by silver staining were all lower for △aroC than for WT and C△aroC. Moreover, qRT-PCR showed that the transcription levels of flagellar synthesis genes, fliA and fliC, were reduced in △aroC. AroC, FliC, or FliA may accompany the motility of △aroC strains. In addition, compared with the WT and C△aroC, the amounts of indole in △aroC were significantly decreased. Notably, the formation of biofilms by these strains could be promoted by exogenous indole. Therefore, the aroC gene could affect the biofilm formation of E. tarda concerning its impact on flagella and indole.

Cancer is a multifactorial disease that involves unique tumor microenvironment (TEM) and abnormal organs with complex structures. Conventional anticancer drugs require high doses and systemic administration, and nonspecific tissue distribution causing serious side effects. Nanomaterials have far-reaching influences in cancer treatment, theranostics and imaging. Specifically, anticancer drug-loading into nanocarriers can avoid unnecessary systemic toxicity and achieve sustained drug release in response to all kinds of exogenous or endogenous stimuli. Nanocarriers can be stimulated by external (light irradiation and magnetic fields) or internal (pH, glutathione, enzymes and oxidative stress) elements, which have attracted growing attention in the field of drug delivery systems. On the basis of this powerful concept, environment-responsive nanocarriers have been designed to ensure delivery of the anticancer drug to the targeting lesion sites and effective release, which can overcome the biological barriers that limit conventional treatments and reduce the administered drug dose. Herein, this review will discuss the fundamentals of environment-responsive nanocarriers to enhance the permeability and retention (EPR) effect, and improve cancer therapy outcomes.

Eha is a virulence regulator in Edwardsiella tarda (E. tarda). The present study examined how Eha regulated its target genes to affect the bacterial survival within the cells. We constructed the reporter a pGEX-4T-ehaflag plasmid expressing Eha tagged at its C terminus with the flag epitope, and introduced the plasmid into an eha mutant ET13 strain, and obtained a Cehaflag strain. The expression and activity of an EhaFlag fusion protein restored the survival of the Cehaflag as the wild type in macrophages by Western blotting and intracellular survival experiments. We used a monoclonal anti-Flag antibody to precipitate EhaFlag-DNA complexes using chromatic immunoprecipitation (ChIP). We then designed primers based on the differentially-expressed genes identified from RNA-sequencing, and identified ten Eha-interacting genes by qPCR. We amplified the promoter regions of the ten genes and the eha gene from ET13 strain by PCR, constructed pBD-PtargetlacZ and pBD-PehalacZ plasmids. The eha gene directly and positively regulated these target genes, and be negatively auto-regulated by Eha in E. tarda, as determined by comparing their β-Galactosidase activities. These target genes were distributed in the categories involved in the bacterial growth, movement and resistance to H2O2 or acid. We further constructed a ETATCC_RS15225 mutant (△dcuA1), a ETATCC_ RS14855 mutant (△flgK) anda ETATCC_RS07650 mutant (ΔtnaA), and a partial complementary strains of △eha-tnaA and △eha-flgK and the complementary strains of CΔflgK and CΔtnaA. The ETATCC_RS15225 gene probably encoded a transporter protein DcuA1 at outer membrane with SDS-PAGE and RT-PCR. The ETATCC _RS14855 gene probably encoded FlgK protein and affected the bacterial motility. The ETATCC_RS07650 gene encoded Tryptophanase, which affected the bacterial survival within macrophages. With the assistance of these above strains, our results showed that the eha gene was able to regulate the ETATCC_RS15225 gene to express its outer membrane protein DcuA1, the ETATCC _RS14855 gene to control the flagellar motility and the ETATCC_RS07650 to affect the bacterial survival within macrophages. With the combination of other functions of above three genes, our results suggested that Eha directly regulates the target genes to affect E. tarda to survive within the cells.

