Mitochondrial lysyl-tRNA synthetase independent import of tRNA lysine into yeast mitochondria

Aminoacyl tRNA synthetases play a central role in protein synthesis by charging tRNAs with amino acids. Yeast mitochondrial lysyl tRNA synthetase (Msk1), in addition to the aminoacylation of mitochondrial tRNA, also functions as a chaperone to facilitate the import of cytosolic lysyl tRNA. In this report, we show that human mitochondrial Kars (lysyl tRNA synthetase) can complement the growth defect associated with the loss of yeast Msk1 and can additionally facilitate the in vitro import of tRNA into mitochondria. Surprisingly, the import of lysyl tRNA can occur independent of Msk1 in vivo. This suggests that an alternative mechanism is present for the import of lysyl tRNA in yeast.     

Subfamily I Treponema pallidum repeat protein family: sequence variation and immunity

A 12-membered Treponema pallidum repeat (Tpr) protein family has been identified in T. pallidum subsp. pallidum, the causative agent of syphilis. The subfamily I Tpr proteins (C, D, F, and I) possess conserved sequence at the N- and C-termini and central regions that differentiate the members. These proteins may be important in the immune response during syphilis infection and in protective immunity. Strong antibody responses have been observed toward some of the subfamily I Tpr proteins during infection with different syphilis isolates. Some sequence variation has also been identified in one subfamily I Tpr member, TprD, among T. pallidum subsp. pallidum isolates. In this study, we examined sequences in the remaining subfamily I Tpr proteins among strains. Both TprF and TprI were conserved among T. pallidum subsp. pallidum isolates.While some heterogeneity was identified in TprC. We further examined the immune response and protective capacity of TprF protein in this paper. We demonstrate that the N-terminal conserved region of the subfamily I Tpr proteins elicits strong antibody and T-cell responses during infection, and immunization with this region attenuates syphilitic lesion development upon infectious challenge.     

The sequence of the acidic repeat protein (arp) gene differentiates venereal from nonvenereal Treponema pallidum subspecies, and the gene has evolved under strong positive selection in the subspecies that causes syphilis

Despite the completion of the Treponema pallidum genome project, only minor genetic differences have been found between the subspecies that cause venereal syphilis (ssp. pallidum) and the nonvenereal diseases yaws (ssp. pertenue) and bejel (ssp. endemicum). In this paper, we describe sequence variation in the arp gene which allows straightforward differentiation of ssp. pallidum from the nonvenereal subspecies. We also present evidence that this region is subject to positive selection in ssp. pallidum, consistent with pressure from the immune system. Finally, the presence of multiple, but distinct, repeat motifs in both ssp. pallidum and Treponema paraluiscuniculi (the pathogen responsible for rabbit syphilis) suggests that a diverse repertoire of repeat motifs is associated with sexual transmission. This study suggests that variations in the number and sequence of repeat motifs in the arp gene have clinical, epidemiological, and evolutionary significance.     

梅毒螺旋体疫苗的研究进展

梅毒是由密螺旋体苍白亚种( Treponema pallidum subsp. pallidum , Tp)感染引起的慢性系统性性传播疾病,流行于中低等收入国家。越来越多的临床病例表明清除Tp感染需要加强公共卫生筛查和治疗,而接种疫苗是预防Tp感染极有价值和首选的方法。本文概述了研制Tp疫苗的必要性,总结疫苗研究过程中候选抗原的相关信息及递送系统,分析Tp疫苗发展策略。     

梅毒检测技术研究进展

悔毒是由悔毒螺旋体引起的一种性传播疾病,传染性很强,目前已成为世界性的公共安全问题。、早发现早诊断对于悔毒的治疗极为重要．．梅毒的诊断主要依靠实验室检测。我们从病原体、抗体、基因等3个方面对近几年悔毒检测技术和方法进行了综述,论述了其基本原理、研究进展及各自的优缺点。     

Complete genome sequence of Treponema paraluiscuniculi, strain Cuniculi A: the loss of infectivity to humans is associated with genome decay

