F0F1-ATPase分子马达技术对单核细胞增生李斯特菌检测的研究

利用载色体chromatophore上的F₀F₁-ATPase分子马达生物传感器,建立了应用在食品检测中快速检测方法。首先从嗜热菌中提取载色体后,标记荧光探针F-DHPE,合成生物素化单增李氏菌prfA探针,在已标记F-DHPE的载色体ATP合酶的ε亚基上连接ε亚基抗体-生物素-链霉亲和素-生物素-prfA探针,将待测单核细胞增生李斯特菌标准菌株和阴性对照分别与此生物传感器结合,通过环境H+量测定ATP产生量,进而对单核细胞增生李斯特菌DNA进行检测。结果表明,chro prfA(连接在载色体chro上的prfA探针)的浓度在0.052 mg/mL,单核细胞增生李斯特菌DNA浓度在40 ng/mL为最适检测条件。通过传统检测方法及PCR检测方法对照,本方法具有良好的检测符合性。     

片球菌素PediocinPA-1抑菌活性检测

目的检测通过基因工程获得的片球菌素Pediocin PA-1抑菌活性。方法采用琼脂扩散法检测片球菌素Pediocin PA-1对单核细胞增生李斯特杆菌、金黄色葡萄球菌、铜绿假单胞菌、沙门菌和大肠埃希菌O157的抑菌活性。结果片球菌素Pediocin PA-1对单核细胞增生李斯特杆菌、金黄色葡萄球菌、沙门菌、铜绿假单胞菌和大肠埃希菌O157等均有抑制作用。其中对单核细胞增生李斯特杆菌、沙门菌、大肠埃希菌和金黄色葡萄球菌的抑制作用效果明显,对铜绿假单胞菌有微弱的抑制作用。结论通过基因工程获得的片球菌素Pediocin PA-1具有抑菌活性。     

2016-2018年辽宁省食品中单核细胞增生李斯特菌污染情况及毒力基因的检测

目的了解辽宁省食品中单核细胞增生李斯特菌毒力基因携带特点,对该省食品中单核细胞增生李斯特菌的污染情况进行调查。方法依据GB 4789.30-2016《食品安全国家标准 食品微生物学检验 单核细胞增生李斯特氏菌检验》及采用PCR扩增技术检测的方法对2016-2018年采自该省15个监测点、收集的3 310份食品检出的47株单核细胞增生李斯特菌进行9种毒力基因检测。结果食品中单核细胞增生李斯特菌检出率为1.42%(47/3310),食品中单核细胞增生李斯特菌最少携带3种毒力基因,其中携带prfA、plcA、hly、mpl、plcB、inlA、inlB和iap八种毒力基因是该省食品中单核细胞增生李斯特菌的主要毒力基因型,达到检出菌总数的65.96%。结论研究结果证实辽宁省食品中存在单核细胞增生李斯特菌污染情况,应严格监控食品中单核细胞增生李斯特菌的携带情况。     

单核细胞增生李斯特氏菌PCR-DHPLC检测新技术的建立

应用PCR结合变性高效液相色谱(DHPLC)技术建立食品中单核细胞增生李斯特氏菌fimY的快速检测方法。根据单核细胞增生李斯特氏菌prfA和hlyA基因序列的特点设计特异性引物,PCR扩增的产物经DHPLC技术进行快速检测。以单核细胞增生李斯特氏菌等61株参考菌株做特异性试验;单核细胞增生李斯特氏菌菌株稀释成不同梯度,进行灵敏度试验。试验结果表明该方法具有很好的特异性,灵敏度较高,检测低限可达到为181CFU/ml。可以快速、准确检测单核细胞增生李斯特氏菌,是食品中致病菌快速检测的新技术。     

植物乳杆菌素L-1对单核细胞增生李斯特氏菌作用机理的研究

以凝胶层析纯化的植物乳杆菌素作用单核细胞增生李斯特氏菌,结果表明该细菌素可以导致能量化的敏感细胞胞内K 、无机磷离子、乳酸脱氢酶、紫外吸收物质和ATP发生不同程度的泄漏,相应地破坏了膜Δψ和部分ΔpH,引起PMF的耗散,结果导致细胞的死亡。综合所测指标,可以推测植物乳杆菌素L-1对单增李斯特氏菌的作用目标主要是细胞膜,通过形成非选择性孔洞使得选择性离子和小分子生命物质外泄,从而打破原有平衡,最终引起细胞的衰亡。     

