问号钩体粘附侵袭相关基因特征分析

使用NCBI ,Swissprot/TrEMBL ,ProDom ,Pfam ,Tmpred ,SignalP ,ClustW等网络资源和软件 ,根据问号钩体黄疸出血型赖株粘附侵袭相关基因诠释结果 ,对mce ,invA ,mviN和atsE四个粘附侵袭相关基因编码蛋白的结构域、跨膜区域和信号肽等进行了详细分析 ,并使用Bioedit,Mega2软件进行氨基酸多重序列比较并绘制系统发生树。结果显示 ,mce和mviN为穿膜蛋白 ,invA和atsE为菌体内蛋白质 ;许多对哺乳动物和对植物致病的微生物具有mce ,invA ,mviN和atsE四个粘附侵袭相关基因 ,其表达的蛋白质在感染宿主过程中起重要作用 ,钩体的粘附侵袭相关蛋白与它们在一级结构上有较高相似性。据生物信息学结果推测 ,问号钩体黄疸出血型赖株粘附侵袭相关基因和钩体致病性间有密切关系 ,其编码蛋白在致病过程中可能起重要作用     

The number of large ribosomal RNA genes in Leptospira interrogans and Leptospira biflexa

We determined the number of large ribosomal RNA genes in five strains of Leptospira by hybridization of 15 restriction endonuclease digests of genomic DNA to the [32P]-labeled fragment of 23s rRNA gene. Almost all the restriction gels gave two radioactive bands. The conclusion from these results is that there are at least two rRNA genes in these leptospiral strains. Furthermore, the hybridization patterns of L. icterohaemorrhagiae strains Ictero No. I and RGA are almost identical. The number of rRNA genes and taxonomic relationships of these leptospires were discussed.     

Genome conservation in isolates of Leptospira interrogans.

Reference strains for each of the 23 serogroups of Leptospira interrogans yielded different pulsed-field gel electrophoresis patterns of NotI digestion products. This was also the case for the 14 serovars belonging to serogroup Icterohaemorrhagiae (with one exception). The NotI restriction patterns of 45 clinical leptospiral isolates belonging to serovar icterohaemorrhagiae were analyzed and compared with those of type strains. No differences were observed between isolates from countries of different continents, namely, France, French Guiana, New Caledonia, and Tahiti. The pattern was indistinguishable from that of the reference strain of serovar icterohaemorrhagiae.     

Signaling mechanisms involved in the response to genotoxic stress and regulating lifespan

Ageing is defined by the loss of functional reserve over time, leading to a decreased capacity to maintain homeostasis under stress and increased risk of morbidity and mortality. Ageing is extremely heterogeneous between individuals and even between tissues within an organism, making it challenging to identify the molecular basis of ageing. Much of our current understanding of ageing comes from genetic studies in model organisms seeking genes that either accelerate or decelerate the ageing process. These studies revealed not only causes of ageing, but also signaling mechanisms that both promote and protect against ageing. In all cases, the signaling pathways that influence lifespan are familiar mechanisms that regulate cellular metabolism, growth, proliferation, differentiation and survival. This review highlights the significant overlap in signaling mechanisms implicated in both the cellular response to genotoxic stress and regulation of organism lifespan.     

PIASy mediates NEMO sumoylation and NF-kappaB activation in response to genotoxic stress

