福氏志贺菌O-抗原磷酸乙醇胺修饰研究进展

福氏志贺菌是发展中国家引起痢疾的主要致病菌。福氏志贺菌O-抗原除噬菌体介导的糖基化和(或)乙酰化外,最近发现一种新的修饰方式磷酸乙醇胺修饰,其机制为由质粒携带的opt基因编码磷酸乙醇胺转移酶在O-抗原的鼠李糖II或(和)鼠李糖III上添加磷酸乙醇胺基团,从而形成血清型Xv、4av和Yv福氏志贺菌,并表达MASF IV-1抗原。人工转化携带opt基因的质粒到无MASF IV-1抗原表达的不同血清型福氏志贺菌中,转化菌株都能表达MASF IV-1抗原。在某些血清型福氏志贺菌中,O-抗原的磷酸乙醇胺修饰与糖基化、乙酰化修饰之间相互作用。现对福氏志贺菌的O-抗原磷酸乙醇胺修饰机制及其与糖基化、乙酰化修饰间的相互作用进行了综述。     

应用蛋白质组学方法完善福氏志贺菌基因组注释的研究

常规的应用计算机软件注释基因存在缺陷,目前对基因组的准确注释依然是一项富有挑战性的任务本研究旨在应用蛋白质基因组学(proteogenomics)方法完善福氏志贺菌的基因组注释。提取福氏2a志贺菌301株(Sf2a301)的全菌蛋白,胰蛋白酶水解的肤混合物经二维液相色谱分离、在线ESI串联质谱分析,质谱数据检索Sf2a301的6个读码框数据库,鉴定结果进一步经过生物信息学分析和实验验证。本研究共验证了729个Sf2a301已注释基因的蛋白编码产物,鉴定蛋白在分子量、等电点和疏水性等理化性质方面的分布与Sf2a301基因组已注释蛋白的趋势一致。共发现了6个未注释的新基因,新基因得到了RT-PCR在转录水平上的进一步验证。蛋白质基因组学能够有效的完善志贺菌的基因组注释,不仅验证了已注释基因,而且能够发现新的基因补充其原有基因组注释库,这种策略有望被推广到其他经过测序的生物体基因组注释工作中。     

基因芯片技术探究肺炎克雷伯菌RcsB的转录谱

肺炎克雷伯菌(Klebsiella pneumoniae,KP)是临床上重要的条件致病菌,主要引起肺炎、泌尿系统疾病、肝脓肿以及菌血症等临床疾病。Rcs B是磷酸信号转导系统中的核心调控子,有证据证明Rcs B可调控部分肠杆菌毒力的表达。本研究利用基因芯片技术筛选出Rcs B调控的靶基因并进行生物信息学分析,同时实时荧光定量PCR技术验证基因芯片结果。通过基因芯片技术共筛选出223个差异基因,其中表达下调基因有132个,表达上调基因有91个。q RT-PCR实验显示,挑选的8个基因的q RT-PCR结果均与芯片结果相符。223个差异基因的COG分析表明Rcs B调控的靶基因主要影响能量的产生和转换,碳、氨基酸的转运和代谢以及细胞壁、外膜的生成等细胞途径。GO富集分析表明Rcs B调控的靶基因主要参与碳水化合物衍生物代谢,酶的催化活性,膜合成等生理生化过程。本研究通过全基因组芯片技术对肺炎克雷伯菌Rcs B的转录谱进行了探究,明确了Rcs B调控的靶基因的相关功能及其调控机制,为通过探究Rcs B的靶基因来进一步深入了解Rcs磷酸信号转导系统对肺炎克雷伯菌毒力的影响奠定了基础。     

