Halicyclamine A, a marine spongean alkaloid as a lead for anti-tuberculosis agent

In the course of our search for anti-microbial agents against dormant Mycobacterium tuberculosis, halicyclamine A was re-discovered as a lead for anti-tuberculosis agent from a marine sponge of Haliclona sp. on the guidance of the constructed bioassay. Halicyclamine A showed growth inhibition against Mycobacterium smegmatis, Mycobacterium bovis BCG, and M. tuberculosis H37Ra with MICs in the range of 1.0-5.0microg/ml under both aerobic condition and hypoxic condition inducing dormant state. The growth-inhibitory activity of halicyclamine A was bactericidal, and halicyclamine A did not exhibit cross-resistance with the currently used anti-tuberculosis drugs of isoniazid, ethambutol, rifampicin, and streptomycin. Halicyclamine A has been isolated originally as one of the active constituents inhibiting inosine 5'-monophosphate dehydrogenase (IMPDH). Then, in order to elucidate action-mechanism of halicyclamine A, we prepared IMPDH over-expressing strains of M. smegmatis. However, IMPDH was not target for halicyclamine A, because halicyclamine A showed same MIC value against the wild-type M. smegmatis and IMPDH over-expressing strains.     

绿茶粗提物对耻垢分枝杆菌的生长抑制研究

采用乙酸乙酯在中性条件下萃取绿茶,得到含有表没食子儿茶素没食子酸酯(Epigallocatechin gallate,EGCG)的粗提物。通过纸片琼脂扩散法和细菌生长曲线来评价绿茶粗提物对耻垢分枝杆菌的抑菌效果,利用透射电子显微镜(Transmission electron microscopy,TEM)观察其对耻垢分枝杆菌细胞壁结构的影响。结果显示,绿茶粗提物对耻垢分枝杆菌生长产生明显抑制作用,且抑菌作用随着浓度的升高逐渐加强;经绿茶粗提物处理的耻垢分枝杆菌细胞壁呈现膨出、变形等形态学变化。因此,绿茶粗提物具有抑制耻垢分枝杆菌生长的功能,其作用机制可能与其主要成分EGCG影响细胞壁肽聚糖的生物合成有关。     

Nature of the Receptor Substance of Mycobacterium smegmatis for D4 Bacteriophage Adsorption

The acetone-soluble fraction extracted from lyophilized cells of Mycobacterium smegmatis ATCC 607 inhibited D4, a species-specific phage active against M. smegmatis. Evidence is presented indicating that the D4 inhibition was caused by the phage receptor substance(s) contained in this fraction. A fraction eluted from silicic acid with chloroform-methanol (95:5, v/v) showed the strongest inhibition of D4 phage. This fraction contained sugars and amino acids, and its infrared absorption spectrum was practically identical with those of the mycoside C isolated from the other species of mycobacteria. Further fractionation revealed that the active material was a mixture of several closely related peptidoglycolipids all of which showed, more or less, the phage inhibition. One of the compounds was purified and partially characterized; it contains three different amino acids, allo-threonine, alanine, and phenylalanine, at a molar ratio of 1:1:1, and also three different deoxyhexoses, probably 6-deoxytalose, 3,4-di-o-methylrhamnose, and 2,3,4-tri-o-methylrhamnose. A tentative name of "mycoside C(sm)" is proposed for this substance which possesses a slightly different structure from the known types of mycoside C and is probably specific for the species of M. smegmatis. A fraction extracted from the D4-resistant mutant of M. smegmatis ATCC 607 by acetone and then by chloroform-methanol (95:5, v/v) showed no phage inhibition and had no sugar component. In addition, this fraction contained lysine, serine, and a small amount of both glycine and an unidentified amino acid.     

Porins are required for uptake of phosphates by Mycobacterium smegmatis

Phosphorus is an essential nutrient, but how phosphates cross the mycobacterial cell wall is unknown. Phosphatase activity in whole cells of Mycobacterium smegmatis was significantly lower than that in lysed cells, indicating that access to the substrate was restricted. The loss of the outer membrane (OM) porin MspA also reduced the phosphatase activity in whole cells compared to that in lysed cells. A similar result was obtained for M. smegmatis that overexpressed endogenous alkaline phosphatase, indicating that PhoA is not a surface protein, contrary to a previous report. The uptake of phosphate by a mutant lacking the porins MspA and MspC was twofold lower than that by wild-type M. smegmatis. Strikingly, the loss of these porins resulted in a severe growth defect of M. smegmatis on low-phosphate plates. We concluded that the OM of M. smegmatis represents a permeability barrier for phosphates and that Msp porins are the only OM channels for the diffusion of phosphate in M. smegmatis. However, phosphate diffusion through Msp pores is rather inefficient as shown by the 10-fold lower permeability of M. smegmatis for phosphate compared to that for glucose. This is likely due to the negative charges in the constriction zone of Msp porins. The phosphatase activity in whole cells of Mycobacterium bovis BCG was significantly less than that in lysed cells, indicating a similar uptake pathway for phosphates in slow-growing mycobacteria. However, porins that could mediate the diffusion of phosphates across the OM of M. bovis BCG and Mycobacterium tuberculosis are unknown.     