单核细胞增生李斯特菌抗酸应激机制

单核细胞增生李斯特菌(Listeria monocytogenes,简称单增李斯特菌)是重要的人畜共患食源性病原,在青贮饲料、发酵食品、宿主胃内以及巨噬细胞吞噬体内都会遭遇酸应激。该菌有多种抗酸应激系统,如F0F1-ATPase、谷氨酸脱羧酶(Glutamate decarboxylase system,GAD)、精氨酸脱亚胺酶(Arginine deiminase,ADI)、鲱氨酸脱亚胺酶(Agmatine deiminase,Ag DI)系统等。在环境pH(pHex)4.5条件下可维持其细胞内pH(pHi)稳态,在pHex 3.5时仍能存活;用温和酸应激(pHex 4.5)预处理单增李斯特菌,可以通过酸耐受反应(Acid tolerance response)提高其在致死性酸性环境中的存活率,这一过程受σB正调控,即σB激活可以保护单增李斯特菌应对多种环境应激。因此,σB可以作为新型抗菌药物的靶标。更为重要的是,弱酸性发酵食品要严格控制李斯特菌的污染,以降低消费者的感染风险。     

单核细胞增生李斯特菌的检测技术进展

单核细胞增生李斯特菌（Listeria monocytogenes）是一类人畜共患的食源性致病菌。近年来其检测技术取得了迅猛的发展,本文对目前使用的基于培养、免疫学和分子生物学技术的三大类单核细胞增生李斯特菌检测方法进行了综述,同时对单核细胞增生李斯特菌检测的新策略进行了展望。     

InlA和InlB介导单核细胞增生李斯特菌入侵宿主细胞分子机制的研究进展

单核细胞增生李斯特菌(Listeria monocytogenes)是一种革兰氏阳性食源性致病菌。在造成宿主食源性感染的过程中, 单核细胞增生李斯特菌能凭借其独特的表面蛋白入侵宿主的非吞噬细胞。内化素蛋白家族(Internalins)是介导单核细胞增生李斯特菌入侵宿主非吞噬细胞的主要因子。本文根据国内外一些最新的研究成果, 结合作者近几年的工作, 综述了在侵染宿主的过程中, 单核细胞增生李斯特菌主要的内化素蛋白InlA和InlB介导细菌入侵宿主细胞的分子机制, 以期为阐明食源性致病菌致病机理、预防和治疗食源性疾病提供理论基础。     

凉拌豆制品中单核细胞增生李斯特菌生长模型建立的研究

单核细胞增生李斯特菌是凉拌豆制品中检出率较高的一种致病菌。本研究考察了温度(4℃、15℃、25℃、30℃和37℃)对凉拌豆制品中单核细胞增生李斯特菌生长的影响,并采用SGompertz和SLogistic模型对不同温度下单核细胞增生李斯特菌的生长数据进行拟合;在此基础上,以拟合度(R2)、准确因子(A_f)和偏差因子(B_f)为指标,比较并建立了凉拌豆制品中单核细胞增生李斯特菌的二级生长模型。结果表明,SGompertz模型能更好的拟合凉拌豆制品中单核细胞增生李斯特菌在不同温度下的生长数据,平方根模型能够较准确地预测凉拌豆制品中单核细胞增生李斯特菌的生长状况,因此依次选择此两种模型作为单核细胞增生李斯特菌在凉拌豆制品中的一级和二级生长模型,且模型具有可靠性。研究结果可为凉拌豆制品中单核细胞增生李斯特菌的定量风险评估提供依据。     

单核细胞增生李斯特菌单克隆抗体的制备与鉴定

以单核细胞增生李斯特菌细胞碎片免疫BALB/c小鼠,间接ELISA法成功筛选获得2株稳定分泌抗LM的单克隆杂交瘤细胞株4A7、4H11.抗体效价为1∶160 000以及1∶20 000,亚型为IgG1、IgG2a,Dot-ELISA结果表明4A7和4H11单克隆抗体具有很好的属特异性,Western blot分析表明4A7、4H11抗体分别与单核细胞增生李斯特菌62 kDa以及32 kDa外膜蛋白抗原表位结合,胶体金免疫电镜实验进一步确证以上抗体可有效识别单核细胞增生李斯特菌细胞表面抗原.     

