结核分支杆菌H37Rv株菌体抗原单克隆抗体制备、特性分析及纯化

目的　利用单克隆抗体技术分析分支杆菌多肽抗原的共性和个性 ,建立纯化单克隆抗体的最佳方法。方法　按常规方法制备单克隆抗体。分泌单克隆抗体阳性杂交细胞株的筛选采用ELISA法 ,确认采用免疫印迹法。 2 4种分支杆菌菌株均生长于改良罗氏培养基上 ,H3 7Rv株分别生长于改良罗氏培养基和苏通液体培养基上。单克隆抗体的纯化采用硫酸铵沉淀法、辛酸沉淀法和离子交换法。结果　经筛选获得 14株单克隆抗体杂交细胞瘤。其中 ,C2 、7C12 单克隆抗体所抗的 76 0 0 0、16 0 0 0抗原成份为分泌抗原 ,76 0 0 0抗原存在于 2 4种分支杆菌菌体中 ,为共同抗原。 16 0 0 0抗原仅存在于H3 7Rv ,H3 7Ra,牛分支杆菌 ,BCG株中。单克隆抗体免疫印迹结果显示 ,分支杆菌H3 7Rv株在不同培养条件下 ,其 40 0 0 0 / 380 0 0多肽抗原表达不同。单克隆抗体纯化以硫酸铵沉淀法回收率最高 ,离子交换法获得的单抗纯度最高 ,辛酸沉淀法可以有效地去除白蛋白 ,并具有简单、省时等优点。单克隆抗体的相对亲和力均在 10 -4 ～ 10 -7之间。结论　C2 、7C12 单克隆抗体均抗结核分支杆菌H3 7Rv株的分泌抗原成份。 40 0 0 0 / 380 0 0多肽抗原为结核分支杆菌H3 7Rv株固体培养菌体特有。单抗纯化结果显示离子交换层析法适用于科研分析 ,而辛酸     

Population genomics of Mycobacterium tuberculosis in the Inuit

Nunavik, Québec suffers from epidemic tuberculosis (TB), with an incidence 50-fold higher than the Canadian average. Molecular studies in this region have documented limited bacterial genetic diversity among Mycobacterium tuberculosis isolates, consistent with a founder strain and/or ongoing spread. We have used whole-genome sequencing on 163 M. tuberculosis isolates from 11 geographically isolated villages to provide a high-resolution portrait of bacterial genetic diversity in this setting. All isolates were lineage 4 (Euro-American), with two sublineages present (major, n = 153; minor, n = 10). Among major sublineage isolates, there was a median of 46 pairwise single-nucleotide polymorphisms (SNPs), and the most recent common ancestor (MRCA) was in the early 20th century. Pairs of isolates within a village had significantly fewer SNPs than pairs from different villages (median: 6 vs. 47, P < 0.00005), indicating that most transmission occurs within villages. There was an excess of nonsynonymous SNPs after the diversification of M. tuberculosis within Nunavik: The ratio of nonsynonymous to synonymous substitution rates (dN/dS) was 0.534 before the MRCA but 0.777 subsequently (P = 0.010). Nonsynonymous SNPs were detected across all gene categories, arguing against positive selection and toward genetic drift with relaxation of purifying selection. Supporting the latter possibility, 28 genes were partially or completely deleted since the MRCA, including genes previously reported to be essential for M. tuberculosis growth. Our findings indicate that the epidemiologic success of M. tuberculosis in this region is more likely due to an environment conducive to TB transmission than a particularly well-adapted strain.The tubercule bacillus, Mycobacterium tuberculosis, is a highly successful, medically important human-adapted pathogen. Studies of diverse strain collections reveal a geographic aggregation of the principal M. tuberculosis lineages (1) consistent with a dissemination of this organism around the world with the paleo migration (2). Ancient DNA studies also support the notion that M. tuberculosis has caused disease in humans for thousands of years. Thus, it can be inferred that M. tuberculosis has evolved in step with its human host, successfully responding to changes in the host and its environment that could affect the capacity to cause transmissible disease.In contrast to the global diversity of M. tuberculosis strains (1–3), we have previously observed limited genetic diversity in the Nunavik region of Québec (4). One possible explanation is a founder strain, wherein genetic similarity is due to a single recent introduction of a bacterium and may not necessarily represent ongoing spread between communities. In this scenario, isolates might have indistinguishable genotypes by conventional genotyping modalities (restriction fragment length polymorphism, mycobacterial interspersed repetitive units, spoligotyping) but distinct genotypes when assessed using a higher-resolution method, namely whole-genome sequencing (WGS) (5). An additional explanation is that a single clone of M. tuberculosis is currently spreading both within and between villages; however, the great distances between these communities that are not linked by roads make intervillage spread less likely. These possible explanations need not be mutually exclusive.To evaluate these possibilities, we conducted WGS on M. tuberculosis isolates from Nunavik isolated over 23 y. Estimation of the divergence date of the most recent common ancestor (MRCA) provided evidence that tuberculosis (TB) was introduced into this region in the early 20th century, following which time there has been substantial ongoing transmission, predominantly within villages. This setting provides a unique opportunity to study the genomic characteristics of an epidemiologically successful strain of M. tuberculosis over time.     

