结核分枝杆菌蛋白Rv3425对细菌表型及毒力影响的研究

为探索蛋白Rv3425在结核分枝杆菌(Mycobacterium tuberculosis,M. tuberculosis)中的功能,本研究以耻垢分枝杆菌(Mycobacterium smegmatis,M. smegmatis)为模式菌株,构建重组了耻垢分枝杆菌Ms-Rv3425。分别将构建菌株(Ms-Rv3425)、野生株(Ms)及空载对照(Ms-Pact)接种于7H9-OADC培养基中37 ℃培养,观察Ms-Rv3425与Ms及Ms-Pact之间在生长速率、菌落形态、生物膜以及聚集度方面的差异。分别用低pH值以及含有十二烷基磺酸钠(sodium dodecyl sulfate,SDS)、氨苄西林、异烟肼及利福平的培养基进行培养,计算存活率以分析抗逆和抗药能力;用上述压力条件培养结核分枝杆菌标准株H37Ra,分析Rv3425内源表达量的变化;进行THP-1细胞感染和BALB/c小鼠攻毒实验分析菌株的毒性变化。结果显示,与Ms及Ms-Pact相比,Ms-Rv3425的菌落形态更为粗糙且隆起,成膜及聚集能力增强;在压力条件下,Ms-Rv3425表现出更高的抗逆和抗药能力,H37Ra中Rv3425的表达量也显著上调;胞内存活率及小鼠致死率更高,各脏器病理损伤更为严重。综上所述,过表达Rv3425能够改变耻垢分枝杆菌的表型,提高抗逆性、抗药性和毒力。深入探讨PPE家族蛋白Rv3425的功能,将为结核病的防治带来新的视角。     

Neutral-red reaction is related to virulence and cell wall methyl-branched lipids in Mycobacterium tuberculosis

Searching for virulence marking tests for Mycobacterium tuberculosis, Dubos and Middlebrook reported in 1948 that in an alkaline aqueous solution of neutral-red, the cells of the virulent H37Rv M. tuberculosis strain fixed the dye and became red in color, whereas the cells of the avirulent H37Ra M. tuberculosis strain remained unstained. In the 1950 and 1960s, fresh isolates of M. tuberculosis were tested for this neutral-red cytochemical reaction and it was reported that they were neutral-red positive, whereas other mycobacteria of diverse environmental origins that were non-pathogenic for guinea pigs were neutral-red negative. However, neutral-red has not really been proven to be a virulence marker. To test if virulence is in fact correlated to neutral-red, we studied a clinical isolate of M. tuberculosis that was originally neutral-red positive but, after more than 1 year passing through culture mediums, turned neutral-red negative. We found that, in comparison to the original neutral-red positive strain, this neutral-red negative variant was attenuated in two murine models of experimental tuberculosis. Lipid analysis showed that this neutral-red negative natural mutant lost the capacity to synthesize pthiocerol dimycocerosates, a cell wall methyl-branched lipid that has been related to virulence in M. tuberculosis. We also studied the neutral-red of different gene-targeted M. tuberculosis mutants unable to produce pthiocerol dimycocerosates or other cell wall methyl-branched lipids such as sulfolipids, and polyacyltrehaloses. We found a negative neutral-red reaction in mutants that were deficient in more than one type of methyl-branched lipids. We conclude that neutral-red is indeed a marker of virulence and it indicates important perturbations in the external surface of M. tuberculosis cells.     

结核杆菌活动期高表达基因Rv1776c的克隆及其在耻垢分枝杆菌中的表达

目的构建表达结核分枝杆菌Rv1776c基因的重组耻垢分支杆菌,并鉴定该基因在重组耻垢分支杆菌中的活性。方法采用PCR技术克隆结核分枝杆菌Rv1776c基因,构建大肠埃希菌-分支杆菌穿梭表达质粒pMV-Rv1776c,通过酶切和测序鉴定其正确性,用电穿孔法将重组质粒转染到耻垢分支杆菌mc^2155中。以SDS-PAGE及Western blot检测证实Rv1776c蛋白在重组耻垢分支杆菌内的表达。结果重组耻垢分支杆菌构建成功,生长曲线说明重组质粒不会影响耻垢分支杆菌的体外生长;SDSPAGE及Western blot检测证实Rv1776c在耻垢分枝杆菌内表达出相对分子量约56kD的Rv1776c蛋白。结论成功构建了Rv1776c基因的穿梭质粒pMV-Rv1776c,且该质粒在耻垢分枝杆菌内具有生物活性,为进一步研究其表达产物的功能提供基础。     