Activation and inhibition of the Escherichia coli F1-ATPase by monoclonal antibodies which recognize the epsilon subunit

The properties of two monoclonal antibodies which recognize the epsilon subunit of Escherichia coli F1-ATPase were studied in detail. The epsilon subunit is a tightly bound but dissociable inhibitor of the ATPase activity of soluble F1-ATPase. Antibody epsilon-1 binds free epsilon with a dissociation constant of 2.4 nM but cannot bind epsilon when it is associated with F1-ATPase. Likewise epsilon cannot associate with F1-ATPase in the presence of high concentrations of epsilon-1. Thus epsilon-1 activates F1-ATPase which contains the epsilon subunit, and prevents added epsilon from inhibiting the enzyme. Epsilon-1 cannot bind to membrane-bound F1-ATPase. The epsilon-4 antibody binds free epsilon with a dissociation constant of 26 nM. Epsilon-4 can bind to the F1-ATPase complex, but, like epsilon-1, it reverses the inhibition of F1-ATPase by the epsilon subunit. The epsilon subunit remains crosslinkable to both the beta and gamma subunits in the presence of epsilon-4, indicating that it is not grossly displaced from its normal position by the antibody. Presumably the activation arises from more subtle conformational effects. Antibodies epsilon-4 and delta-2, which recognizes the delta subunit, both bind to F1F0 in E. coli membrane vesicles, indicating that these subunits are substantially exposed in the membrane-bound complex. Epsilon-4 inhibits the ATPase activity of the membrane-bound enzyme by about 50%, and Fab prepared from epsilon-4 inhibits by about 40%. This inhibition is not associated with any substantial change in the major apparent Km for ATP. These results suggest that inhibition of membrane-bound F1-ATPase arises from steric effects of the antibody.     

Mechanically driven proton conduction in single delta-free F0F1-ATPase

In order to observe mechanically driven proton flux in F(0)F(1)-ATPase coupled with artificial driven rotation on F(1) simultaneously, a double channel observation system was established. An artificial delta-free F(0)F(1)-ATPase was constructed with alpha(3), beta(3), epsilon, gamma, and c(n) subunits as rotator and a, b(2) as stator. The chromatophore was immobilized on the glass surface through biotin-streptavidin-biotin system, and the magnetic bead was attached to the beta subunit of delta-free F(0)F(1)-ATPase. The mechanically driven proton flux was indicated by the fluorescence intensity change of fluorescein reference standard (F1300) and recorded by a cooled digital CCD camera. The mechanochemical coupling stoichiometry between F(0) and F(1) is about 4.15 +/- 0.2H(+)/rev when the magnetic field rotated at 0.33 Hz (rps).     

Mitochondrial adenosine triphosphatase in mit- mutants of Saccharomyces cerevisiase with defective subunit 6 of the enzyme complex

mit- Mutants carrying genetically defined mutations in the oli2 region of the mitochondrial DNA were analysed. Most of these mutants demonstrated either the absence of subunit 6 or its replacement by shorter mitochondrial translation products which could be shown to be structurally related to subunit 6 by using a rabbit anti F1F0-antiserum, and by limited proteolytic mapping of the new mitochondrial translation products. Three representative oli2 mit- strains were analysed for the effects of a grossly altered subunit 6 or of a complete absence of this subunit on the activity and assembly of the H+-ATPase. Our results suggest that this subunit is not required for the assembly of the proton channel of the enzyme complex. Thus, in the absence of subunit 6, the mitochondrial respiratory activities in the oli2 mutants were found to be still sensitive to oligomycin, a specific inhibitor of the H+-ATPase proton channel. Immunoprecipitation of the assembled H+-ATPase subunits from these mutant strains using a monoclonal anti-beta-subunit antibody indicates that subunit 6 is also not essential for the assembly of most F1 subunits to components of the F0 sector.     

Mitochondrial H+-ATPase in mutants of Saccharomyces cerevisiae with defective subunit 8 of the enzyme complex

Mutants of Saccharomyces cerevisiae carrying defined lesions in the mitochondrial aap1 gene, coding for membrane subunit 8 of the H+-ATPase, have been investigated to examine the consequence of the mutations on the function and assembly of the enzyme complex. These include three mit- mutants, which cannot grow by oxidative metabolism due to their inability to synthesize full-length subunit 8, and three partial revertants of one of the mutants. The mutations in these strains have been previously characterized by DNA sequencing. The use of a monoclonal antibody to the beta subunit of the H+-ATPase as a probe of assembly defect revealed that the presence of subunit 8 is essential for the assembly of subunit 6 to the enzyme complex. Mitochondria isolated from the mit- mutants have negligible [32Pi]ATP exchange activity and they exhibited ATPase activity which is not sensitive to inhibition by oligomycin, indicating a defective membrane F0 sector. Normal assembly of subunit 8 (and subunit 6) was observed in the revertant strains, despite 8-9 amino-acid substitutions in the membrane-spanning region of the H+-ATPase subunit 8 in two of the strains. The assembled complex, however, exhibited reduced [32Pi]ATP exchange activity and low sensitivity to oligomycin, indicating that the product of the aap1 gene is a functional subunit of the mitochondrial H+-ATPase.     