结核分枝杆菌分泌蛋白MPT64单克隆抗体的制备及其特异性

目的 制备结核分枝杆菌(Mycobacterium tuberculosis,Mtb)分泌蛋白MPT64的单克隆抗体(Monoclonal antibody,mAb),并分析mAb的特异性。方法 PCR扩增mpt64基因并克隆入pET28a(+)构建原核表达载体;将获得的重组菌株在IPTG作用下诱导表达MPT64蛋白,Western blot验证蛋白表达;亲和层析法纯化目的蛋白。重组MPT64蛋白免疫小鼠,取脾细胞与杂交瘤细胞SP2/0融合,筛选得到阳性杂交瘤细胞系。以该杂交瘤细胞制备小鼠腹水,中压液相色谱仪纯化腹水获得mAb。ELISA法检测获得的mAb的相对亲和力和亚类,Western blot检测mAb的特异性。结果 本研究成功构建原核表达载体pET28a(+)-mpt64并诱导表达了MPT64蛋白。亲和层析法获得纯化的重组MPT64蛋白。该重组蛋白免疫小鼠的脾细胞和SP2/0细胞融合后,经筛选获得能够稳定分泌抗MPT64 mAb的杂交瘤细胞系MPT64-A5B2。中压液相色谱仪纯化小鼠腹水中的MPT64-A5B2 mAb。该mAb的亲和力为2.813×10^-7 g/mL,属于IgG 1亚类。MPT64-A5B2 mAb能特异性识别Mtb中的MPT64蛋白。结论 本研究制备了MPT64重组蛋白,并获得了抗MPT64 mAb,为MPT64用于结核病诊断和治疗制剂的研制奠定了基础。     

Monoclonal antibodies to Mycobacterium tuberculosis CDC 1551 reveal subcellular localization of MPT51

Mycobacterium tuberculosis CDC 1551, a highly immunogenic outbreak strain, previously reported to have unique surface distribution of capsular polysaccharide, was used to generate novel monoclonal antibodies (mabs) to surface mycobacterial targets. Two immunoglobulin G1 (IgG1) mAbs, 16a1 and 16a6 were generated. The mAbs originated from the same B cell, bound strongly to whole cell M. tuberculosis CDC1551 and to its cell wall, membrane and cytosol fractions recognizing a 90kDa protein. Immunoprecipitation using mAb 16a1 isolated a protein with amino acid peptide sequences matching MPT51 from the cytosol. This immunogenic protein of unknown function was previously reported only in culture filtrates of M. tuberculosis. Our findings suggest for the first time that this protein is found within the M. tuberculosis cell.     

Structural analysis of the dodecameric proteasome activator PafE in Mycobacterium tuberculosis