目的 Eha是一种重要转录调控因子,它可以影响迟缓爱德华氏菌(Et)的胞内存活,本实验探究Eha直接调控的靶基因如何抵御巨噬细胞杀灭细菌的分子机制.方法 构建pGEX-4T-ehaflag重组质粒,电击导入eha基因缺失株的ET-13菌,得到Cehaflag ET13重组菌;用Western blot检测重组菌Eha-Flag融合蛋白的表达,并用细菌胞内存活实验检测细菌EhaFlag融合蛋白的活性;我们采用染色质免疫共沉淀(CHIP)技术,用抗Flag标签抗体对靶基因沉淀Eha DNA片段,除去结合的蛋白并纯化DNA片段;以RNA-Sequencing的差异表达基因设计引物,以CHIP得到的DNA样品为模板,进行qRTPCR;PCR扩增靶基因的启动子区域,构建了pBAD-P靶 lac Z重组质粒,电击分别导入ET 13野生株和eha基因缺失株,比较它们β-半乳糖苷酶活性的差异;SDS-PAGE电泳比较野生株和缺失株外膜蛋白、厌氧C4二羧酸转运蛋白DcuA1和鞭毛钩蛋白FlgK表达的差异,并用细菌胞内存活实验比较野生株和缺失株的差异.结果 Western blot表明,Cehaflag ET13重组菌能够表达Eha-Flag融合蛋白;细菌胞内存活实验表明,该融合蛋白完全可以恢复缺失株在胞内降低的生存能力,Flag融合标签不影响Eha蛋白的功能;通过qPCR鉴定CHIP,富集到Eha的结合靶点,最终筛选出1 0个Eha直接结合的基因;通过野生株和缺失株中β半乳糖苷酶活性的差异,证明eha基因对5个靶基因启动子有直接调控作用;通过检测野生株和缺失株表型的差异,证明eha基因对靶蛋白DcuA1,TnaA和FlgK的表达和活性有调控作用.结论 Eha可以通过直接调控这些靶基因,使Et菌在巨噬细胞内的存活受到影响.

Introduction: The targeted delivery of anti-cancer drugs to tumor tissue has been recognized as a promising strategy to increase their therapeutic efficacy and reduce side effects. Mesoporous silica-coated superparamagnetic Fe3O4 nanoparticles (NH2-MSNs), a kind of nanocarrier, can passively enter tumor tissues to enhance the permeability and retention of drugs. However, NH2-MSNs do not specifically bind to cancer cells. This drawback encouraged us to develop a more efficient nanocarrier for cancer therapy. Methods: Herein, we describe the development of an effective nanocarrier based on NH2-MSNs, which were modified with hyaluronic acid on their surface (HA-MSNs) and loaded with doxorubicin (DOX). We have successfully fabricated uniform spherical HA-MSNs nanocarriers. The targeting ability of this delivery system was evaluated through specific uptake by cells and IVIS imaging. Results: DOX-HA-MSNs nanocarriers displayed more dramatic cytotoxic activity against 4T1 breast cancer cells compared to GES-1 gastric mucosa cells. In vivo results revealed that once DOX-HA-MSNs nanocarriers are exposed to an external magnetic field, they could be rapidly attracted to the magnet and effectively cross the cytoplasmic membrane via CD44 receptor-mediated transcytosis. This allows them to access the cancer cell cytoplasm and release DOX based on changes in the physiological environment. Both in vitro and in vivo results demonstrated that the HA-MSNs nanocarriers provided better therapeutic efficacy. Conclusion: The HA-MSNs nanocarriers represent an effective new paradigm to treat cancers due to active targeting to the tumor cells. Moreover, the specific uptake by the tumor effectively protects normal tissues to reduce off-target side effects. The reported findings support further investigation of HA-MSNs for cancer therapy.

Bacterial small non-coding RNAs (sRNAs) are gene expression modulators that respond to environmental changes and pathogenic conditions. In this study, 13 novel sRNAs were identified in the intracellular pathogen, Edwardsiella tarda (E. tarda) ET13 strain, based on RNA sequencing and bioinformatic analyses. Eight of the 13 putative sRNAs from the ET13 strain were transcribed (as indicated by RT-PCR) following exposure to different stresses. The transcription levels of three sRNAs (EsR128, EsR139 and EsR240) were all highly induced under these stress conditions. Northern blot hybridization was employed to verify that EsR240 was expressed in the ET13 strain under both logarithmic and stationary growth phases, and that it formed a single copy transcript in the chromosomes of the ET13 strain. The precise start and end points of EsR240 were determined using 5'and 3' RACE. The conservation of EsR240 was in agreement with the characteristics of sRNA, as indicated by a BLAST analysis. Furthermore, the survival rates of EsR240 mutant were lower than the rates of the wild type ET13 under stress conditions. When the infection time was extended 4 or 6 h, the CFUs of the wild type bacteria increased more significantly within macrophages compared to the mutant. When the intra-peritoneal (i.p.) route of infection was used in mice, the bacterial loads of the tissues in the mice infected with the wild type bacteria were significantly higher than in the mice infected with the mutants. The virulence of the EsR240 mutant was 6.79-fold lower than the wild type bacterium based on the LD50. In addition, the IntaRNA program was used to predict the target genes of EsR240. Out of the top 10 predicted target genes, 9 genes were regulated by EsR240. These target genes may encode FtsH protease modulator YccA, Na+ and H+ antiporters, FtsX-like permease family protein, glycoside hydrolases or various other proteins. Therefore, EsR240 may positively regulate its target genes in E. tarda to maintain intracellular survival within host macrophages and to increase its virulence.