汉中市中心医院细菌性痢疾志贺菌16S rRNA序列分析、毒力基因与耐药检测

目的对汉中市中心医院细菌性痢疾患者分离的70株志贺菌进行16S rRNA序列分析、毒力基因与耐药检测。方法对2018年6月至2018年12月我院收治的细菌性痢疾患者进行病原菌分离鉴定,并进行16S rRNA遗传进化分析;PCR法检测其6种毒力基因;K-B法检测其对8种抗生素的敏感性。结果 16S rRNA测序结果显示,福氏志贺菌感染率为57.14%(40/70),宋内志贺菌感染率为42.86%(30/70)。16S rRNA系统进化分析结果显示,40株福氏志贺菌与参考菌株(NR_026331.1)同源性为95.2%～99.0%,30株宋内志贺菌与参考菌株(NR_104826.1)同源性为96.3%～99.9%。毒力基因检测结果表明,70株志贺菌均可以检测出6个相关毒力基因set1A、set1B、sen、Ial、ipaH、virA,毒力基因检出率最高的为ipaH,达到100.00%,其次为Ial、virA,均达到90.00%;其中福氏志贺菌set1A、set1B、Ial基因的检出率高于宋内志贺菌,sen和virA基因的检出率低于宋内志贺菌,二者ipaH基因的检出率都为100.00%。药敏试验结果表明,70株志贺菌耐药率最高的抗生素为氨苄西林,耐药率达到80.00%,其次为强力霉素,耐药率达到75.71%,耐药率最低的为头孢吡肟,耐药率为20.00%;其中40株福氏志贺菌耐药率最高的抗生素为强力霉素,其次为氨苄西林,最低的为头孢吡肟;30株宋内志贺菌耐药率最高的抗生素为氨苄西林,其次为哌拉西林,最低的为氧氟沙星。结论 2018年下半年汉中市中心医院从细菌性痢疾患者身上分离得到的志贺菌主要为福氏志贺菌与宋内志贺菌,毒力基因检测和耐药性试验结果显示分离菌株有较强致病性,提示对细菌性痢疾的监测、防控与治疗应进一步加强。     

肺炎克雷伯菌调控蛋白RcsAB的构建表达及活性鉴定

为了探讨转录调控子Rcs AB对靶基因的转录调控作用,构建肺炎克雷伯菌RcsA和RcsB的重组质粒,之后诱导蛋白表达,提取纯化后测定其活性。PCR得到rcsA、rcsB片段,分别将两DNA片段克隆至表达载体pMAL-C5X、pET28a,构建重组质粒。再将重组质粒导入E. coli BL21(DE3)菌株中,经IPTG诱导后收集菌体,超声破碎。破碎后的上清过柱、透析纯化,得到高纯度的蛋白,通过EMSA进行蛋白活性鉴定。RcsA,RcsB蛋白成功表达纯化,并能够与靶基因结合,初步证明蛋白具有生物学活性。成功制备有生物学活性的RcsA、RcsB蛋白,为进一步研究RcsAB蛋白复合物特异的生物学功能提供物质基础。     

志贺菌耐药性与非编码RNA的关系研究

采用RNA-seq技术对志贺菌耐药性与非编码RNA之间的关系进行研究。首先采用梯度剂量的环丙沙星诱导福氏志贺菌2a标准菌株产生耐药性而获得耐药菌株,然后提取标准菌株与耐药菌株的总RNA进行RNA-seq分析。结果显示,在福氏志贺菌的2个样品中共发现100个候选sRNA,并与sRNA的数据库比对得到注释信息,每个候选sRNA都对应着很多个靶标基因,而且靶标基因的表达也有明显差异。预测这些sRNA可能会通过调控耐药基因和一些与细胞增殖或凋亡相关的基因发挥作用,因此还需要挑选合适的候选sRNA并对其用荧光定量PCR实验进行验证。     

铜绿假单胞菌外毒素A的调控基因

铜绿假单胞菌(绿脓杆菌)外毒素A(PEA)为该菌最主要的致病物质,由toxA基因编码.本文综述PEA的调控基因,包括正调控基因regA、regB、lasR、ptxR、vfr和负调控基因fur、ptxS.regA是影响toxA转录的最主要基因.除lasR外,其余其因均在转录水平上通过regA调控PEA的产量.     