The RD1 proteins of Mycobacterium tuberculosis: expression in Mycobacterium smegmatis and biochemical characterization

A 9.5-kb section of DNA called region of deletion 1 (RD1) is present in virulent Mycobacterium tuberculosis strains but is deleted in all attenuated Mycobacterium bovis BCG vaccine strains. This region codes for at least nine genes. Some or all RD1 gene products may be involved in virulence and pathogenesis, and at least two, ESAT-6 and CFP-10, represent potent T- and B-cell antigens. In order to produce the entire set of RD1 proteins with their natural posttranslational modifications, a robust expression system for M. tuberculosis proteins in the fast-growing saprophytic strain Mycobacterium smegmatis was developed. Our system employs the inducible acetamidase promoter and allows translational fusion of recombinant M. tuberculosis proteins with polyhistidine or influenza hemagglutinin epitope tags for affinity purification. Using eGFP as reporter gene, we showed that the acetamidase promoter is tightly regulated in M. smegmatis and that this promoter is much stronger than the widely used constitutive groEL2 promoter. We then cloned 11 open reading frames (ORFs) found within RD1 and successfully expressed and purified the respective proteins. Sera from tuberculosis patients and M. tuberculosis-infected mice reacted with 10 purified RD1 proteins, thus demonstrating that Rv3871, Rv3872, Rv3873, CFP-10, ESAT-6, Rv3876, Rv3878, Rv3879c and ORF-14 are expressed in vivo. Finally, glycosylation of the RD1 proteins was analyzed. We present preliminary evidence that the PPE protein Rv3873 is glycosylated at its C terminus, thus highlighting the ability of M. smegmatis to produce M. tuberculosis proteins bearing posttranslational modifications.     

Molecular dissection of the role of two methyltransferases in the biosynthesis of phenolglycolipids and phthiocerol dimycoserosate in the Mycobacterium tuberculosis complex

A few mycobacterial species, most of which are pathogenic for humans, produce dimycocerosates of phthiocerol (DIM) and of glycosylated phenolphthiocerol, also called phenolglycolipid (PGL), two groups of molecules shown to be important virulence factors. The biosynthesis of these molecules is a very complex pathway that involves more than 15 enzymatic steps and has just begun to be elucidated. Most of the genes known to be involved in these pathways are clustered on the chromosome of M. tuberculosis. Based on their amino acid sequences, we hypothesized that the proteins encoded by Rv2952 and Rv2959c, two open reading frames of this locus, are involved in the transfer of methyl groups onto various hydroxyl functions during the biosynthesis of DIM, PGL, and related p-hydroxybenzoic acid derivatives (p-HBAD). Using allelic exchange and site-specific recombination, we produced three recombinant strains of Mycobacterium tuberculosis carrying insertions in Rv2952 or Rv2959c. Analysis of these mutants revealed that (i) the protein encoded by Rv2952 is a methyltransferase catalyzing the transfer of a methyl group onto the lipid moiety of phthiotriol and glycosylated phenolphthiotriol dimycocerosates to form DIM and PGL, respectively, (ii) Rv2959c is part of an operon including the newly characterized Rv2958c gene that encodes a glycosyltransferase also involved in PGL and p-HBAD biosynthesis, and (iii) the enzyme encoded by Rv2959c catalyzes the O-methylation of the hydroxyl group located on carbon 2 of the rhamnosyl residue linked to the phenolic group of PGL and p-HBAD produced by M. tuberculosis. These data further extend our understanding of the biosynthesis of important mycobacterial virulence factors and provide additional tools to decipher the molecular mechanisms of action of these molecules during the pathogenesis of tuberculosis.     