Protein modification by SUMO (small ubiquitin-like modifier) is an important regulatory mechanism for multiple cellular processes. SUMO-1 modification of NEMO (NF-kappaB essential modulator), the IkappaB kinase (IKK) regulatory subunit, is critical for activation of NF-kappaB by genotoxic agents. However, the SUMO ligase, and the mechanisms involved in NEMO sumoylation, remain unknown. Here, we demonstrate that although small interfering RNAs (siRNAs) against PIASy (protein inhibitor of activated STATy) inhibit NEMO sumoylation and NF-kappaB activation in response to genotoxic agents, overexpression of PIASy enhances these events. PIASy preferentially stimulates site-selective modification of NEMO by SUMO-1, but not SUMO-2 and SUMO-3, in vitro. PIASy-NEMO interaction is increased by genotoxic stress and occurs in the nucleus in a manner mutually exclusive with IKK interaction. In addition, hydrogen peroxide (H2O2) also increases PIASy-NEMO interaction and NEMO sumoylation, whereas antioxidants prevent these events induced by DNA-damaging agents. Our findings demonstrate that PIASy is the first SUMO ligase for NEMO whose substrate specificity seems to be controlled by IKK interaction, subcellular targeting and oxidative stress conditions.     

Separation of recombination and SOS response in Escherichia coli RecA suggests LexA interaction sites

RecA plays a key role in homologous recombination, the induction of the DNA damage response through LexA cleavage and the activity of error-prone polymerase in Escherichia coli. RecA interacts with multiple partners to achieve this pleiotropic role, but the structural location and sequence determinants involved in these multiple interactions remain mostly unknown. Here, in a first application to prokaryotes, Evolutionary Trace (ET) analysis identifies clusters of evolutionarily important surface amino acids involved in RecA functions. Some of these clusters match the known ATP binding, DNA binding, and RecA-RecA homo-dimerization sites, but others are novel. Mutation analysis at these sites disrupted either recombination or LexA cleavage. This highlights distinct functional sites specific for recombination and DNA damage response induction. Finally, our analysis reveals a composite site for LexA binding and cleavage, which is formed only on the active RecA filament. These new sites can provide new drug targets to modulate one or more RecA functions, with the potential to address the problem of evolution of antibiotic resistance at its root.     

Systemic understanding of Lactococcus lactis response to acid stress using transcriptomics approaches

Journal of Industrial Microbiology & Biotechnology - During fermentation, acid stress caused by the accumulation of acidic metabolites seriously affects the metabolic activity and production...     

问号钩端螺旋体基因组DNA芯片的研制

目的 研制并初步评估问号钩端螺旋体(简称钩体)赖型赖株的基因组DNA芯片。方法 利用Primegens引物设计软件筛选出问号钩体赖型赖株全基因组中的特异性基因进行引物设计。对成功设计出相应引物的3 290个基因用聚合酶链反应方法进行扩增,以纯化后的产物点样制备芯片。并用双色荧光杂交策略对芯片质量进行了初步平估。结果 共获得3 290个基因产物用于点样。参考株自身杂交实验结果表明:该芯片有较高的点一致性、信噪比和较低的假阳性率。结论 成功制备了包含问号钩体赖型赖株3 290个目的基因的基因组DNA芯片,并可用于基于该芯片的问号钩体比较基因组学的研究。     

Development of a Multiple-Locus Variable number of tandem repeat Analysis (MLVA) for Leptospira interrogans and its application to Leptospira interrogans serovar Australis isolates from Far North Queensland, Australia

The human gastric pathogen Helicobacter pylori causes chronic gastritis, peptic ulcer disease, gastric carcinoma, and mucosa-associated lymphoid tissue (MALT) lymphoma. It infects over 50% of the worlds' population, however, only a small subset of infected people experience H. pylori-associated illnesses. Associations with disease-specific factors remain enigmatic years after the genome sequences were deciphered. Infection with strains of Helicobacter pylori that carry the cytotoxin-associated antigen A (cagA) gene is associated with gastric carcinoma. Recent studies revealed mechanisms through which the cagA protein triggers oncopathogenic activities. Other candidate genes such as some members of the so-called plasticity region cluster are also implicated to be associated with carcinoma of stomach. Study of the evolution of polymorphisms and sequence variation in H. pylori populations on a global basis has provided a window into the history of human population migration and co-evolution of this pathogen with its host. Possible symbiotic relationships were debated since the discovery of this pathogen. The debate has been further intensified as some studies have posed the possibility that H. pylori infection may be beneficial in some humans. This assumption is based on increased incidence of gastro-oesophageal reflux disease (GERD), Barrett's oesophagus and adenocarcinoma of the oesophagus following H. pylori eradication in some countries. The contribution of comparative genomics to our understanding of the genome organisation and diversity of H. pylori and its pathophysiological importance to human healthcare is exemplified in this review.     