单增李斯特菌二硫键形成蛋白DsbG介导酸耐受及鞭毛运动性调控北大核心

【目的】本研究旨在通过构建单增李斯特菌(Listeria monocytogenes)二硫键形成蛋白编码基因dsbG缺失株和回补株,探究该蛋白在酸耐受和鞭毛介导的运动性中发挥的生物学功能。【方法】利用同源重组方法构建单增李斯特菌dsbG缺失株和回补株,比较野生株和突变株在不同pH梯度培养基条件下的生长速率和存活率;通过实时荧光定量PCR方法,比较致死酸应激条件下野生株和缺失株酸耐受基因转录水平。通过半固体培养基、荧光定量PCR方法和鞭毛负染色透射电镜观察,比较野生株和突变株的运动能力、鞭毛相关基因转录水平变化和鞭毛形态差异。【结果】与野生株相比,dsbG缺失株的生长能力和速率差异不显著;在pH 3.5(盐酸和柠檬酸)培养条件下,存活率显著降低;精氨酸合成途径基因argD和argF转录水平分别下调2.4和3.7倍。同时,dsbG缺失株的运动能力减弱,且鞭毛形成相关的flaA、flgB和flgD等基因转录水平显著下调(分别为29.7、6.7和6.9倍),鞭毛形成能力减弱。【结论】本研究首次证实了单增李斯特菌二硫键形成蛋白DsbG能感应低pH应激,并形成耐受;证实了DsbG通过调控鞭毛相关基因的转录进而影响细菌的鞭毛形成和运动性。本研究有助于深入了解二硫键形成蛋白家族介导单增李斯特菌环境适应的分子机制,为食源性致病菌的防控提供理论基础。     

粗糙脉孢菌转录因子LAH-3参与渗透压调控

【目的】通过构建转录因子lah-3基因缺失突变体,研究lah-3基因缺失突变体菌株的渗透压表型,进而探究lah-3基因在渗透压调控中的作用。【方法】采用同源基因重组敲除技术构建lah-3基因缺失突变体。用4%NaCl和1 mol/L Sorbitol进行渗透压处理。利用Northern blot检测渗透压应答基因的表达。利用Westhern blot检测LAH-3蛋白磷酸化修饰水平,OS-2蛋白的表达水平及其磷酸化修饰水平。【结果】在转录因子lah-3基因缺失突变体中,渗透压应答基因gcy-1、stl-1以及pck-1的表达水平都明显降低,而且在渗透压刺激下,LAH-3蛋白磷酸化修饰水平升高。LAH-3的磷酸化修饰不受OS-2调控。lah-3基因的缺失既不影响OS-2蛋白的表达水平,也不影响其在渗透压刺激后的磷酸化修饰。【结论】粗糙脉孢菌中转录因子LAH-3参与调控渗透压应答基因的转录,但其响应过程不依赖于OS-2 MAPK信号通路。     

不同生长时期鼠疫耶尔森菌调控子Fis转录谱的比较分析

目的:进行不同生长时期鼠疫耶尔森菌(鼠疫菌)野生株和fis突变株转录谱的比较分析。方法:基于Red重组系统缺失替换鼠疫菌的fis基因;用鼠疫菌全基因组DNA芯片转录谱技术,比较不同生长时期鼠疫菌野生株和fis突变株在转录水平上的差异;用实时定量RT-PCR对转录谱结果进行验证。结果:构建了鼠疫菌fis::Km突变株,芯片杂交数据与RT-PCR验证的比较结果表明二者有较高的相关性,相关系数r=0.93。结论:无论生长对数期还是稳定期,Fis都能够激活或抑制鼠疫菌一些重要基因的转录,如Ⅲ型分泌系统效应蛋白编码基因、psaAB、pla、rovA等,表明Fis可能与其他调控子一起,在鼠疫菌的代谢及毒力因子转录的协调控制上起关键作用。     