Rv3303c of Mycobacterium tuberculosis protects tubercle bacilli against oxidative stress in vivo and contributes to virulence in mice

Ability of Mycobacterium tuberculosis to survive under oxidative stress in vivo is an important aspect of pathogenesis. Rv3303c gene from M. tuberculosis encodes an NAD(P)H quinone reductase. These enzymes have been shown to manage oxidative stress in other pathogenic bacteria. We have hypothesized that Rv3303c protein will remove reactive oxygen species released by the host and hence reduce oxidative stress to M. tuberculosis. rv3303c was PCR cloned and the purified recombinant enzyme reduced superoxide generator menadione. Antisense and sense RNA constructs of rv3303c were electroporated in M. tuberculosis H37Rv. The transformants were characterized by difference in expression of specific mRNA and protein. Antisense transformants were markedly reduced in virulence as compared to sense transformants as judged by several parameters such as weight and survival of infected mice, growth in vivo, colonization and histopathology of lungs. In the presence of menadione, the sense transformant was more resistant to killing in vitro than the antisense transformant. It may be concluded that the rv3303c gene contributes to virulence of M. tuberculosis in vivo and this might be mediated in part by increased resistance to reactive oxygen intermediates thereby enhancing intracellular growth and colonization.     

p-Hydroxybenzoic acid synthesis in Mycobacterium tuberculosis

Glycosylated p-hydroxybenzoic acid methyl esters and structurally related phenolphthiocerol glycolipids are important virulence factors of Mycobacterium tuberculosis. Although both types of molecules are thought to be derived from p-hydroxybenzoic acid, the origin of this putative biosynthetic precursor in mycobacteria remained to be established. We describe the characterization of a transposon mutant of M. tuberculosis deficient in the production of all forms of p-hydroxybenzoic acid derivatives. The transposon was found to be inserted in Rv2949c, a gene located in the vicinity of the polyketide synthase gene pks15/1, involved in the elongation of p-hydroxybenzoate to phenolphthiocerol in phenolic glycolipid-producing strains. A recombinant form of the Rv2949c enzyme was produced in the fast-growing non-pathogenic Mycobacterium smegmatis and purified to near homogeneity. The recombinant enzyme catalyzed the removal of the pyruvyl moiety of chorismate to form p-hydroxybenzoate with an apparent K(m) value for chorismate of 19.7 microm and a k(cat) value of 0.102 s(-1). Strong inhibition of the reaction by p-hydroxybenzoate but not by pyruvate was observed. These results establish Rv2949c as a chorismate pyruvate-lyase responsible for the direct conversion of chorismate to p-hydroxybenzoate and identify Rv2949c as the sole enzymatic source of p-hydroxybenzoic acid in M. tuberculosis.     

结核分枝杆菌Rv3425蛋白的原核表达纯化及其血清学诊断价值

目的：用原核系统表达结核分枝杆菌Rv3425蛋白并纯化,评价该重组蛋白在结核病血清学诊断方面的价值。方法：以结核分枝杆菌H37Rv株基因组为模板,PCR扩增得到Rv3425基因序列,克隆至表达载体pET-28a中,转入大肠杆菌BL21（DE3）进行诱导、表达后纯化,用Western印迹和ELISA法进行抗原性初步评价。结果：在原核系统内经IPTG诱导表达后,Rv3425蛋白主要以包涵体形式存在,经复性和镍柱层析纯化后,纯度达95%以上;Western印迹和ELISA结果证明重组Rv3425具有较强的抗原活性;用纯化的Rv3425蛋白做抗原,临床诊断结核病人血清,阳性率达50%。结论：高纯度的Rv3425蛋白在结核病诊断方面具有很高的应用价值,可作为结核病诊断的备选抗原。     

Characterising Mycobacterium tuberculosis Rv1510c protein and determining its sequences that specifically bind to two target cell lines

The process of Mycobacterium tuberculosis infection of the macrophage implies a very little-known initial recognition and adherence step, important for mycobacterial survival; many proteins even remain like hypothetical. The Rv1510c gene, encoding a putatively conserved membrane protein, was investigated by analysing the M. tuberculosis genome sequence data reported by Cole et al. and a previous report that used PCR assays to show that the Rv1510 gene was only present in M. tuberculosis. This article confirmed all the above and identified the transcribed gene in M. tuberculosis, Mycobacterium africanum, and in M. tuberculosis clinical isolates. Antibodies raised against peptides from this protein recognised a 44 kDa band, corresponding to Rv1510c theoretical mass (44,294 Da). Assays involving synthetic peptides covering the whole protein binding to U937 and A549 cell lines led to recognising five high activity binding peptides in the Rv1510 protein: 11094, 11095, 11105, 11108, and 11111. Their affinity constants and Hill coefficients were determined by using U937 cells. Cross-linking assays performed with some of these HABPs showed that they specifically bound to a U937 cell line 51 kDa protein, but not to Hep G2 or red blood cell proteins, showing this interaction's specificity.     

