Expression ofMycobacterium tuberculosis genes inEscherichia coli

TwoEscherichia coli clones expressingMycobacterium tuberculosis antigens were isolated from a gene-bank in the plasmid vector pBR 325. ‘Western blot’ analysis revealed the presence of a unique protein band of molecular weight 68,000 and 38,000, respectively in cellextracts from each clone. The 68,000 dalton antigen was found to be expressed onEscherichia coli outer surface. Plasmid DNA from a third clone could confer leucine independence on two differentleu B mutants ofEscherichia coli but not on mutants in otherleu genes, pointing to the possibility ofgenetic complementation. Thus,Mycobacterium tuberculosis DNA is capable of expression inEscherichia coli.     

Immunodiagnosis of pulmonary tuberculosis by concomitant detection of antigen and antibodies of excretory-secretory protein ofMycobacterium tuberculosis H37Ra

With a view to diagnosing tuberculosis in populations in endemic areas, excretory-secretory antigen fraction(Mtb EST-6) of purifiedMycobacterium tuberculosis H₃₇Ra and affinity purified polyclonal antibodies againstMtb EST were used to detect both antibodies and circulating antigen in the sera of patients and disease-free individuals. Indirect stick penicillinase ELISA system usingMtb EST-6 detected antigen-specific IgG antibody in 84% of sputum positive, 77% of sputum negative pulmonary tuberculosis patients and 7% of healthy and 11% of subjects with nontub~rculosis diseases. Similarly, a sandwich penicillinase ELISA system using affinity purified antiMtb EST antibodies detected circulating antigen in 83% and 61% of sputum positive and negative pulmonary tuberculosis subjects. In contrast only 24% of healthy and 18% of disease controls showed seropositivity. Antibody assay showed higher sensitivity and specificity (83% and 91% respectively) compared to antigen detection (sensitivity of 79% and specificity of 79%). However, by concomitant use of both assays it was possible to enhance the specificity of detection to 98%, though sensitivity was reduced marginally to 70%. The present study confirms the presence of both antigen and specific antibodies in the circulation during clinical disease and draws attention to the utility ofMtb EST-6 as a diagnostic marker of pulmonary tuberculosis.     

Potential of Mycobacterium tuberculosis resuscitation-promoting factors as antigens in novel tuberculosis sub-unit vaccines

Novel vaccines are needed to control tuberculosis (TB), the bacterial infectious disease that together with malaria and HIV is worldwide responsible for high levels of morbidity and mortality. TB can result from the reactivation of an initially controlled latent infection by Mycobacterium tuberculosis (Mtb). Mtb proteins for which a possible role in this reactivation process has been hypothesized are the five homologs of the resuscitation-promoting factor of Micrococcus luteus, namely Mtb Rv0867c (rpfA), Rv1009 (rpfB), Rv1884c (rpfC), Rv2389c (rpfD) and Rv2450c (rpfE). Analysis of the immune recognition of these 5 proteins following Mtb infection or Mycobacterium bovis BCG vaccination of mice showed that Rv1009 (rpfB) and Rv2389c (rpfD) are the most antigenic in the tested models. We therefore selected rpfB and rpfD for testing their vaccine potential as plasmid DNA vaccines. Elevated cellular immune responses and modest but significant protection against intra-tracheal Mtb challenge were induced by immunization with the rpfB encoding DNA vaccine. The results indicate that rpfB is the most promising candidate of the five rpf-like proteins of Mtb in terms of its immunogenicity and protective efficacy and warrants further analysis for inclusion as an antigen in novel TB vaccines.     