目的:探讨非编码小RNA(sRNA_EsR240)调控迟缓爱德华菌(Edwardsiella tarda,Et)胞内生存和毒力的作用.方法:根据转录组测序,结合生物信息学方法分析ET13菌sRNA.采用RT-PCR检测在不同应激(低酸、营养缺乏、氧化和高盐)条件下sRNA的转录水平,筛选高表达的sRNA;Northern blotting和RACE试验检测EsR240的长度及转录起始和终止位点.采用BLAST软件对EsR240的保守性进行分析;IntaRNA软件预测EsR240靶基因;qRT-PCR检测这些靶基因的表达水平.采用自杀质粒同源重组的方法构建ET13菌的EsR240缺失株和互补株.比较野生株ET13、EsR240缺失株和互补株在胞外不同应激条件下的存活率,及其在巨噬细胞胞内存活能力和对小鼠毒力的差异.结果:根据ET13菌RNA测序的结果,与蛋白库注释基因相比对;比对不上的候选sRNA与sRNA库比对;用软件发现13个具有启动子和ρ-非依赖型终止子新的sRNA,RT-PCR显示其中8个sRNA在各种应激条件下的转录水平不一,EsR128、EsR139和EsR240转录水平均较高.Northern blotting验证了EsR240在Et生长期和稳定期均转录,大小是596 bp.RACE试验确定了EsR240的转录起始和终止位点.BLAST软件分析显示,EsR240在Et中普遍存在,符合sRNA的特征.IntaRNA软件预测EsR240靶基因及qRT-PCR结果显示,EsR240调控大部分预测靶基因的表达,这些靶基因注释可能为三磷酸腺苷结合盒(ATP binding cassette,ABC)-F转运体、FtsH蛋白酶的调控子YccA、Na+/H+反向转运体和糖苷水解酶等;在不同条件下,EsR240调控靶基因数目不同.EsR240的缺失明显影响Et在胞外不同应激条件下存活率和胞内生存能力,而且影响Et对小鼠的毒力.结论:EsR240通过调控靶基因影响Et的胞内生存,是一个正调控Et对小鼠毒力的sRNA.

目的 Ⅲ型分泌系统(T3SS)是迟缓爱德华菌(Edwardsiella tarda,简称Et)一种重要的毒力因子,EseC是T3SS一个转位因子.通过缺失eseC基因,探索Et菌T3SS与该菌感染细胞和组织后,诱导宿主产生炎症反应的关系.方法 构建Et.CD菌eseC基因缺失株及其互补株;用菌落计数法比较两菌株在RAW264.7巨噬细胞胞内存活数目;用CCK-8法比较两菌株感染巨噬细胞后的细胞存活率;利用流式比较野生株和缺失株诱发巨噬细胞的细胞坏死.分别将野生株和缺失株注射小鼠腹腔,用菌落计数法比较两菌株在肝脏、脾脏、肺脏和肾脏中细菌CFU/mL,以及通过观察这些器官的大小和HE染色切片组织,比较两菌株对这些小鼠组织的炎症反应;用ELISA比较这些小鼠血清促炎性细胞因子IL-1β和TNF-α的浓度;用RT-PCR,比较野生株和缺失株效应蛋白基因转录水平.结果 eseC基因缺失后,Et感染巨噬细胞后,胞内细菌数目和细胞存活率均明显降低,说明野生株感染细胞后,可通过其T3SS引起更多细胞的死亡,释放出更多细菌成份;野生株可通过其T3SS诱导更多巨噬细胞发生坏死,伴有大量促炎症因子的释放;T3SS有助于野生株在肝、肺、脾和肾中组织中的大量增殖;野生株感染小鼠后,和eseC基因缺失株相比,它们的肝脏,脾脏和肺脏外观明显肿大,肝、肺、脾和肾组织中有明显的急性炎症反应,野生株诱发产生的促炎性细胞因子TNF-α和IL-1β浓度明显高于缺失株;RT-PCR结果显示,eseC基因的缺失使得Et菌的T3SS效应蛋白的eseJ和eseE基因转录水平明显下降.结论 eseC基因可以通过T3SS影响Et在组织和细胞的致炎作用.