F1F0-ATPase from Escherichia coli with mutant F0 subunits. Partial purification and immunoprecipitation of F1F0 complexes

Previously identified mutations in subunits a and b of the F0 sector of the F1F0-ATPase from Escherichia coli are further characterized by isolating detergent-solubilized, partially purified F1F0 complexes from cells bearing these mutations. The composition of the various F1F0 complexes was judged by quantitating the amount of each subunit present in the detergent-solubilized preparations. The composition of the F0 sectors containing altered polypeptides was determined by quantitating the F0 subunits that were immunoprecipitated by antibodies directed against the F1 portion. In this way, the relative amounts of F0 subunits (a, b, c) which survived the isolation procedure bound to F1 were determined for each mutation. This analysis indicates that both missense mutations in subunit a (aser206----leu and ahis245----tyr) resulted in the isolation of F1F0 complexes with normal subunit composition. The nonsense mutation in subunit a (atyr235----end) resulted in isolation of a complex containing the b and c subunits. The bgly131----asp mutation in the b subunit results in an F0 complex which does not assemble or survive the isolation. The isolated F1F0 complex containing the mutation bgly9----asp in the b subunit was defective in two regards: first, a reduction in F1 content relative to F0 and second, the absence of the a subunit. Immunoprecipitations of this preparation demonstrated that F1 interacts with both c and mutant b subunits. A strain carrying the mutation, bgly9----asp, and the compensating suppressor mutation apro240----leu (previously shown to be partially unc+) yielded an F1F0 ++ complex that remained partially defective in F1 binding to F0 but normal in the subunit composition of the F0 sector. The assembly, structure, and function of the F1F0-ATPase is discussed.     

Structure of the yeast mitochondrial adenosine triphosphatase. Results of trypsin degradation

A comparison was made of the subunit sensitivities of the F1-ATPase and the Triton-solubilized ATPase complex to trypsin degradation. The dissociation of the F1-ATPase from ATPase complex increased the trypsin sensitivity of subunit 3 by a factor of 2 and increased the sensitivity of a particular trypsin site (or group of sites) on subunit 1 by 7-fold. The overall degradation of subunits 1 and 2 appears to be the same in solubilized ATPase complex and the F1-ATPase. Implications of these findings for structural models of the ATPase complex are discussed.     

Epsilon subunit, an endogenous inhibitor of bacterial F(1)-ATPase, also inhibits F(0)F(1)-ATPase

Since the report by Sternweis and Smith (Sternweis, P. C., and Smith, J. B. (1980) Biochemistry 19, 526-531), the epsilon subunit, an endogenous inhibitor of bacterial F(1)-ATPase, has long been thought not to inhibit activity of the holo-enzyme, F(0)F(1)-ATPase. However, we report here that the epsilon subunit is exerting inhibition in F(0)F(1)-ATPase. We prepared a C-terminal half-truncated epsilon subunit (epsilon(DeltaC)) of the thermophilic Bacillus PS3 F(0)F(1)-ATPase and reconstituted F(1)- and F(0)F(1)-ATPase containing epsilon(DeltaC). Compared with F(1)- and F(0)F(1)-ATPase containing intact epsilon, those containing epsilon(DeltaC) showed uninhibited activity; severalfold higher rate of ATP hydrolysis at low ATP concentration and the start of ATP hydrolysis without an initial lag at high ATP concentration. The F(0)F(1)-ATPase containing epsilon(DeltaC) was capable of ATP-driven H(+) pumping. The time-course of pumping at low ATP concentration was faster than that by the F(0)F(1)-ATPase containing intact epsilon. Thus, the comparison with noninhibitory epsilon(DeltaC) mutant shed light on the inhibitory role of the intact epsilon subunit in F(0)F(1)-ATPase.     

Isolated epsilon subunit of thermophilic F1-ATPase binds ATP

F1-ATPase, a soluble part of the F0F1-ATP synthase, has subunit structure alpha3beta3gammadeltaepsilon in which nucleotide-binding sites are located in the alpha and beta subunits and, as believed, in none of the other subunits. However, we report here that the isolated epsilon subunit of F1-ATPase from thermophilic Bacillus strain PS3 can bind ATP. The binding was directly demonstrated by isolating the epsilon subunit-ATP complex with gel filtration chromatography. The binding was not dependent on Mg2+ but was highly specific for ATP; however, ADP, GTP, UTP, and CTP failed to bind. The epsilon subunit lacking the C-terminal helical hairpin was unable to bind ATP. Although ATP binding to the isolated epsilon subunits from other organisms has not been detected under the same conditions, a possibility emerges that the epsilon subunit acts as a built in cellular ATP level sensor of F0F1-ATP synthase.