Antigenic relationship between reactivity to hepatitis B e antigen and 19 kDa protein ofMycobacterium tuberculosis among the Tibetan settlers in Karnataka

McGlynn and her co-workers have reported that among the Vietnamese refugees in Philadelphia and among Alaskan natives who are hepatitis B carriers, there is a statistically significant association between anegative tuberculin test and the presence of hepatitis B e antigen. A repetition of this work among the population of Bangalore did not yield any significant results because of the very low incidence of hepatitis found among this population. However, on the basis of available data that hepatitis B infection is more prevalent among the Mongolian population than among people of other populations, the work was repeated among Tibetans who had settled down in Karnataka. This set of experiments showed that, contrary to the report of McGlynnet al, there is a statistically significant association between apositive tuberculin test and the presence of hepatitis B e antigen and that those individuals who showed the presence of hepatitis B e antigen exhibited less severe form of the disease than those who were negative to this antigen. These findings suggested that immunity to tuberculosis and hepatitis B infections may have a common underlying principle. Data bank search revealed a stretch of amino acid sequences which is common to hepatitis B e antigen and 19 kDa antigen ofMycobacterium tuberculosis. The significance of these results is discussed.     

N-[2-Naphthyl]-glycine hydrazide,a potent inhibitor of DNA-dependent RNA polymerase ofMycobacterium tuberculosis H37RV

N-[2-Naphthyl]-glycine hydrazide has been shown for the first time as a potent inhibitor of the DNA-dependent RNA polymerase (EC 2.7.7.6) ofMycobacterium tuberculosis H₃₇R_v. At a concentration of 10^-9 M, the compound shows maximum inhibition of the enzyme, the inhibition being less at higher concentrations. It is suggested that the novel type of inhibition pattern may be due to hydrophobic interactions occurring between the molecules of the compound at higher concentrations. The finding that there is a shift in the λ_max of the compound could also account for this phenomenon. The effect of this compound was also tested on DNA-dependent RNA polymerases from an eukaryotic fungus,Microsporum canis. At a concentration of 10⁻⁹ M it inhibits RNA polymerase II (32%) but not RNA polymerasesI andIII     

Purification and characterization of Mycobacterium tuberculosis KatG, KatG(S315T), and Mycobacterium bovis KatG(R463L)

Isoniazid, a first-line antibiotic used for the treatment of tuberculosis, is a prodrug that requires activation by the Mycobacterium tuberculosis enzyme KatG. The KatG(S315T) mutation causes isoniazid resistance while the KatG(R463L) variation is thought to be a polymorphism. Much of the work to date focused on isoniazid activation by KatG has utilized recombinant enzyme overexpressed in Escherichia coli. In this work, native KatG and KatG(S315T) were purified from M. tuberculosis, and KatG(R463L) was purified from Mycobacterium bovis. The native molecular weight, enzymatic activity, optical, resonance Raman, and EPR spectra, K(D) for isoniazid binding, and isoniazid oxidation rates were measured and compared for each native enzyme. Further, the properties of the native enzymes were compared and contrasted with those reported for recombinant KatG, KatG(S315T), and KatG(R463L) in order to assess the ability of the recombinant enzymes to act as good models for the native enzymes.     

Effect of oral exposure ofMycobacterium avium intracellular on the protective immunity induced by BCG

The relative protective efficacy of oral administration of mycobacteria as compared to the conventional intradermal route of vaccination has been assessed in guinea pigs. Skin test reactivity to partially purified protein derivative and protective immunity to challenge with virulentMycobacterium tuberculosis were used as parameters of protective immunity. Oral immunisation of guinea pigs either with BCG or withMycobacterium avium intracellulare induces skin test reactivity and protective immunity comparable to that induced by intradermal route of vaccination. Oral exposure ofMycobacterium avium intracellulare prior to oral or intradermal dose of BCG did not interfere with the protective immunity induced by BCG in guinea pigs challenged withMycobacterium tuberculosis H₃₇Rv.     

An approach for studying the mediators of pathogenesis inMycobacterium tuberculosis

Mycobacterium tuberculosis is an example of an intracellular pathogen that mediates the disease state through complex interactions with the host’s immune system. Not only does this organism replicate in the hostile environment prevailing within the infected macrophage, but it has also developed intricate mechanisms to inhibit several defence mechanisms of the host’s immune system. It is postulated here that the mediators of these interactions with the host are products of differentially expressed genes in the pathogen. B and T cell responses of the host are hence to be used as tools to identify such gene products from an expression library of theMycobacterium tuberculosis genome. The various pathways of generating a productive immune response that may be targeted by the pathogen are discussed     