Regulation of Rfa2 phosphorylation in response to genotoxic stress in Candida albicans

Successful pathogens must be able to swiftly respond to and repair DNA damages inflicted by the host defence. The replication protein A (RPA) complex plays multiple roles in DNA damage response and is regulated by phosphorylation. However, the regulators of RPA phosphorylation remain unclear. Here, we investigated Rfa2 phosphorylation in the pathogenic fungus Candida albicans. Rfa2, a RFA subunit, is phosphorylated when DNA replication is inhibited by hydroxyurea and dephosphorylated during the recovery. By screening a phosphatase mutant library, we found that Pph3 associates with different regulatory subunits to differentially control Rfa2 dephosphorylation in stressed and unstressed cells. Site‐directed mutagenesis revealed T11, S18, S29, and S30 being critical for Rfa2 phosphorylation in response to genotoxic insult. We obtained evidence that the genome integrity checkpoint kinase Mec1 and the cyclin‐dependent kinase Clb2–Cdc28 mediate Rfa2 phosphorylation. Although cells expressing either a phosphomimetic or a non‐phosphorylatable version of Rfa2 had defects, the latter exhibited greater sensitivity to genotoxic challenge, failure to repair DNA damages and to deactivate Rad53‐mediated checkpoint pathways in a dosage‐dependent manner. These mutants were also less virulent in mice. Our results provide important new insights into the regulatory mechanism and biological significance of Rfa2 phosphorylation in C. albicans.     

问号钩端螺旋体中一个新的OmpA家族蛋白抗原性及保守性研究

目的克隆表达和鉴定问号钩端螺旋体(L.interrogans)黄疸出血群赖型赖株中一个新的外膜蛋白(Omp)A家族基因LA0301,研究LA0301编码蛋白的抗原性和在15个钩端螺旋体(简称钩体)血清群代表株中的保守性,探讨其在疫苗研究中的意义。方法生物信息学软件分析预测LA0301的特征。构建原核表达重组体pQE31-LA0301,经IPTG诱导后用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)及蛋白质印迹法(Western blot)鉴定表达情况。用表达的重组蛋白免疫BALB/c小鼠,Western印迹检测其免疫反应性和在不同血清型钩体中的保守性。酶联免疫吸附试验(ELISA)和Western印迹检测兔抗钩体全菌血清中的LA0301编码蛋白的抗体。结果生物信息学预测结果显示,LA0301具有OmpA家族的结构域。克隆表达了重组质粒pQE31-LA0301,重组蛋白能刺激BALB/c小鼠产生特异性抗体,效价为1:32000。在兔抗钩体全菌血清中检测到特异的LA0301蛋白抗体,并在15个血清群的代表株钩体中均可检测到LA0301蛋白。结论LA0301蛋白是问号钩体中一个新的OmpA家族蛋白,具有良好的抗原性和保守性,并且能在钩体感染的过程中刺激机体产生相应的抗体。为进一步研究钩体新型疫苗候选基因奠定了基础。     

Genetic differences among the LPS biosynthetic loci of serovars of Leptospira interrogans and Leptospira borgpetersenii