Treponema paraluiscuniculi is the causative agent of rabbit venereal spirochetosis. It is not infectious to humans, although its genome structure is very closely related to other pathogenic Treponema species including Treponema pallidum subspecies pallidum, the etiological agent of syphilis. In this study, the genome sequence of Treponema paraluiscuniculi, strain Cuniculi A, was determined by a combination of several high-throughput sequencing strategies. Whereas the overall size (1,133,390 bp), arrangement, and gene content of the Cuniculi A genome closely resembled those of the T. pallidum genome, the T. paraluiscuniculi genome contained a markedly higher number of pseudogenes and gene fragments (51). In addition to pseudogenes, 33 divergent genes were also found in the T. paraluiscuniculi genome. A set of 32 (out of 84) affected genes encoded proteins of known or predicted function in the Nichols genome. These proteins included virulence factors, gene regulators and components of DNA repair and recombination. The majority (52 or 61.9%) of the Cuniculi A pseudogenes and divergent genes were of unknown function. Our results indicate that T. paraluiscuniculi has evolved from a T. pallidum-like ancestor and adapted to a specialized host-associated niche (rabbits) during loss of infectivity to humans. The genes that are inactivated or altered in T. paraluiscuniculi are candidates for virulence factors important in the infectivity and pathogenesis of T. pallidum subspecies.     

梅毒疫苗的研究进展

梅毒是由梅毒螺旋体(Tp)感染所引起的性传播疾病。梅毒感染可引发机体多系统慢性持续性损害,可垂直传播,还可明显增加感染和传播艾滋病的危险性。尽管付出相当的努力,但Tp不同寻常的生物学特征及当前梅毒疫苗方法学的不足严重阻碍了梅毒疫苗的研究进程,迄今为止,国内外仍然没有切实有效的疫苗能预防Tp感染。简要介绍了梅毒感染后机体免疫应答特点,综述了梅毒疫苗的类型及疫苗发展中可能存在的障碍,并进一步提出了今后梅毒疫苗设计的新方向。     

An engineered class I transfer RNA with a class II tertiary fold

Structure-based engineering of the tertiary fold of Escherichia coli tRNA(Gln)2 has enabled conversion of this transfer RNA to a class II structure while retaining recognition properties of a class I glutamine tRNA. The new tRNA possesses the 20-nt variable stem-loop of Thermus thermophilus tRNA(Ser). Enlargement of the D-loop appears essential to maintaining a stable tertiary structure in this species, while rearrangement of a base triple in the augmented D-stem is critical for efficient glutaminylation. These data provide new insight into structural determinants distinguishing the class I and class II tRNA folds, and demonstrate a marked sensitivity of glutaminyl-tRNA synthetase to alteration of tRNA tertiary structure.     

TpF1 from Treponema pallidum activates inflammasome and promotes the development of regulatory T cells

Human syphilis is a multistage disease, with diverse and wide-ranging manifestations caused by Treponema pallidum. Despite the fact that a cell-mediated immune response takes part in the course of syphilis, T. pallidum often manages to evade host immunity and, in untreated individuals, may trigger chronic infection. With this study, we demonstrate for the first time, to our knowledge, that Treponema pallidum induces a regulatory T (Treg) response in patients with secondary syphilis and we found that the miniferritin TpF1, produced by the bacterium, is able to expand this response and promote the production of TGF-β. Accordingly, TpF1 stimulates monocytes to release IL-10 and TGF-β, the key cytokines in driving Treg cell differentiation. Interestingly, we also found that TpF1 stimulates monocytes to synthesize and release several proinflammatory cytokines, such as TNF-α, IL-6, and IL-1β, the latter following the activation of the multiprotein complex inflammasome. Collectively, these data strongly support a central role for TpF1 both in the inflammation process, which occurs in particular during the early stage of syphilis, and in the long-term persistence of the spirochete within the host by promoting Treg response and TGF-β production.     

Multiple alleles of Treponema pallidum repeat gene D in Treponema pallidum isolates

Two new tprD alleles have been identified in Treponema pallidum: tprD2 is found in 7 of 12 T. pallidum subsp. pallidum isolates and 7 of 8 non-pallidum isolates, and tprD3 is found in one T. pallidum subsp. pertenue isolate. Antibodies against TprD2 are found in persons with syphilis, demonstrating that tprD2 is expressed during infection.     

检测梅毒螺旋体抗体的纤维膜条方法的建立及其在诊断中的应用

目的：建立以纤维膜为载体的检测梅毒螺旋体抗体的方法,检查病人血清中对梅毒螺旋体多种抗原的抗体,用于梅毒感染的诊断。方法：将基因工程表达及纯化的梅毒螺旋体蛋白tp15、tp17、tp42和tp47分别结合在纤维膜上,用载抗原的纤维膜条检查血清中的抗体,抗体阳性者在相应抗原位置显示出特异条带。结果：梅毒螺旋体感染者血清中存在特异性抗体,在检查的460份临床诊断的患者血清中,对tp15、tp17、tp42和tp47抗原的抗体检出率分别为41.3%、100%、98.7%和51.7%;134份献血员血清抗体阴性。结论：建立的检测梅毒螺旋体感染的方法可同时检查对多种抗原的抗体,以纤维膜条作为诊断条检测血清抗体方法简便,用于临床诊断更特异、更敏感。     