结核分枝杆菌吡嗪酰胺耐药性检测方法的研究进展

吡嗪酰胺(pyrazinamide,PZA)是重要的一线抗结核药物,与异烟肼、利福平和乙胺丁醇构成治疗方案的核心。因其疗效较好,被广泛应用于结核病的治疗过程。然而,近年来随着耐多药结核病的出现,PZA耐药导致部分患者治疗失败,因此常规开展PZA药物敏感性试验对于减少耐药性的发生显得极为重要。由于PZA在酸性条件下才能发挥作用,而结核分枝杆菌在酸性环境下生长不良,故PZA耐药性检测一直是临床中的难题。本文结合国内外最新研究进展,就结核分枝杆菌PZA耐药性检测方法的研究进行阐述,期望能为更有效地诊治结核病提供新思路。     

Formation of Nonculturable Cells of Mycobacterium tuberculosis and Their Resuscitation

Nonculturable cells were found to occur in populations of Mycobacterium tuberculosis cells during the long poststationary phase. These cells were small (0.6–0.8 m) ovoid and coccoid forms with intact cell walls and negligible respiratory activity, which allows them to be regarded as dormant cells. Nonculturable cells were characterized by low viability after plating onto solid medium; a minor part of the population of these cells could be cultivated in liquid medium. Cell-free culture liquid of an exponential-phase Mycobacterium tuberculosisculture or the bacterial growth factor Rpf exerted a resuscitating effect, increasing substantially the growth capacity of the nonculturable cells in liquid medium. During resuscitation of nonculturable cells, a transition from ovoid to rodlike cell shape occurred. At early stages of resuscitation, ovoid cells formed small aggregates. The recovery of culturability was associated with the formation of rod-shaped cells in the culture. The data obtained demonstrate the in vitro formation of dormant cells of Mycobacterium tuberculosis, which do not grow on solid media but can be resuscitated in liquid medium under the effect of substance(s) secreted by actively growing cells.     

结核分枝杆菌抗原蛋白的研究及其应用新进展

结核病(tuberculosis, TB)是由结核分枝杆菌(Mycobacterium tuberculosis, MTB)感染引起的慢性传染病,是仅次于正在暴发的新型冠状病毒肺炎(COVID-19)的第二大单一感染致死病因。COVID-19的大流行对TB的诊断及治疗造成了破坏性的影响,全球实现终结TB目标的进展偏离了轨道。因此,早诊断、早治疗依然是防控TB蔓延的关键。TB精准诊断一直受MTB抗原特异性、检测技术特异性和灵敏度的影响,因此亟需挖掘高特异性新抗原、开发新检测技术。随着蛋白质基因组学(proteogenomics)和质谱技术的快速发展,从临床体液、组织样本中高效、精准靶向检测MTB特异性已知、甚至新抗原的表达,以及监测治疗过程中的抗原表达量的动态变化,是TB诊断及治疗的发展趋势。在MTB标准菌株H37Rv的4 008个注释基因中(NC＿000 962.3, NCBI),国内外报道的已注释抗原虽有140多个,但仅有极少的抗原应用于TB的筛查及辅助诊断,离世界卫生组织(World Health Organization, WHO)的诊断标准尚远。本文通过对MTB已报道抗原以及基...     

CRISPR/Cas系统与结核病防控新措施相关性研究

结核分枝杆菌(Mycobacterium tuberculosis,MTB,以下简称结核杆菌)感染引起的结核病仍然是严重影响人类健康全球性重大传染病。全球约1/4人口是结核杆菌的潜伏感染者。2019年,世界卫生组织(Worldhealthorganization,WHO)报道全球约150万人死于结核病。深入研究结核杆菌生物学有望为结核病防控提供新工具。成簇规律性间隔短回文重复(Clusteredregularlyinterspacedshort palindromic repeats,CRISPR)/Cas是细菌免疫系统的重要成分,在结核杆菌等分枝杆菌中也广泛存在,同时,也是分枝杆菌基因编辑的重要工具。本文结合课题组研究工作,综述了结核杆菌III-A型CRISPR/Cas系统各组分的生物学功能以及与致病的相关性,CRISPR/Cas编辑工具在诊断治疗耐药结核杆菌和结核病防控新措施中的进展。     