The gene organization in the lipopolysaccharide biosynthetic (rfb) locus was analyzed in seven Leptospira interrogans serovars within serogroup Icterohemorrhagiae, seven non-Icterohemorrhagiae serovars and one Leptospira borgpetersenii serovar. Two groups of loci were delineated based on DNA hybridization and sequence analysis. Group 1 contained the two Hardjo subtypes, Hardjoprajitno and Hardjobovis. Group 2 (containing Copenhageni, Pomona, Naam, Mwogolo, Smithi, Lai, Canicola, Autumnalis, Pyrogenes, Australis and Icterohemorrhagiae) differed from Group 1 in its organization upstream of orf11, where five ORFs (32, 33, 34, 35, 37) were identified that were not contained in the Group 1 loci. These ORFs encoded a putative epimerase (orf32), a glycosyltransferase (orf33), two integral membrane proteins (orfs 34 and 35), and a galactosyltransferase (orf37). Serovars Australis, Pomona and Autumnalis did not contain orf37. Serovar Bataviae was excluded from the grouping because of its unique genetic organization upstream of orf13. In the Group 2 loci, comparison of the genetic layout at the 5' end revealed differences which included mutations disrupting reading frames in either or both orf34 and orf35 and apparent allelic differences between orf33 homologs that may be sufficient to account for the genetic basis of serovar identity.     

Axial filament involvement in the motility of Leptospira interrogans.

Motility mutants of Leptospira interrogans serovar illini were isolated and analyzed by dark-field and electron microscopy. Mutants were obtained by screening for small colonies after nitrosoguanidine treatment. One class of mutants did not have hook- or spiral-shaped ends. In addition, the axial filaments from these mutants were not coiled. An analysis of revertants of two of the mutants in this class indicated that the mutations were pleiotropic with respect to motility, hook- and spiral-shaped ends, and axial filament coiling. We conclude that the axial filaments and the hook- and spiral-shaped ends are involved in L. interrogans motility.     

Surface proteomics and label-free quantification of Leptospira interrogans serovar Pomona

Leptospirosis is a re-emerging zoonosis with a global distribution. Surface-exposed outer membrane proteins (SE-OMPs) are crucial for bacterial–host interactions. SE-OMPs locate and expose their epitope on cell surface where is easily accessed by host molecules. This study aimed to screen for surface-exposed proteins and their abundance profile of pathogenic Leptospira interrogans serovar Pomona. Two complementary approaches, surface biotinylation and surface proteolytic shaving, followed by liquid chromatography tandem-mass spectrometry (LC-MS/MS) were employed to identify SE-OMPs of intact leptospires. For quantitative comparison, in-depth label-free analysis of SE-OMPs obtained from each method was performed using MaxQuant. The total number of proteins identified was 1,001 and 238 for surface biotinylation and proteinase K shaving, respectively. Among these, 39 were previously known SE-OMPs and 68 were predicted to be localized on the leptospiral surface. Based on MaxQuant analysis for relative quantification, six known SE-OMPs including EF- Tu, LipL21, LipL41, LipL46, Loa22, and OmpL36, and one predicted SE-OMPs, LipL71 were found in the 20 most abundant proteins, in which LipL41 was the highest abundant SE-OMP. Moreover, uncharacterized LIC14011 protein (LIP3228 ortholog in serovar Pomona) was identified as a novel predicted surface βb-OMP. High-abundance leptospiral SE-OMPs identified in this study may play roles in virulence and infection and are potential targets for development of vaccine or diagnostic tests for leptospirosis.     

Immunodominant antigens recognized by the human immune response to infection by organisms of the species Leptospira interrogans serogroup Australis

Abstract Serum samples from patients infected by organisms of Leptospira interrogans serogroup Australis were tested by Western blot to determine the nature of major antigens that are involved in the immune response. Although there was some patient-to-patient variability, immunodominant genus-specific antigens were found to be proteins of apparent molecular ratio 68, 46 and 35-kDa, and lipopolysaccharide (LPS) sub-units in the 35-14-kDa region. Serogroup epitopes specific for Australis were exclusively saccharides of about 32 and 24 kDa: a serovar-specific antigen for serovar lora was of 38–40 kDa and behaved like a protein. Antibodies to the LPS serogroup-specific antigens and to the 38–40 kDa protein were long-lasting and consequently suggest that these immunodominant epitopes are important in resistance to re-infection.