The Rcs phosphorelay system is essential for pathogenicity in Erwinia amylovora

The Rcs phosphorelay system is a modified two-component signal transduction system found exclusively in Enterobacteriaceae . In this study, we characterized the roles of the Rcs system in Erwinia amylovora , a highly virulent and necrogenic enterobacterium causing fire blight disease on rosaceous plants. Our results showed that rcsB , rcsC , rcsD and rcsBD mutants were non-pathogenic on immature pear fruit. The bacterial growth of these mutants was also greatly reduced compared with that of the wild-type strain in immature pear fruit. In an in vitro amylovoran assay, rcsB and rcsD mutants were deficient in amylovoran production, whereas the rcsC mutant exhibited higher amylovoran production than that of the wild-type. Consistent with amylovoran production, expression of the amylovoran biosynthetic gene amsG , using green fluorescent protein as a reporter, was not detectable in rcsB , rcsD and rcsBD mutants both in vitro and in vivo . The expression of amsG in vitro was higher in the rcsC mutant than in the wild-type, whereas its expression in vivo was higher in the wild-type than in the rcsC mutant. In addition, rcs mutants were more susceptible to polymyxin B treatment than the wild-type, suggesting that the Rcs system conferred some level of resistance to polymyxin B. Furthermore, rcs mutants showed irregular and slightly reduced motility on swarming plates. Together, these results indicate that the Rcs system plays a major role in virulence and survival of E. amylovora in immature pear fruit.     

The Rcs phosphorelay modulates the expression of plant cell wall degrading enzymes and virulence in Pectobacterium carotovorum ssp. carotovorum

Production of plant cell wall degrading enzymes, the major virulence factors of soft-rot Pectobacterium species, is controlled by many regulatory factors. Pectobacterium carotovorum ssp. carotovorum SCC3193 encodes an Rcs phosphorelay system that involves two sensor kinases, RcsC(Pcc) and RcsD(Pcc), and a response regulator RcsB(Pcc) as key components of this system, and an additional small lipoprotein RcsF(Pcc). This study indicates that inactivation of rcsC(Pcc), rcsD(Pcc) and rcsB(Pcc) enhances production of virulence factors with the highest effect detected for rcsB(Pcc). Interestingly, mutation of rcsF(Pcc) has no effect on virulence factors synthesis. These results suggest that in SCC3193 a parallel phosphorylation mechanism may activate the RcsB(Pcc) response regulator, which acts as a repressor suppressing the plant cell wall degrading enzyme production. Enhanced production of virulence factors in Rcs mutants is more pronounced when bacteria are growing in the absence of plant signal components.     

Acetyl phosphate-sensitive regulation of flagellar biogenesis and capsular biosynthesis depends on the Rcs phosphorelay

As part of our attempt to map the impact of acetyl phosphate (acetyl approximately P) on the entire network of two-component signal transduction pathways in Escherichia coli, we asked whether the influence of acetyl approximately P on capsular biosynthesis and flagellar biogenesis depends on the Rcs phosphorelay. To do so, we performed a series of epistasis experiments: mutations in the components of the pathway that controls acetyl approximately P levels were combined with mutations in components of the Rcs phosphorelay. Cells that did not synthesize acetyl approximately P produced no capsule under normally permissive conditions, while those that accumulated acetyl approximately P synthesized capsule under conditions previously considered to be non-permissive. Acetyl approximately P-dependent capsular biosynthesis required both RcsB and RcsA, while the lack of RcsC restored capsular biosynthesis to acetyl approximately P-deficient cells. Similarly, acetyl approximately P-sensitive repression of flagellar biogenesis was suppressed by the loss of RcsB (but not of RcsA), while it was enhanced by the lack of RcsC. Taken together, these results show that both acetyl approximately P-sensitive activation of capsular biosynthesis and acetyl approximately P-sensitive repression of flagellar biogenesis require the Rcs phosphorelay. Moreover, they provide strong genetic support for the hypothesis that RcsC can function as either a kinase or a phosphatase dependent on environmental conditions. Finally, we learned that RcsB and RcsC inversely regulated the timing of flagellar biogenesis: rcsB mutants elaborated flagella prematurely, while rcsC mutants delayed their display of flagella. Temporal control of flagella biogenesis implicates the Rcs phosphorelay (and, by extension, acetyl approximately P) in the transition of motile, planktonic individuals into sessile biofilm communities.     

Acid and base resistance in Escherichia coli and Shigella flexneri: role of rpoS and growth pH.