The crystal structure of a Thermus thermophilus tRNA(Gly) acceptor stem microhelix at 1.6 Å resolution

tRNAs are aminoacylated with the correct amino acid by the cognate aminoacyl-tRNA synthetase. The tRNA/synthetase systems can be divided into two classes: class I and class II. Within class I, the tRNA identity elements that enable the specificity consist of complex sequence and structure motifs, whereas in class II the identity elements are assured by few and simple determinants, which are mostly located in the tRNA acceptor stem. The tRNA(Gly)/glycyl-tRNA-synthetase (GlyRS) system is a special case regarding evolutionary aspects. There exist two different types of GlyRS, namely an archaebacterial/human type and an eubacterial type, reflecting the evolutionary divergence within this system. We previously reported the crystal structures of an Escherichia coli and of a human tRNA(Gly) acceptor stem microhelix. Here we present the crystal structure of a thermophilic tRNA(Gly) aminoacyl stem from Thermus thermophilus at 1.6? resolution and provide insight into the RNA geometry and hydration.     

Immunity in experimental syphilis. IV. Serological reactivity of antigens extracted from gamm-irradiated Treponema pallidum and Treponema reiteri

Miller, James N. (University of California School of Medicine, Los Angeles), J. H. De Bruijn, and J. H. Bekker. Immunity in experimental syphilis. IV. Serological reactivity of antigens extracted from gamma-irradiated Treponema pallidum and Treponema reiteri. J. Bacteriol. 91:583-587. 1966.-Ultrasonic lysate preparations extracted from virulent Treponema pallidum, Nichols strain, suspensions exposed to 652,800 R of gamma-irradiation exhibited a loss in the serological reactivity of their heat-labile antigens; the heat-stable components of both the lysate and residue antigens were unaffected. The activity of heat-stable, cardiolipin T. pallidum complement-fixing antigen obtained from similarly irradiated organisms was also unaltered. gamma-Irradiation of the cultivable Treponema reiteri with dosages as high as 6,500,000 R failed to alter serologically either the heat-labile or heat-stable component of its lipopolysaccharide-protein (Reiter protein) antigen. The reactivity of the lipopolysaccharide portion of the Reiter protein complex with an antiserum to T. pallidum Nichols indicates previously unsuspected antigenic differences between the rabbit-adapted Nichols strain of the organism and so-called "wild" human strains of T. pallidum in which this antigen is generally absent.     

Physical map of the genome of Treponema pallidum subsp. pallidum (Nichols).

A physical map of the chromosome of Treponema pallidum subsp. pallidum (Nichols), the causative agent of syphilis, was constructed from restriction fragments produced by NotI, SfiI, and SrfI. These rare-cutting restriction endonucleases cleaved the T. pallidum genome into 16, 8, and 15 fragments, respectively. Summation of the physical lengths of the fragments indicates that the chromosome of T. pallidum subsp. pallidum is approximately 1,030 to 1,080 kbp in size. The physical map was constructed by hybridizing a variety of probes to Southern blots of single and double digests of T. pallidum genomic DNA separated by contour-clamped homogeneous electric field electrophoresis. Probes included cosmid clones constructed from T. pallidum subsp. pallidum genomic DNA, restriction fragments excised from gels, and selected genes. Physical mapping confirmed that the chromosome of T. pallidum subsp. pallidum is circular, as the SfiI and SrfI maps formed complete circles. A total of 13 genes, including those encoding five membrane lipoproteins (tpn47, tpn41, tpn29-35, tpn17, and tpn15), a putative outer membrane porin (tpn50), the flagellar sheath and hook proteins (flaA and flgE), the cytoplasmic filament protein (cfpA), 16S rRNA (rrnA), a major sigma factor (rpoD), and a homolog of cysteinyl-tRNA synthetase (cysS), have been localized in the physical map as a first step toward studying the genetic organization of this noncultivable pathogen.     

Use of the polymerase chain reaction to detect DNA sequences specific to pathogenic treponemes in cerebrospinal fluid

The polymerase chain reaction was used to detect Treponema pallidum in specimens of cerebrospinal fluid (CSF), as a means of diagnosing syphilis. Segments of the TmpA and 4D genes were amplified to provide an estimated threshold sensitivity of approximately 65 organisms in 0.5 ml. A spectrum of pathogens known to cause meningitis, and several non-pathogenic treponemes were unreactive. Treponema pertenue, and only one of 30 control specimens of CSF were positive. In contrast, 10 of 19 CSFs from patients being evaluated for latent or tertiary syphilis were positive, as were 7 of 28 specimens from HIV-positive patients.     