The human pathogen Mycobacterium tuberculosis (Mtb) requires a proteasome system to cause lethal infections in mice. We recently found that proteasome accessory factor E (PafE, Rv3780) activates proteolysis by the Mtb proteasome independently of adenosine triphosphate (ATP). Moreover, PafE contributes to the heat-shock response and virulence of Mtb. Here, we show that PafE subunits formed four-helix bundles similar to those of the eukaryotic ATP-independent proteasome activator subunits of PA26 and PA28. However, unlike any other known proteasome activator, PafE formed dodecamers with 12-fold symmetry, which required a glycine-XXX-glycine-XXX-glycine motif that is not found in previously described activators. Intriguingly, the truncation of the PafE carboxyl-terminus resulted in the robust binding of PafE rings to native proteasome core particles and substantially increased proteasomal activity, suggesting that the extended carboxyl-terminus of this cofactor confers suboptimal binding to the proteasome core particle. Collectively, our data show that proteasomal activation is not limited to hexameric ATPases in bacteria.Although the ubiquitin proteasome pathway plays essential roles in eukaryotes (reviewed in refs. 1 and 2), most bacterial species do not have proteasome systems and instead degrade proteins using ATP-dependent proteases like ClpP, Lon, and HslUV (reviewed in refs. 3 and 4). However, bacteria of the orders Actinomycetales and Nitrospirales also encode proteasomes that are structurally highly similar to eukaryotic and archaeal proteasomes (reviewed in refs. 5 and 6). Importantly, the human pathogen Mycobacterium tuberculosis (Mtb), an Actinomycete, requires proteasomal function to cause lethal infections in mice (7). Ablation of proteasomal degradation sensitizes bacteria to nitric oxide, an antimicrobial free radical made by macrophages and other cell types, and attenuates bacterial growth in mice (7–9). The potential to target persistent or latent bacteria has made the Mtb proteasome system a prioritized target for the development of antituberculosis drugs (10, 11). Indeed, Mtb-specific proteasome inhibitors have been identified that may provide a promising lead for new drugs to treat tuberculosis (12, 13).There are numerous similarities and differences between eukaryotic and bacterial proteasomes. The 20S proteasome core particle (20S CP), which consists of two seven-membered β-rings between two seven-membered α-rings, is highly conserved structurally between prokaryotes and eukaryotes (14–16). However, the accessory factors that associate with the 20S CPs quickly diverge among the domains of life. Both bacteria and eukaryotes use a covalent small protein modification to mark substrate proteins for degradation; however, the eukaryotic ubiquitin tag is a well-folded protein whereas the Mtb Pup (prokaryotic ubiquitin-like protein) tag is intrinsically disordered (17, 18). Furthermore, degradation of ubiquitylated proteins by eukaryotic 20S CPs largely relies on a complex regulatory particle that caps one or both ends of the 20S CP and includes a heterohexameric ring of adenosine triphosphatases (ATPases) for substrate recognition and unfolding (reviewed in refs. 19 and 20). In contrast, the mycobacterial 20S CP uses a homohexameric ATPase ring called Mpa (mycobacterial proteasome ATPase) for both the recognition and unfolding of pupylated proteins (18, 21, 22).In addition to the ATPase activators, proteolysis by eukaryotic proteasomes can also be stimulated by several ATP-independent factors, such as the 11S activators PA26 and PA28, as well as Blm10 (23–28). We and another group recently discovered that Mtb has an analogous factor encoded by Rv3780 that we call PafE (proteasome accessory factor E; also known as Bpa for bacterial proteasome activator), which stimulates the degradation of small peptides and β-casein in vitro (29, 30). Both studies also showed that a carboxyl (C)-terminal glycine-glutamine-tyrosine-leucine (GQYL) motif is essential for interacting with and activating 20S CPs, and the penultimate tyrosine residue contributes to activation similarly to tyrosines observed in the “HbYX” (hydrophobic-tyrosine-any amino acid) motif in other characterized proteasome activators (reviewed in ref. 28). Our work further showed that PafE promotes the degradation of at least one native Mtb protein substrate, heat-shock protein repressor (HspR), and that an Mtb pafE mutant is sensitive to heat shock and is attenuated for growth in mice (30). Importantly, PafE-mediated degradation does not require pupylation. Thus, there appear to be at least two independent paths for targeting proteins to the mycobacterial proteasome for degradation.Like the eukaryotic 11S proteasome activators, PafE does not require ATP to stimulate proteolysis. However, it was unknown if PafE formed heptameric complexes like PA26 or PA28. In this work, we show that PafE monomers assume a four-helix bundle structure that is similar to that found in 11S activators, but assemble differently into an unprecedented dodecameric ring structure with 12-fold symmetry. We used isothermal titration calorimetry, cryo-electron microscopy (cryo-EM), and X-ray crystallography to analyze interactions between PafE and 20S core particles, and found that PafE binding induces a larger gate-opening change than has been described for other organisms. We also found that PafE has an extended C terminus that limits the ability of PafE to activate proteasomal degradation in vitro and in vivo.     

Dispersal of Mycobacterium tuberculosis via the Canadian fur trade

Patterns of gene flow can have marked effects on the evolution of populations. To better understand the migration dynamics of Mycobacterium tuberculosis, we studied genetic data from European M. tuberculosis lineages currently circulating in Aboriginal and French Canadian communities. A single M. tuberculosis lineage, characterized by the DS6(Quebec) genomic deletion, is at highest frequency among Aboriginal populations in Ontario, Saskatchewan, and Alberta; this bacterial lineage is also dominant among tuberculosis (TB) cases in French Canadians resident in Quebec. Substantial contact between these human populations is limited to a specific historical era (1710-1870), during which individuals from these populations met to barter furs. Statistical analyses of extant M. tuberculosis minisatellite data are consistent with Quebec as a source population for M. tuberculosis gene flow into Aboriginal populations during the fur trade era. Historical and genetic analyses suggest that tiny M. tuberculosis populations persisted for ～100 y among indigenous populations and subsequently expanded in the late 19th century after environmental changes favoring the pathogen. Our study suggests that spread of TB can occur by two asynchronous processes: (i) dispersal of M. tuberculosis by minimal numbers of human migrants, during which small pathogen populations are sustained by ongoing migration and slow disease dynamics, and (ii) expansion of the M. tuberculosis population facilitated by shifts in host ecology. If generalizable, these migration dynamics can help explain the low DNA sequence diversity observed among isolates of M. tuberculosis and the difficulties in global elimination of tuberculosis, as small, widely dispersed pathogen populations are difficult both to detect and to eradicate.     