目的 比较高毒力肺炎克雷伯菌与经典肺炎克雷伯茵的毒力特性,探讨不同毒力的肺炎克雷伯茵对视网膜色素上皮细胞炎症相关因子表达的影响.方法 收集8株高毒力肺炎克雷伯菌及8株经典肺炎克雷伯菌,分为高毒力肺炎克雷伯菌组和经典肺炎克雷伯茵组,通过黏液丝实验、PCR扩增rmpA基因检测、细菌抗血清杀伤实验及细菌抗吞噬实验比较两组肺炎克雷伯菌的毒力特性;两组细菌分别感染视网膜色素上皮细胞,通过ELISA检测视网膜色素上皮细胞内单核细胞趋化蛋白-1(monocyte chemotactic protein-1,MCP-1)及白细胞介素-6(interleukin-6,IL-6)表达水平.结果 高毒力肺炎克雷伯菌组细菌的高黏液表型、rmpA基因表达水平、抗血清杀伤能力及抗巨噬细胞吞噬能力均显著高于经典肺炎克雷伯菌组;两组细菌分别感染视网膜色素上皮细胞后,高毒力肺炎克雷伯菌组MCP-1和IL-6含量分别为(1177.38±227.17)ng·L-1、(118.63±40.39)ng·L-1,经典肺炎克雷伯菌组分别为(669.50±62.01)ng· L-1、(53.75±14.04) ng·L-1;视网膜色素上皮细胞MCP-1和IL-6表达水平高毒力肺炎克雷伯菌组分别是经典肺炎克雷伯菌组的1.76倍和2.20倍,差异均有统计学意义(均为P＜0.05).结论 相对于经典肺炎克雷伯菌,高毒力肺炎克雷伯菌具有高黏液表型、抗血清杀伤能力及抗巨噬细胞吞噬能力强的特性,且感染视网膜色素上皮细胞后,可引起更严重的炎症反应.

Our studies tried to demonstrate Eha (Et haemolysin activator) could regulate the resistance of the bacterium against acidification to survive in the macrophage and explain its underlying molecular mechanism.When the bacteria infected the macrophages at time intervals,intracellular survival rate in bafilomycin-treated macrophages was higher than that with untreated cells,and the rate of wild type ET 13 was higher than that of its eha mutant,respectively (P＜0.05).The survival rate of the wild type was higher than that of the mutant under acid treatment (P＜0.05).To determine the conditions that induced the highest eha expression,we constructed a pMP220-Peha LacZ plasmid and determined the lacZ expression under different conditions.After exposure of pH6.3 medium for 2 h time,we performed the whole transcriptomic profiles of the wild type and mutant by RNA-sequencing.We identified 147 differentially-expressed genes ([log2 ratio| ≥1),113 and 34 of which were significantly up-and down-regulated,respectively in the mutant,comparing with the wild type.These findings were validated by qRT-PCR.GO functional analysis revealed that these genes were divided into 25 categories,including the bacterial catalysis,cellular composition,combination,localization,metabolism,processing,and transportation.Based on the KEGG database,these genes were distributed in 55 pathways,such as two-component system,ABC transporters,and microbial metabolism in diverse environments.Overall,Eha is an important regulator to affect all kinds of target genes and pathways for E.tarda to adapt to an acid environment.These results could be helpful for further investigations of the mechanisms by which E.tarda survives in macrophages.