Growth ofMycobacterium smegmatis in minimal and complete media

The growth patterns ofMycobacterium smegmatis SN2 in a minimal medium and in nutrient broth have been compared. The growth was monitored by absorbancy (Klett readings), colony forming units, wet weight and content of DNA, RNA and protein. During the early part of the growth cycle, the bacteria had higher wet weight and macromolecular content in nutrient broth than in minimal media. During the latter half of the growth cycle however, biosynthesis stopped much earlier in nutrient broth and the bacteria had a much lower content of macromolecules than in the minimal medium. In both the media, a general pattern of completing biosynthesis rapidly in the initial phase and a certain amount of cell division at a later time involving the distribution of preformed macromolecules was seen. The possible adaptive significance of this observation has been discussed.     

Deoxyribonucleic acid replication time in Mycobacterium tuberculosis H37 Rv

The DNA increment method, designed for measuring the increment in the amount of DNA after inhibition of initiation of fresh rounds of replication initiation was employed to measure the rate of deoxyribonucleic acid (DNA) chain growth in Mycobacterium tuberculosis H37Rv growing in Youman and Karlson's medium at 37°C with a generation time of 24 h and also in relatively fast growing species like Mycobacterium smegmatis and Escherichia coli. From the results obtained, the time required for a DNA replication fork to traverse the chromosome from origin to terminus (C period) was calculated. The chain elongation rates of DNA of the three organisms was determined from the C period and the known genome sizes assuming that all these genomes have a single replication origin and bidirectional replication fork. The rate for M. tuberculosis was 3,200 nucleotides per min about 11 times slower than that of M. smegmatis and about 13–18 times slower than that of E. coli.Abbreviations DNA deoxyribonucleic acid - td delay in initiation - OD optical density - CAM chloramphenicol - RIF rifampicin     

Functional characterization of the phospholipase C activity of Rv3487c and its localization on the cell wall of Mycobacterium tuberculosis

Mycobacterium tuberculosis survives and persists for prolonged periods within its host in an asymptomatic,latent state and can reactivate years later if the host's immune system weakens. The dormant bacilli synthesize and accumulate triacylglycerol, reputed to be an energy source during latency. Among the phospholipases, phospholipase C plays an important role in the pathogenesis. Mutations in a known phospholipase C, plcC, of M.tuberculosis attenuate its growth during the late phase of infection in mice. Hydrolysis of phospholipids by phospholipase C generates diacylglycerol, a well-known signalling molecule that participates in the activation of extracellular signal-regulated kinases (ERK) through protein kinase C leading to macrophage activation. In the present study, we show that M.tuberculosis possesses an additional cell wall-associated protein, Rv3487c, with phospholipase C activity. The recombinant Rv3487c hydrolyses the substrate phosphatidylcholine and generates diacylglycerol by removing the phosphocholine. Furthermore, Rv3487c is expressed during infection as it exhibits significant humoral immunoreactivity with sera from children with tuberculosis, but not with that from adult patients.     

Expression and characterization of Rv2430c, a novel immunodominant antigen of Mycobacterium tuberculosis

About 10% of the coding sequence of Mycobacterium tuberculosis corresponds to hitherto unknown members of the PE and PPE protein families which display significant sequence and length variation at their C-terminal region. It has been suggested that this could possibly represent a rich source of antigenic variation within the pathogen. We describe the purification and biophysical characterization of the recombinant PPE protein coded by hypothetical ORF Rv2430c, a member of the PPE gene family that was earlier shown to induce a strong B cell response. Expression of the recombinant PPE protein in Escherichia coli led to its localization in inclusion bodies and subsequent refolding using dialysis after its extraction from the same resulted in extensive precipitation. Therefore, an on-column refolding strategy was used, after which the protein was found to be in the soluble form. CD spectrum of the recombinant protein displayed predominantly alpha helical content (81%) which matched significantly with in silico and web-based secondary structure predictions. Furthermore, fluorescence emission spectra revealed that aromatic amino acids are buried inside the protein, which are exposed to aqueous environment under 8M urea. These results, for the first time, provide evidence on the structural features of PPE family protein which, viewed with its reported immunodominant characteristics, have implications for other proteins of the PE/PPE family.     