Nisin A depletes intracellular ATP and acts in bactericidal manner against Mycobacterium smegmatis

Nisin is a bacteriocin produced by many strains of Lactococcus lactis. This study examined the effect of nisin on Mycobacterium smegmatis, a non-pathogenic species of Mycobacterium. Nisin had a minimum inhibitory concentration of 8.0 micrograms ml-1 and a minimum inhibitory dose of 7.5 micrograms ml-1 against Myco. smegmatis. Treatment with 25.0 micrograms ml-1 nisin caused partial inhibition of Myco smegmatis; the survivors were nisin-sensitive when tested in a separate experiment. Mycobacterium smegmatis cells exposed to 50.0 micrograms ml-1 of nisin, lost their viability. the effect of nisin on the growth of Myco. smegmatis was both time- and concentration-dependent. Nisin (10.0 micrograms ml-1) caused 97.7 +/- 2.0% reduction in internal ATP and leakage of intracellular ATP out of Myco. smegmatis cells after several hours of treatment. These data suggest that nisin inhibits Myco. smegmatis by the same mechanism by which it inhibits other bacteria and warrants further investigation as a possible antitubercular agent.     

New anthracycline antibiotics produced by interspecific recombinants of streptomycetes. IV. Antimicrobial activity of iremycin

The in vitro antimicrobial activity of iremycin (10-(alpha-L-rhodosaminyl)-gamma-rhodomycinone) was determined in comparison to that of doxorubicin, a 14-hydroxy-derivative of daunorubicin, which exhibited a strong antitumor activity and is useful in chemotherapy of human tumors. The MIC values determined by means of a standardized agar diffusion plate test indicated a lower antimicrobial activity of iremycin in vitro in comparison to that of doxorubicin. In contrast to doxorubicin, iremycin was highly active against Mycobacterium smegmatis, but five-fold less active than doxorubicin against Staphylococcus aureus, seven-fold less active against Bacillus subtilis, and twenty five-fold less active against Commamonas terrigena. Furthermore, iremycin was hundred-fold less active against a highly sensitive permeation mutant of Pseudomonas aeruginosa. No inducing activity on prophages in lysogenic E. coli cells was demonstrable for iremycin and no growth inhibition in the repair test was observable. In contrast, iremycin inhibited the multiplication of gamma-phages in the BIP test, but the MIC values of violamycin BI, doxorubicin and iremycin in this test system indicated that iremycin is two hundred fifty-fold less active than violamycin BI and ten-fold less active than doxorubicin. No serum binding was demonstrable for iremycin.     

Nitric oxide scavenging and detoxification by the Mycobacterium tuberculosis haemoglobin,HbN in Escherichia coli

Nitric oxide (NO), generated in large amounts within the macrophages, controls and restricts the growth of internalized human pathogen, Mycobacterium tuberculosis H37Rv. The molecular mechanism by which tubercle bacilli survive within macrophages is currently of intense interest. In this work, we have demonstrated that dimeric haemoglobin, HbN, from M. tuberculosis exhibits distinct nitric oxide dioxygenase (NOD) activity and protects growth and cellular respiration of heterologous hosts, Escherichia coli and Mycobacterium smegmatis, from the toxic effect of exogenous NO and the NO-releasing compounds. A flavohaemoglobin (HMP)-deficient mutant of E. coli, unable to metabolize NO, acquired an oxygen-dependent NO consumption activity in the presence of HbN. On the basis of cellular haem content, the specific NOD activity of HbN was nearly 35-fold higher than the single-domain Vitreoscilla haemoglobin (VHb) but was sevenfold lower than the two-domain flavohaemoglobin. HbN-dependent NO consumption was sustained with repeated addition of NO, demonstrating that HbN is catalytically reduced within E. coli. Aerobic growth and respiration of a flavohaemoglobin (HMP) mutant of E. coli was inhibited in the presence of exogenous NO but remained insensitive to NO inhibition when these cells produced HbN, VHb or flavohaemoglobin. M. smegmatis, carrying a native HbN very similar to M. tuberculosis HbN, exhibited a 7.5-fold increase in NO uptake when exposed to gaseous NO, suggesting NO-induced NOD activity in these cells. In addition, expression of plasmid-encoded HbN of M. tuberculosis in M. smegmatis resulted in 100-fold higher NO consumption activity than the isogenic control cells. These results provide strong experimental evidence in support of NO scavenging and detoxification function for the M. tuberculosis HbN. The catalytic NO scavenging by HbN may be highly advantageous for the survival of tubercle bacilli during infection and pathogenesis.     