Horizontal transfer of a virulence operon to the ancestor of Mycobacterium tuberculosis

The contribution of interspecies horizontal gene transfer (HGT) to the evolution and virulence of Mycobacterium tuberculosis, the agent of tuberculosis in humans, has been barely investigated. Here we have studied the evolutionary history of the M. tuberculosis Rv0986-8 virulence operon recently identified, through functional genomics approaches, as playing an important role in parasitism of host phagocytic cells. We showed that among actinobacteria, this operon is specific to the M. tuberculosis complex and to ancestral Mycobacterium prototuberculosis species. These data, together with phylogenetic reconstruction and other in silico analyses, provided strong evidence that this operon has been acquired horizontally by the ancestor of M. tuberculosis, before the recent evolutionary bottleneck that preceded the clonal-like evolution of the M. tuberculosis complex. Genomic signature profiling further suggested that the transfer was plasmid mediated and that the operon originated from a gamma-proteobacterium donor species. Our study points out for the first time the contribution of HGT to the emergence of M. tuberculosis and close relatives as major pathogens. In addition, our data underline the importance of deciphering gene transfer networks in M. tuberculosis in order to better understand the evolutionary mechanisms involved in mycobacterial virulence.     

Characterization of three glycosyltransferases involved in the biosynthesis of the phenolic glycolipid antigens from the Mycobacterium tuberculosis complex

Mycobacterium tuberculosis and Mycobacterium leprae, the two main mycobacterial pathogens in humans, produce highly specific long chain beta-diols, the dimycocerosates of phthiocerol, and structurally related phenolic glycolipid (PGL) antigens, which are important virulence factors. In addition, M. tuberculosis also secretes glycosylated p-hydroxybenzoic acid methyl esters (p-HBAD) that contain the same carbohydrate moiety as the species-specific PGL of M. tuberculosis (PGL-tb). The genes involved in the biosynthesis of these compounds in M. tuberculosis are grouped on a 70-kilobase chromosomal fragment containing three genes encoding putative glycosyltransferases: Rv2957, Rv2958c, and Rv2962c. To determine the functions of these genes, three recombinant M. tuberculosis strains, in which these genes were individually inactivated, were constructed and biochemically characterized. Our results demonstrated that (i) the biosynthesis of PGL-tb and p-HBAD involves common enzymatic steps, (ii) the Rv2957, Rv2958c, and Rv2962c genes are involved in the formation of the glycosyl moiety of the two classes of molecules, and (iii) the product of Rv2962c catalyzes the transfer of a rhamnosyl residue onto p-hydroxybenzoic acid ethyl ester or phenolphthiocerol dimycocerosates, whereas the products of Rv2958c and Rv2957 add a second rhamnosyl unit and a fucosyl residue to form the species-specific triglycosyl appendage of PGL-tb and p-HBAD. The recombinant strains produced provide the tools to study the role of the carbohydrate domain of PGL-tb and p-HBAD in M. tuberculosis pathogenesis.     

Biochemical characterization of a chromosomal toxin-antitoxin system in Mycobacterium tuberculosis

In the present paper, we report the biochemical characterization of a chromosomal toxin-antitoxin (TA) system in Mycobacterium tuberculosis, consisting of the Rv1991c gene and its upstream open reading frame (ORF) termed Rv1991a. Rv1991c was characterized as a toxin with ribonuclease activity and Rv1991a as the antitoxin against Rv1991c. Rv1991a interacted with Rv1991c to form a complex. A promoter located immediately upstream of Rv1991a was identified. Both Rv1991a and the Rv1991a-Rv1991c complex were able to bind to the promoter region of the Rv1991a-Rv1991c operon, indicating that the expression of the Rv1991a-Rv1991c operon can be autoregulated.     

Mycobacterium tuberculosislpdC, Rv0462, induces dendritic cell maturation and Th1 polarization

Mycobacterium tuberculosis, the etiological factor of pulmonary tuberculosis, causes significant morbidity and mortality worldwide. Activation of host immune responses for containment of mycobacterial infections involves participation of innate immune cells, such as dendritic cells (DCs). In this study, we demonstrated that the gene encoding lipoamide dehydrogenase C (lpdC) from M. tuberculosis, Rv0462, induce maturation and activation of DCs involved in the MAPKs signaling pathway. Moreover, Rv0462-treated DCs activated naïve T cells, polarized CD4⁺ and CD8⁺ T cells to secrete IFN-γ in syngeneic mixed lymphocyte reactions, which would be expected to contribute to Th1 polarization of the immune response. Our results suggest that Rv0462 can contribute to the innate and adaptive immune responses during tuberculosis infection, and thus modulate the clinical course of tuberculosis.     