Escherichia coli K-12 strains and Shigella flexneri grown to stationary phase can survive several hours at pH 2 to 3, which is considerably lower than the acid limit for growth (about pH 4.5). A 1.3-kb fragment cloned from S. flexneri conferred acid resistance on acid-sensitive E. coli HB101; sequence data identified the fragment as a homolog of rpoS, the growth phase-dependent sigma factor sigma 38. The clone also conferred acid resistance on S. flexneri rpoS::Tn10 but not on Salmonella typhimurium. E. coli and S. flexneri strains containing wild-type rpoS maintained greater internal pH in the face of a low external pH than strains lacking functional rpoS, but the ability to survive at low pH did not require maintenance of a high transmembrane pH difference. Aerobic stationary-phase cultures of E. coli MC4100 and S. flexneri 3136, grown initially at an external pH range of 5 to 8, were 100% acid resistant (surviving 2 h at pH 2.5). Aerobic log-phase cultures grown at pH 5.0 were acid resistant; survival decreased 10- to 100-fold as the pH of growth was increased to pH 8.0. Extended growth in log phase also decreased acid resistance substantially. Strains containing rpoS::Tn10 showed partial acid resistance when grown at pH 5 to stationary phase; log-phase cultures showed < 0.01% acid resistance. When grown anaerobically at low pH, however, the rpoS::Tn10 strains were acid resistant. E. coli MC4100 also showed resistance at alkaline pH outside the growth range (base resistance). Significant base resistance was observed up to pH 10.2. Base resistance was diminished by rpoS::Tn10 and by the presence of Na+. Base resistance was increased by an order of magnitude for stationary-phase cultures grown in moderate base (pH 8) compared with those grown in moderate acid (pH 5). Anaerobic growth partly restored base resistance in cultures grown at pH 5 but not in those grown at pH 8. Thus, both acid resistance and base resistance show dependence on growth pH and are regulated by rpoS under certain conditions. For acid resistance, and in part for base resistance, the rpoS requirement can be overcome by anaerobic growth in moderate acid.     

Contribution of the lipopolysaccharide to resistance of Shigella flexneri 2a to extreme acidity

Shigella flexneri is endemic in most underdeveloped countries, causing diarrheal disease and dysentery among young children. In order to reach its target site, the colon, Shigella must overcome the acid environment of the stomach. Shigella is able to persist in this stressful environment and, because of this ability it can initiate infection following the ingestion of very small inocula. Thus, acid resistance is considered an important virulence trait of this bacterium. It has been reported that moderate acid conditions regulate the expression of numerous components of the bacterial envelope. Because the lipopolysaccharide (LPS) is the major component of the bacterial surface, here we have addressed the role of LPS in acid resistance of S. flexneri 2a. Defined deletion mutants in genes encoding proteins involved in the synthesis, assembly and length regulation of the LPS O antigen were constructed and assayed for resistance to pH 2.5 after adaptation to pH 5.5. The results showed that a mutant lacking O antigen was significantly more sensitive to extreme acid conditions than the wild type. Not only the presence of polymerized O antigen, but also a particular polymer length (S-OAg) was required for acid resistance. Glucosylation of the O antigen also contributed to this property. In addition, a moderate acidic pH induced changes in the composition of the lipid A domain of LPS. The main modification was the addition of phosphoethanolamine to the 1' phosphate of lipid A. This modification increased resistance of S. flexneri to extreme acid conditions, provide that O antigen was produced. Overall, the results of this work point out to an important role of LPS in resistance of Shigella flexneri to acid stress.     

Acid tolerance of enterohemorrhagic Escherichia coli.

Enterohemorrhagic Escherichia coli (EHEC) strains were tested for their ability to survive in acid pH at 37 degrees C. No loss of viability was observed in an O157:H7 EHEC strain (ATCC 43895) at pH levels of 3.0 and 2.5 for at least 5 h. The level of acid tolerance of most EHEC isolates was very high, similar to that of Shigella flexneri strains. The acid tolerance was dependent on the growth phase and pH of the growth medium.