抗结核分枝杆菌38kD抗原单克隆抗体的制备及荧光抗体检测方法的建立

目的制备抗结核分枝杆菌单克隆抗体，经纯化后标记荧光素，建立直接免疫荧光法用于痰标本的结核分枝杆菌检测。方法常规方法制备单克隆抗体。ELISA法筛选阳性杂交瘤细胞株，用免疫印迹法确认。单克隆抗体采用饱和硫酸铵粗提和SephadexG-50层析纯化。单抗纯化后采用直接法标记异硫氰酸荧光素，标记后的荧光抗体用于临床痰涂片结核分枝杆菌的检测。结果经筛选获得4株单克隆抗体杂交瘤细胞株。ELISA检测小鼠腹水单克隆抗体效价达到1：6400—1：12800。免疫印迹实验表明4株单抗均为抗结核分枝杆菌38kDa蛋白单抗，其中两株产生较强的免疫反应条带。采用异硫氟酸荧光素标记纯化的单抗，建立直接免疫荧光检测法，对41份结核病患者的痰标本进行检测，阳性率为90．24％，与抗酸染色法比较，直接荧光检测法敏感性显著高于抗酸染色法（P〈0．05）。结论应用抗结核分枝杆菌38kDa蛋白的单克隆抗体，建立直接免疫荧光检测法．应用于临床痰标本结核分枝杆菌检测，对于辅助诊断结核病具有一定价值。     

Peptidoglycan synthesis in Mycobacterium tuberculosis is organized into networks with varying drug susceptibility