Correlation of bacterial viability with uptake of [14C] acetate into phenolic glycolipid-1 ofMycobacterium leprae within Schwannoma cells

The viability ofMycobacterium leprae, maintained within 33B Schwannoma cells, was estimated in terms of incorporation of [¹⁴C] acetate into its specific phenolic glycolipid-1. This measure of viability was correlated with two other assays,viz., fluorescein diacetate/ethidium bromide staining and mouse footpad growth. Observation of a 2-fold increase in the number of intracellularMycobacterium leprae over an experimental period of 12 days also corroborated this contention. Furthermore, on addition of anti-leprosy drugs to these intracellularMycobacterium leprae there was significant decrease in phenolic glycolipid-1 synthesis indicative of loss of viability of the organisms. This study also established the importance of the host cell for active bacillary metabolism, asMycobacterium leprae maintained in cell-free conditions showed no incorporation into phenolic glycolipid-1. Moreover, compromising the host’s protein synthesis capacity with cycloheximide, also led to reduction in bacillary metabolism. As this system measures the metabolic synthesis of a uniqueMycobacterium leprae component, it would be useful for development and screening of compounds acting against specific bacillary targets.     

Mycobacterium tuberculosis alters the differentiation of monocytes into macrophages in vitro

This paper shows that in vitro infection of human monocytes by Mycobacterium tuberculosis affected monocyte to macrophage differentiation. Despite the low bacterial load used, M. tuberculosis-infected monocytes had fewer granules, displayed a reduced number of cytoplasmic projections and decreased HLA class II, CD68, CD86 and CD36 expression compared to cells differentiated in the absence of mycobacteria. Infected cells produced less IL-12p70, TNF-α, IL-10, IL-6 and high IL-1β in response to lipopolysaccharide and purified protein M. tuberculosis-derived. Reduced T-cell proliferative response and IFN-γ secretion in response to phytohemagglutinin and culture filtrate proteins from M. tuberculosis was also observed in infected cells when compared to non-infected ones. The ability of monocytes differentiated in the presence of M. tuberculosis to control mycobacterial growth in response to IFN-γ stimulation was attenuated, as determined by bacterial plate count; however, they had a similar ability to uptake fluorescent M. tuberculosis and latex beads compared to non-infected cells. Recombinant IL-1β partially altered monocyte differentiation into macrophages; however, treating M. tuberculosis-infected monocytes with IL-1RA did not reverse the effects of infection during differentiation. The results indicated that M. tuberculosis infection altered monocyte differentiation into macrophages and affected their ability to respond to innate stimuli and activate T-cells.     

Functional phosphoglucose isomerase from Mycobacterium tuberculosis H37Rv: rapid purification with high yield and purity

Phosphoglucose isomerase (PGI) EC 5.3.1.9, is a housekeeping enzyme that catalyzes the reversible isomerization of d-glucopyranose-6-phosphate and d-fructofuranose-6-phosphate. We have previously reported expression and multistep purification of recombinant PGI from Mycobacterium tuberculosis using conventional methods. We now describe an improved and simplified single step approach for purification of functionally active mycobacterial rPGI. The gene encoding PGI from M. tuberculosis H37Rv was cloned in bacterial expression vector pET22b(+). Expression of recombinant PGI with six-histidine-tag protein was observed both in the soluble fraction and inclusion bodies. Approximately 116mg of recombinant enzyme was purified to near homogeneity with approximately 80% yield from the soluble fraction of 1L culture at shake flask level using one step Ni-NTA affinity chromatography. The specific activity of the purified six-histidine-tagged recombinant PGI (rPGI-His(6)) was approximately 800U/mg of protein. The apparent K(m) value of the active recombinant protein followed Michaelis-Menten kinetics and was 0.27+/-0.03mM. K(i) for the competitive inhibitor 6-phosphogluconate was 0.75mM. The enzyme had pH optima in the range of pH 7.6-9.0 and was stable up to 55 degrees C. rPGI-His(6) exhibited enzyme activity almost equal to that of enzyme without histidine tag.     

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.