Edwardsiella tarda is a pathogen with a broad host range that infects both animals and humans. Eha is a new transcriptional regulator identified in ET13, which is involved in the bacterial hemolytic activity. This study explored the effect of the Eha in the pathogenesis of E. tarda and the transcriptional regulation of the bacterial virulence genes (eseC, fliC, pagC and fimA). Our results found that the virulence of the eha mutant was 2.5-fold less than the one of its wild ET13 by LD50 in a murine model of i.p. infection, and the bacterial loads of the mutant displayed a different profile from the one of the wild strain. Most significantly, the mice infected with the mutant have greatly reduced acute inflammation in the liver, spleen and kidney compared to the ones infected with the wild. We further demonstrated that eseC, fliC and pagC were regulated directly by the Eha with qRT-PCR and β-Galactosidase assay, but fimA wasn't done. The promoter regions of the genes modulated and the cly gene reported before had been found to contain a common conserved motif by using software. In addition, we found that the wild strain was more toxic to RAW264.7 macrophages, and induced less the host cell apoptotic responses than the eha mutant did. Altogether, these data suggested that the Eha was required for the bacterial infection and the transcriptive regulation of the important virulence genes of E. tarda.

Edwardsiella tarda is distributed widely in a variety of hosts. Eha has recently been found to be its virulence regulator. In order to explore the mechanism of its regulation, we investigated the survival rates of wild type strain ET13, and its eha mutant and complemented strains in RAW264.7 macrophages under light microscopic observation as well as by counting bacterial CFUs on the plates. All of the different strains could live within the macrophages; however, the intracellular numbers of the wild type were significantly higher than the mutant when the incubation time extended 4 h or 6 h (P < 0.05). Furthermore, more ROS were produced by the mutant-infected cells, indicating that Eha may enhance ET13's capacity to detoxify ROS. In agreement with this, we found that the mutant exhibited more sensitivity by H2O2 disk inhibitory assay and less survival ability with H2O2 treatment. We further demonstrated that the bacterial antioxidant enzymes SodC and KatG were regulated by Eha with qRT-PCR and β-galactosidase assay. Collectively, our data show Eha is required for E. tarda to resist the oxidative stress from the macrophages.

目的 eha基因是E.tarda毒力株ET-13一个重要的转录调控基因,本文研究其对该菌株毒力的影响.方法 利用菌落计数法比较野生株和eha缺失株感染小鼠毒力(LD50)的差异,以及比较两菌株在小鼠每个肝脏、脾脏和肾脏中细菌菌落数目的差异;利用组织HE染色观察两菌株对宿主组织损伤的差异;利用RT-PCR,比较两种细菌毒力基因表达的差异.结果 eha缺失株相比野生株其毒力下降了2.5倍;eha缺失株在宿主体内的生存能力明显降低.小鼠感染野生株后,出现肝细胞水肿和中性粒细胞浸润,脾小体内细胞坏死,肾小管水肿等病变,以及上述脏器外观出现改变.提示野生株对小鼠的肝脏、脾脏和肾脏有毒性作用,而eha缺失株对上述脏器无明显毒性作用.RT-PCR结果显示,eha基因的缺失使得E.tarda菌的Ⅲ型分泌系统分泌蛋白基因eseC和外膜蛋白基因(pagC)的转录水平下降,菌毛蛋白基因(fimA)的转录水平没有改变.结论 eha基因缺失后,E.tarda菌ET-13株的毒力因子表达降低,使该菌毒力明显减弱,对宿主的致病性和病理损伤减轻.因此,eha基因是一个毒力正调控基因.

Objective:To study the effect of hOGG1 on human retinal pigment epithelial cells in H2 O2 induced oxidative stress, by observing pAd-CMV5-DEST-hogg1 transfecting APRE-19.Methods: Construct recombinant adenovirus pAd-CMV5-DEST-hogg1, control adenovirus vector pAd/CMV/V5-GW/lacZ, and transfect them into ARPE-19 cells(RPE-Ad-hogg1, RPE-Ad-lacZ) respectively.The two cell lines,together with a normal ARPE-19 cell strain, were treated by H2 O2 (100μmol· L-1 ,2 h) .The oxygen free radicals ( ROS) production was detected by Flow Cytometry(FCM).Immunohistochemisty assessed 8-oxoguanine(8-oxodG),which is the oxidative damage end products.Results:The construction and packaging of recombinant adenovirus-hogg1 vector were successful,&nbsp;and it could be expressed in ARPE-19 cells.ARPE-19 cells with high hOGG1 expression had a lower ROS and 8-oxodG production(P<0.05) and lower degree of oxidational damage on 8-oxodG.Conclusion:A high expression of hOGG1 in ARPE-19 cells can reduce the degree of oxidative stress induced by H2 O2 .