Peptidoglycan (PG), a complex polymer composed of saccharide chains cross-linked by short peptides, is a critical component of the bacterial cell wall. PG synthesis has been extensively studied in model organisms but remains poorly understood in mycobacteria, a genus that includes the important human pathogen Mycobacterium tuberculosis (Mtb). The principle PG synthetic enzymes have similar and, at times, overlapping functions. To determine how these are functionally organized, we carried out whole-genome transposon mutagenesis screens in Mtb strains deleted for ponA1, ponA2, and ldtB, major PG synthetic enzymes. We identified distinct factors required to sustain bacterial growth in the absence of each of these enzymes. We find that even the homologs PonA1 and PonA2 have unique sets of genetic interactions, suggesting there are distinct PG synthesis pathways in Mtb. Either PonA1 or PonA2 is required for growth of Mtb, but both genetically interact with LdtB, which has its own distinct genetic network. We further provide evidence that each interaction network is differentially susceptible to antibiotics. Thus, Mtb uses alternative pathways to produce PG, each with its own biochemical characteristics and vulnerabilities.One of the leading causes of infectious disease deaths worldwide is tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb). One-third of the human population is thought to harbor Mtb and ∼1.5 million individuals died of TB last year (1). Mtb’s success as a pathogen is due in part to its unusual cell wall, which is notorious for its complexity and is implicated in Mtb’s innate resistance to many commonly used antibiotics (2). A critical component of the bacterial cell wall (including Mtb’s) is peptidoglycan (PG), a complex polymer that provides structural support and counteracts turgor pressure (3). PG is essential for cell survival, and its synthesis is targeted by many potent antibiotics (2).PG consists of long glycan chains composed of two different sugars (Fig. 1A) that are cross-linked via short peptide side chains that extend from the glycan chains. Notably, generation of mature PG occurs outside of the cell membrane and is mediated by enzymes that incorporate new PG subunits, which are formed in the cytoplasm, into the PG polymer. PonA1 and PonA2 are the two enzymes in Mtb that can both polymerize glycan strands and cross-link peptides [known as bifunctional penicillin binding proteins (PBPs), Fig. 1A]. The predominant peptide cross-links in mycobacteria join the third amino acids (3–3 link) of adjacent stem peptides (4, 5), which are synthesized by l,d-transpeptidases (Ldts) such as LdtB, one of the major Ldts in Mtb (Fig. 1A). The peptides can also be joined by cross-linking the fourth and third amino acids (4–3 link) (Fig. 1A) through the action of bifunctional or monofunctional (capable of only peptide cross-linking) PBPs. The activity of these distinct factors must be coordinated to ensure proper cell-wall synthesis. One method of coordination is the use of large protein complexes, the elongation complex and divisome, which mediate cell-wall biogenesis during cell elongation or division, respectively (2). The essential activity of these enzymes makes them prime drug targets; indeed, PBPs and Ldts are inhibited by carbapenems and penicillin (6, 7), which remains one of the most clinically important drugs in use.Open in a separate windowFig. 1.Deletion of PG synthases influences growth and morphology of Mtb. (A) Transglycosylation (TG) and transpeptidation (TP) reactions incorporate new PG subunits into the cell wall. PonA1 and PonA2 carry out both TG and 4–3 TP reactions. LdtB only mediates 3–3 TP reactions. M, N-acetylmuramic acid. G, N-acetylglucosamine. (B) Deletion of either ponA2 or ldtB does not greatly affect Mtb growth during log phase, although loss of ldtB reduces population density in stationary phase. Error bars are often too small to see. (C) ponA2 mutant cells (n = 179) have increased width compared with wild-type cells (n = 153) (approximate P value <0.0001 by the Kolmogorov–Smirnov test). (D) ldtB mutant cells (n = 193) have increased width and decreased length compared with wild-type cells (n = 153) (approximate P value <0.0001 by the Kolmogorov–Smirnov test for both length and width).Although the biosynthesis and structure of PG have been investigated for decades, predominantly in organisms such as Escherichia coli or Bacillus subtilis, the mechanisms that coordinate the biochemical activities required to polymerize and modify the cell wall remain incompletely understood. Moreover, much less is known about PG synthesis in many pathogenic organisms, including Mtb (2). However, previous studies in Mtb suggest that PG synthesis in this pathogen does not strictly conform to the E. coli paradigm. For example, E. coli has three bifunctional PBPs [PBP1A, PBP1B, and PBP1C (3)], whereas Mtb has just two [PonA1 and PonA2 (8)]. Additionally, PBP2 (known as PBPA in mycobacteria) is a monofunctional PBP and is required for cell elongation in E. coli, but instead seems to function in cell septation in mycobacteria (3, 9).The structure of PG is also different in Mtb than in E. coli: Mycobacterial PG has an unusual prevalence of 3–3 peptide linkages. The abundance of 3–3 cross-links in mycobacterial PG throughout different growth stages (5) suggests that Ldts are active during normal growth; however, their cellular roles or regulation during growth and PG biogenesis remain largely unknown. Whereas penicillins and cephalosporins target only enzymes that produce 4–3 cross-links, Ldts can be targeted by carbapenems (7). Recent work suggests that these agents might be far more efficacious against both dividing and nondividing bacteria (10). Although little is known about Mtb’s five encoded Ldts (11), one, LdtB, is implicated in antibiotic tolerance (11–13), is required for normal virulence in a mouse model of TB (12), and is important for normal cell shape (13).Previous studies have also revealed that PG biosynthesis differs between Mtb and the related saprophytic Mycobacterium smegmatis (Msm). As opposed to Mtb, Msm has three bifunctional PBPs: PonA1, PonA2, and PonA3 (14). PonA1 is required for Msm but not Mtb growth in culture (15, 16); however, PonA1 is required for robust growth of Mtb during infection (16). In contrast, Mtb and Msm ponA2 mutants do not have growth defects in culture (14, 17). However, Mtb strains with inactivated ponA1 or ponA2 exhibit similar survival defects during growth in a host (16, 18), suggesting that these two similar bifunctional enzymes have nonredundant and important contributions to PG synthesis during infection. Collectively, the differences in PG synthase functionality may imply that different PG synthetic pathways exist across species, which may have consequences for a pathogen’s virulence during infection.Here, we interrogated PG synthesis in Mtb by investigating the genetic interactions of ponA1, ponA2, and ldtB, which encode three PG synthases critical for Mtb’s growth during infection. To identify these interactions, we performed genome-wide transposon mutagenesis screens in Mtb mutant strains that lacked one of these enzymes. Advances in high-throughput sequencing technology coupled with the power of whole-genome studies provide unique insights into key bacterial processes, such as cell-wall biosynthesis. Such studies have been performed to a limited extent in bacteria, and further work would substantially expand our understanding of the organization of prokaryotic metabolic processes. In this study, we identified diverse genetic interaction networks for ponA1, ponA2, and ldtB, suggesting that these synthases are embedded within distinct cellular networks for assembling Mtb’s PG. We found that either ponA1 or ponA2 is required for cell growth, and that ldtB interacts with both ponA1 and ponA2. Moreover, mutants that lack these enzymes have differential susceptibility to agents that interfere with cell-wall biogenesis. Thus, the Mtb cell wall is synthesized using multiple interacting networks that are both overlapping and unique.     

A functional role of Rv1738 in Mycobacterium tuberculosis persistence suggested by racemic protein crystallography