Rv2190c, an NlpC/P60 Family Protein, Is Required for Full Virulence of Mycobacterium tuberculosis

Mycobacterium tuberculosis, the etiologic agent of tuberculosis (TB) possesses at least five genes predicted to encode proteins with NlpC/P60 hydrolase domains, including the relatively uncharacterized Rv2190c. As NlpC/P60 domain-containing proteins are associated with diverse roles in bacterial physiology, our objective was to characterize Rv2190c in M. tuberculosis growth and virulence. Our data indicate that lack of Rv2190c is associated with impaired growth, both in vitro and during an in vivo mouse model of TB. These growth defects are associated with altered colony morphology and phthiocerol dimycocerosate levels, indicating that Rv2190c is involved in cell wall maintenance and composition. In addition, we have demonstrated that Rv2190c is expressed during active growth phase and that its protein product is immunogenic during infection. Our findings have significant implications, both for better understanding the role of Rv2190c in M. tuberculosis biology and also for translational developments.     

Crystal structure of a novel esterase Rv0045c from Mycobacterium tuberculosis

There are at least 250 enzymes in Mycobacterium tuberculosis (M. tuberculosis) involved in lipid metabolism. Some of the enzymes are required for bacterial survival and full virulence. The esterase Rv0045c shares little amino acid sequence similarity with other members of the esterase/lipase family. Here, we report the 3D structure of Rv0045c. Our studies demonstrated that Rv0045c is a novel member of α/β hydrolase fold family. The structure of esterase Rv0045c contains two distinct domains: the α/β fold domain and the cap domain. The active site of esterase Rv0045c is highly conserved and comprised of two residues: Ser154 and His309. We proposed that Rv0045c probably employs two kinds of enzymatic mechanisms when hydrolyzing C-O ester bonds within substrates. The structure provides insight into the hydrolysis mechanism of the C-O ester bond, and will be helpful in understanding the ester/lipid metabolism in M. tuberculosis.     

In vitro and in vivo identification of a novel cytotoxic T lymphocyte epitope from Rv3425 of Mycobacterium tuberculosis

The identification of novel cytotoxic T lymphocyte (CTL) epitopes is important to analysis of the involvement of CD8(+) T cells in Mycobacterium tuberculosis infection as well as to the development of peptide vaccines. In this study, a novel CTL epitope from region of difference 11 encoded antigen Rv3425 was identified. Epitopes were predicted by the reversal immunology approach. Rv3425-p118 (LIASNVAGV) was identified as having relatively strong binding affinity and stability towards the HLA-A*0201 molecule. Peripheral blood mononuclear cells pulsed by this peptide were able to release interferon-γ in healthy donors (HLA-A*02(+) purified protein derivative(+)). In cytotoxicity assays in vitro and in vivo, Rv3425-p118 induced CTLs to specifically lyse the target cells. Therefore, this epitope could provide a subunit component for designing vaccines against Mycobacterium tuberculosis.     

结核分枝杆菌Rv3871抗原优势肽段的克隆表达及抗原性鉴定

目的:为了提高结核病诊断试剂的特异性和敏感性,克隆表达结核分枝杆菌H37Rv株RD1区Rv3871抗原优势肽段,并应用ELISA法对其抗原性进行初步鉴定。方法:利用Biosun生物信息学软件对Rv3871抗原进行表位分析,通过PCR从结核分枝杆菌H37Rv基因组中扩增Rv3871抗原优势肽段编码基因,在大肠杆菌HB101中进行表达,采用间接ELISA法初步评价其抗原性。结果:Rv3871-1肽段检测的敏感性为32.5%(13/40),特异性为97.2%(35/36);Rv3871-2敏感性为45%(18/40),特异性为100%;Rv3871-3敏感性为37.5%(15/40),特异性为91.6%。结论:结核分枝杆菌RD1区Rv3871抗原优势肽段Rv3871-2有较高的特异性和敏感性,有望作为候选抗原用于结核病患者的血清学检测。