Human tryptophanyl-tRNA synthetase is switched to a tRNA-dependent mode for tryptophan activation by mutations at V85 and I311

For most aminoacyl-tRNA synthetases (aaRS), their cognate tRNA is not obligatory to catalyze amino acid activation, with the exception of four class I (aaRS): arginyl-tRNA synthetase, glutamyl-tRNA synthetase, glutaminyl-tRNA synthetase and class I lysyl-tRNA synthetase. Furthermore, for arginyl-, glutamyl- and glutaminyl-tRNA synthetase, the integrated 3' end of the tRNA is necessary to activate the ATP-PPi exchange reaction. Tryptophanyl-tRNA synthetase is a class I aaRS that catalyzes tryptophan activation in the absence of its cognate tRNA. Here we describe mutations located at the appended beta1-beta2 hairpin and the AIDQ sequence of human tryptophanyl-tRNA synthetase that switch this enzyme to a tRNA-dependent mode in the tryptophan activation step. For some mutant enzymes, ATP-PPi exchange activity was completely lacking in the absence of tRNA(Trp), which could be partially rescued by adding tRNA(Trp), even if it had been oxidized by sodium periodate. Therefore, these mutant enzymes have strong similarity to arginyl-tRNA synthetase, glutaminyl-tRNA synthetase and glutamyl-tRNA synthetase in their mode of amino acid activation. The results suggest that an aaRS that does not normally require tRNA for amino acid activation can be switched to a tRNA-dependent mode.     

Immuno-PCR法诊断早期梅毒的方法学建立及其应用

目的建立Immuno-PCR法诊断早期梅毒的方法学,评价其灵敏度、特异性、重复性及其临床应用。方法利用基因重组TpN47抗原免疫新西兰兔,制备抗体并用Weston blotting检测;利用抗TpN47抗体作为捕获抗体与血清中TpN47抗原结合,通过链霉亲和素、生物素化抗体、生物素化DNA和PCR扩增等建立Immuno-PCR法检测梅毒螺旋体抗原TpN47体系;评价该方法的灵敏度、特异性和重复性;收集200例临床标本通过Immuno-PCR法、ELISA、TPPA和TURST法进行临床应用比较。结果 Weston blotting结果显示TpN47抗体阳性;Immuno-PCR比ELISA法敏感性强103倍,比TPPA、TURST强105倍;特异性高,重复性好。临床标本中Immuno-PCR法敏感性和特异性分别为86.00%（P〈0.05）和100.00%,ELISA法为71.00%和98.00%,TPPA法为65.00%和100.00%,TRUST法为68.00%和95.00%。结论 Immuno-PCR法检测梅毒螺旋体TpN47抗原敏感性高,特异性强,重复性好,可作为梅毒螺旋体感染的早期诊断方法 。     

Molecular evolution of the tprC, D, I, K, G, and J genes in the pathogenic genus Treponema

We investigated the evolution of 6 genes from the Treponema pallidum repeat (tpr) gene family, which encode potential virulence factors and are assumed to have evolved through gene duplication and gene conversion events. The 6 loci (tprC, D, G, J, I, and K) were sequenced and analyzed in several members of the genus Treponema, including the 3 subspecies of human T. pallidum (T. pallidum subsp. pallidum, pertenue, and endemicum), Treponema paraluiscuniculi (rabbit syphilis), and the unclassified Fribourg-Blanc (simian) isolate. Phylogenetic methods, recombination analysis, and measures of nucleotide diversity were used to investigate the evolutionary history of the tpr genes. Numerous instances of gene conversion were detected by all 3 methods including both homogenizing gene conversion that involved the entire length of the sequence as well as site-specific conversions that affected smaller regions. We determined the relative age and directionality of the gene conversion events whenever possible. Our data are also relevant to a discussion of the evolution of the treponemes themselves. Higher levels of variation exist between the human subspecies than within them, supporting the classification of the human treponemes into 3 subspecies. In contrast to published theories, the divergence and diversity of T. pallidum subsp. pertenue relative to the other subspecies does not support a much older origin of yaws at the emergence of modern human, nor is the level of divergence seen in T. pallidum subsp. pallidum consistent with a very recent (< 500 years) origin of this subspecies. In general, our results demonstrate that intragenomic recombination has played a significant role in the evolution of the studied tpr genes and emphasize that efforts to infer evolutionary history of the treponemes can be complicated if past recombination events are not recognized.