Protein 3D structure can be a powerful predictor of function, but it often faces a critical roadblock at the crystallization step. Rv1738, a protein from Mycobacterium tuberculosis that is strongly implicated in the onset of nonreplicating persistence, and thereby latent tuberculosis, resisted extensive attempts at crystallization. Chemical synthesis of the l- and d-enantiomeric forms of Rv1738 enabled facile crystallization of the d/l-racemic mixture. The structure was solved by an ab initio approach that took advantage of the quantized phases characteristic of diffraction by centrosymmetric crystals. The structure, containing l- and d-dimers in a centrosymmetric space group, revealed unexpected homology with bacterial hibernation-promoting factors that bind to ribosomes and suppress translation. This suggests that the functional role of Rv1738 is to contribute to the shutdown of ribosomal protein synthesis during the onset of nonreplicating persistence of M. tuberculosis.Tuberculosis is caused by the bacterium Mycobacterium tuberculosis (Mtb), currently estimated to infect one-third of the world’s population (1). A major reason for this high rate of infection is the ability of Mtb to enter a dormant state known as nonreplicating persistence (2, 3). This occurs in response to engulfment of the bacterium by activated macrophages. A proportion of bacteria survives the immune system’s antimicrobial onslaught and persists in a dormant form, but can reemerge many years later as an active infection. In the nonreplicating persistent state, protein synthesis is drastically shut down (4), and the bacteria use host lipids, notably cholesterol, as a carbon source (5). Bacteria in this state are largely resistant to current drugs.Microarray studies have identified a number of genes that are highly up-regulated under conditions thought to trigger the onset of dormancy, including hypoxia (low oxygen concentration) and exposure to nitric oxide (6, 7). The most highly up-regulated gene in hypoxic conditions, and the second-highest in response to NO, is Rv1738, suggesting this gene to be a key element in the onset of persistence. Rv1738 is also classed as an essential gene in the genome-wide transposon mutagenesis study of Sassetti et al. (8). The Rv1738 gene encodes a predicted 94-residue protein of unknown function.In this research, we set out to determine the structure of the protein encoded by the ORF Rv1738 of Mtb H37Rv. Our hypothesis was that important clues to the function of the Rv1738 protein might be obtained from its 3D structure, as structure is much more highly conserved than sequence during evolution, and can reveal unsuspected functional and evolutionary relationships. There have been both failures and successes from this approach, but on discovering that Rv1738 could readily be expressed in soluble form in Escherichia coli, we initiated structural studies. Unfortunately, all attempts to crystallize recombinant Rv1738 failed, and the protein also proved unsuitable for structural analysis by NMR because of a tendency to aggregate.Because of our inability to obtain crystals using protein expressed in E. coli, we turned to racemic protein crystallography, a recently introduced approach that can facilitate crystallization of recalcitrant proteins (9). In this method, a racemic mixture comprising equal amounts of the d-protein and l-protein forms of a target molecule are used for crystallization. A protein racemate can crystallize in centrosymmetric space groups, and can thus access many additional space groups, including some predicted to be highly favored for protein crystallization (10). In contrast, natural proteins are chiral, built only from l-amino acids and the achiral amino acid glycine, and thus cannot be incorporated into crystal lattices that include mirror planes or inversion centers. Evidence is accumulating that racemic protein mixtures can be much easier to crystallize in cases in which the natural l-protein is refractory to crystallization (9). d-proteins can only be made by total chemical synthesis (11, 12), but as Rv1738 is a small protein, we considered it a good candidate for total chemical synthesis using modern ligation methods.Here we describe the total chemical synthesis of the d- and l-forms of Rv1738 and the successful crystallization of the {d-Rv1738 + l-Rv1738} protein racemate, in striking contrast to the complete failure of conventional crystallization approaches with the recombinant protein. The racemic crystals diffracted to good resolution, and the structure was solved using an ab initio approach that benefited from the quantized nature of centrosymmetric phases (13). Moreover, the structure of Rv1738 revealed a surprising similarity to a family of stress proteins known as hibernation-promoting factors (HPFs). This suggests that the functional role of the up-regulated Rv1738 protein in nonreplicating persistence of Mtb is to contribute to the shutdown of ribosomal protein synthesis.     

结核分支杆菌铁调控蛋白FurA的基因表达及单克隆抗体的制备

目的表达结核分支杆菌FurA蛋白,并制备针对该蛋白的单克隆抗体(mAb)。方法将重组质粒转化入E.coliBL21,挑出阳性克隆,IPTG诱导表达重组的6×His融合蛋白。采用小鼠腹股沟皮下NC膜免疫的方法免疫小鼠,并进行细胞融合、克隆化制备抗FurAmAb,用ELISA法初步鉴定其特异性位点和相对亲和力。结果获得了高表达的融合蛋白,融合蛋白经SDS-PAGE分析,在相对分子量Mr为23×103处有特异的蛋白条带,为目的蛋白。用该融合蛋白免疫小鼠后,获得了1株抗FurAmAb。结论所获得的抗FurAmAb特异性强、效价高,对进一步研究FurA在结核分支杆菌铁代谢及致病中的作用提供了有力的工具。     

血清抗结核分支杆菌抗体对结核病的诊断价值

目的研究血清抗结核分支杆菌抗体在活动性结核病诊断和病情变化的意义。方法观察血清抗结核分支杆菌抗体在各类结核患者中的敏感性和特异性，与痰涂片及ＰＰＤ皮试的一致性，以及与疾病转归的关系。结果血清抗结核分支杆菌抗体诊断结核病的敏感性为７１．９％，特异性为９１．９％，与痰涂片阳性和ＰＰＤ皮肤试验阳性的一致率分别为８０．２％和６１．６％。抗结核分支杆菌抗体水平与病情变化有一定的相关性。结论血清抗结核分支杆菌抗体可用来诊断活动性结核病及评估结核病的转归。     

Isolation of conditional expression mutants in Mycobacterium tuberculosis by transposon mutagenesis

In Mycobacterium tuberculosis identification of essential genes has been hampered by the scarcity of suitable genetic tools for genome wide screenings. We constructed two Himar1 transposon derivatives in which the Streptomyces pristinamycin I-inducible ptr promoter was inserted at one transposon end in outward orientation. These transposons, Tn-pip/pptr (which harbours the promoter and its repressor pip gene) and Tn-pptr (which depends on a host expressing the pip gene), were inserted in the thermosensitive mycobacteriophage phAE87. After transduction into M. tuberculosis H37Rv, hygromycin resistant clones were selected in the presence of pristinamycin, screened for inducer dependent growth, and the transposon insertion point mapped by sequencing. Out of 3530 Hyg(R) mutants tested, we obtained 14 (0.4%) single insertion conditional mutants. In three (leuA, mazE6, rne) pptr was located upstream of genes whose function had been assessed by experimental evidence, whereas in seven the transposon targeted genes (ftsK, glf, infB, metC, pyrD, secY, and tuf) whose function had been assigned by similarity with homologous genes and four ORFs of unknown function (Rv0883c, Rv1478, Rv2050 and Rv2204c). These results validate our mutagenesis system and provide previously unavailable conditional expression mutants in genes of known, putative and unknown functions for genetic and physiological studies.     

A comparison between the efficiency of the Xpert MTB/RIF assay and nested PCR in identifying Mycobacterium tuberculosis during routine clinical practice

Objectives

Polymerase chain reaction (PCR) for the detection of Mycobacterium tuberculosis (MTB) is more sensitive, specific, and rapid than the conventional methods of acid-fast bacilli (AFB) smear and culture. The aim of this study was to determine if the Xpert MTB/rifampicin (RIF) assay had additional advantages over nested PCR for the detection of MTB in a geographical area with intermediate tuberculosis (TB) incidence.

Methods

Between February and December 2013, the Xpert MTB/RIF assay and MTB nested PCR, as well as AFB smear and culture, were simultaneously performed on 198 clinical samples (160 pulmonary and 38 non-pulmonary specimens) collected from 171 patients hospitalized at Hallym University Medical Center for possible TB. The accuracy of the diagnosis of MTB culture-positive TB and the turnaround time of reporting laboratory results were calculated and compared. Rifampin resistance by the Xpert MTB/RIF assay was reviewed with that of conventional drug susceptibility testing (DST).

Results

The sensitivity, specificity, and positive and negative predictive values of the Xpert MTB/RIF assay and MTB nested PCR for diagnosis of MTB culture-positive pulmonary TB were 86.1% vs. 69.4% (P=0.1563), 97.8% vs. 94.1% (P=0.2173), 91.2% vs. 75.8% (P=0.1695), and 96.4% vs. 92.0% (P=0.2032), respectively. The median turnaround times of the Xpert MTB/RIF assay and MTB nested PCR were 0 [0-4] days and 4 [1-11] days, respectively (P<0.001). Two cases of rifampin resistance, as determined by the Xpert MTB/RIF assay, were found to be multi-drug resistant (MDR) pulmonary TB by DST.

Conclusions

The Xpert MTB/RIF assay seemed to be sensitive, specific, and comparable to nested PCR for identifying MTB among clinically suspected TB patients, and the assay can be valuable in giving a timely identification of resistance to rifampin.     

Reciprocal seasonal variation in vitamin D status and tuberculosis notifications in Cape Town, South Africa

Vitamin D deficiency is associated with susceptibility to tuberculosis (TB) in HIV-uninfected people in Europe, but it is not known whether such an association exists among HIV-infected people in subtropical Africa. We conducted a cross-sectional study to determine whether vitamin D deficiency was associated with susceptibility to active TB in HIV-uninfected (n = 196) and HIV-infected (n = 174) black Africans in Cape Town, South Africa. We also investigated whether there was evidence of seasonal variation in vitamin D status and TB notifications in this setting over an 8-y period. Vitamin D deficiency (serum 25-hydroxyvitamin D [25(OH)D] <50 nmol/L) was present in 232 (62.7%) of 370 participants and was associated with active TB in both HIV-uninfected (odds ratio = 5.2, 95% confidence interval: 2.8-9.7; P < 0.001) and HIV-infected (odds ratio = 5.6, 95% confidence interval: 2.7-11.6; P < 0.001) people. Vitamin D status varied according to season: The mean serum 25(OH)D concentration was highest in January through March and lowest in July through September (56.8 vs. 30.7 nmol/L, respectively; P < 0.001). Reciprocal seasonal variation in TB notifications was observed: The mean number of TB notifications per quarter for Cape Town in 2003 to 2010 was lowest in April through June and highest in October through December (4,222 vs. 5,080; P < 0.001). Vitamin D deficiency is highly prevalent among black Africans in Cape Town and is associated with susceptibility to active TB both in the presence and absence of HIV infection. Reciprocal seasonal variation in serum 25(OH)D concentration and TB notifications suggests that seasonal variations in vitamin D status and TB incidence in this setting are causally related.     

Epidemiological trends and clinical comparisons of Mycobacterium tuberculosis lineages in Thai TB meningitis

Mycobacterium tuberculosis (MTB) strains were isolated from cerebrospinal fluids collected from individual tuberculous meningitis (TBM) patients from 1996 to 2007 (n = 184) and characterised based on IS6110-restriction fragment length polymorphism (RFLP), spoligotyping, Mycobacterium interspersed repetitive unit-variable number of tandem repeat (MIRU-VNTR) and large sequence polymorphisms (LSPs). Beijing strains were found to possess the highest transmissibility and proportion in clustered isolates. Beijing strain predomination and stability, at 56% of the genotypic proportion, as well as association with drug resistance in TBM patients, was demonstrated. The proportion of Beijing sublineages revealed that the modern Beijing sublineage showed an increasing trend, whereas the ancestral Beijing sublineage showed a decreasing trend across the three periods. In contrast, there were neither clustered nor multidrug-resistance (MDR) isolates from the Euro-American (EuA) lineage, and the lineage genotypic proportion trend was also decreased. Based on LSPs, only the Beijing, Indo-Oceanic and Euro-American lineages were identified from TBM patients in Thailand. TBM mortality rates were not associated with either drug resistance or significantly different among MTB lineages. This study may support the Beijing genotype strain as most pathogenic causing TBM, with the EuA lineage genotype as the most benign of the strain genotypes tested. The analysis of drug susceptibility also revealed the trend of increasing drug resistance, especially MDR, in TBM patients in Thailand.     

Production and characterization of a monoclonal antibody against 28.5 kDa tegument antigen of Fasciola gigantica

A monoclonal antibody (MoAb) against the 28.5 kDa tegumental antigen of Fasciola gigantica was produced by the hybridoma technique using spleen cells from BALB/c mice immunized with the tegumental extract from adult F. gigantica. This MoAb was found to be of the isotype IgG(1), kappa-light chain, and shown by immunoblotting to specifically react with the 28.5 kDa antigen present in the tegument, excretion-secretion material of the adult, whole-body extracts of newly excysted juveniles, 5-week-old juvenile and adult parasites. It did not cross-react with antigens from other trematode parasites, including Schistosoma mansoni, Eurytrema pancreaticum and Paramphistomum spp. Immunolocalization of this antigen by indirect immunofluorescence indicated that it was present as a major component of the adult tegument, particularly in its outer rim, tegumental cells, and their processes. Furthermore, the epithelium linings of the oral sucker, buccal tube, pharynx, caecal bifurcation, both male and female genital canals, which were the continuation of the tegumental-type epithelium, were also positively stained with this MoAb. A similar pattern of immunolocalization, but with weaker staining intensity, was observed in newly excysted, 5- and 7-week-old juveniles. Thus this antigen is expressed in all developmental stages of the parasite, and it could be a strong candidate for immunodiagnosis and vaccine development.     

Diagnosis of pulmonary tuberculosis using MTB12 and 38-kDa antigens

Background and objective:   Mycobacterium tuberculosis MTB12 protein plays an essential role in pro-inflammatory responses during the early stages of human pulmonary tuberculosis (TB), even though the T-cell immunoreactivity of MTB12 is weaker than that of the 30-kDa antigen (Ag). The objective of this study was to evaluate the humoral immune responses induced by MTB12 Ag during human TB.
Methods:   Using an ELISA, anti-MTB12 IgG levels in the sera of TB patients and healthy controls were compared with those induced by the 30-kDa Ag and 38-kDa Ag, or both.
Results:   In TB patients, the sensitivity and specificity of MTB12 Ag were similar to those of other antigens at 53.0% and 95.4%, respectively. However, the sensitivity increased to 73.0% when the combination of MTB12 and 38-kDa Ag was measured. Specificity remained high when a combination of the individual antigens was used. ELISA results showed that after anti-tuberculosis treatment, the mean IgG levels against MTB12 alone or MTB12 plus 38-kDa Ag were significantly increased in the TB patients, while those against MTB12 plus 30-kDa Ag were not ( P  < 0.05).
Conclusions:   Collectively, these data suggest that MTB12, in combination with 38-kDa Ag, can be used to increase the accuracy of pulmonary TB diagnosis.