Characterization of a conserved interaction between DNA glycosylase and ParA in Mycobacterium smegmatis and M. tuberculosis

The chromosome partitioning proteins, ParAB, ensure accurate segregation of genetic materials into daughter cells and most bacterial species contain their homologs. However, little is known about the regulation of ParAB proteins. In this study, we found that 3-methyladenine DNA glycosylase I MsTAG(Ms5082) regulates bacterial growth and cell morphology by directly interacting with MsParA (Ms6939) and inhibiting its ATPase activity in Mycobacterium smegmatis. Using bacterial two-hybrid and pull-down techniques in combination with co-immunoprecipitation assays, we show that MsTAG physically interacts with MsParA both in vitro and in vivo. Expression of MsTAG under conditions of DNA damage induction exhibited similar inhibition of growth as the deletion of the parA gene in M. smegmatis. Further, the effect of MsTAG on mycobacterial growth was found to be independent of its DNA glycosylase activity, and to result instead from direct inhibition of the ATPase activity of MsParA. Co-expression of these two proteins could counteract the growth defect phenotypes observed in strains overexpressing MsTAG alone in response to DNA damage induction. Based on protein co-expression and fluorescent co-localization assays, MsParA and MsTAG were further found to co-localize in mycobacterial cells. In addition, the interaction between the DNA glycosylase and ParA, and the regulation of ParA by the glycosylase were conserved in M. tuberculosis and M. smegmatis. Our findings provide important new insights into the regulatory mechanism of cell growth and division in mycobacteria.     

A protein kinase inhibitor as an antimycobacterial agent

The protein kinase inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7) was found to inhibit the growth of two different mycobacterial strains, the slow-growing Mycobacterium bovis Bacille Calmette Guerin (BCG) and the fast-growing saprophyte Mycobacterium smegmatis mc2 155, in a dose-dependent manner. While screening for the effect of kinase inhibitors on mycobacterial growth, millimolar concentrations of H7 induced a 40% decrease in the growth of M. bovis BCG when measured as a function of oxidative phosphorylation. This H7-induced decrease in growth was shown to involve a 2-log fold decrease in the viable counts of M. smegmatis within a 48-h period and a 50% reduction in the number of BCG viable counts within a 10-day period. Micromolar concentrations of H7 compound induced a significant decrease in the activity of the Mycobacterium tuberculosis protein serine/threonine kinase (PSTK) PknB. The inhibition of mycobacterial growth as well as the inhibition of a representative M. tuberculosis protein serine/threonine kinase PknB suggests that conventional PSTK inhibitors can be used to study the role that the mycobacterial PSTK family plays in controlling bacterial growth.     

Cloning and expression of the trehalose-phosphate phosphatase of Mycobacterium tuberculosis: comparison to the enzyme from Mycobacterium smegmatis

Two open reading frames in the Mycobacterium tuberculosis genome, Rv3372 and Rv2006, have about 25% sequence identity at the amino acid level to the trehalose-phosphate phosphatase (TPP) purified from Mycobacterium smegmatis. However, the protein produced from the cloned Rv3372 gene has a molecular weight of about 45kDa whereas the trehalose-P phosphatase purified from M. smegmatis has a molecular weight of about 27kDa. We expressed the Rv3372 protein in Escherichia coli and show here that it is a trehalose-P phosphatase with very similar properties to the M. smegmatis TPP, i.e., complete specificity for trehalose-phosphate as the substrate, an almost absolute requirement for Mg(2+), and a pH optimum of 7-7.5. On the other hand, in contrast to the M. smegmatis enzyme, the Rv3372 protein was much less stable to heat and much less sensitive to inhibition by diumycin and moenomycin. In fact, both of these antibiotics stimulate enzyme activity at low concentrations and only inhibit the activity at higher antibiotic concentrations. Antibody prepared against the 27kDa TPP does not cross react with the 45kDa TPP nor does antibody against the 45kDa TPP cross react with the 27kDa TPP. Nevertheless, studies of secondary structure by circular dichroism indicate that the two enzymes are quite similar in structure. The product of the other gene, Rv2006, is a 159kDa protein with no detectable phosphatase activity. Thus, its function is currently unknown.     

Interaction of rifampicin with nonprotein thiols in mycobacterium smegmatis

In this study, the interaction of Rifampicin (RIF) with cellular glutathione (GSH) in Mycobacterium smegmatis has been investigated. Minimum inhibitory concentration of RIF for M. smegmatis was demonstrated to be 17 micrograms ml-1 medium. Three subinhibitory concentrations viz. 5, 10 and 15 micrograms RIF ml-1 medium were used to study its interaction with cellular non protein thiols (NPSH). Maximum depletion (57.8%) in NPSH levels [5, 5'-dithiobis (2-nitrobenzoic acid) assay] was observed on second day when the cells were grown in the presence of 15 micrograms RIF ml-1 medium. When the same samples were assayed for GSH levels (glyoxylase assay) the depletion of GSH levels by RIF was still observed, confirming the earlier findings. GSH depletion paralleled with growth inhibition and reached to normal level on 5th day of growth. Cellular depletion of GSH was also observed when 3 day grown cells of M. smegmatis were exposed to various concentrations of RIF (20, 40 and 60 micrograms ml-1 medium) for different time intervals. Maximum depletion of NPSH levels was observed when 3 day grown cultures were treated with 60 micrograms RIF ml-1 medium for a period of 6 h. The results of this study clearly demonstrate that RIF depletes cellular GSH levels regardless of the fact that the drug is included in the medium before inoculating it or after the cells have been grown for a period of three days. The depletion of cellular GSH levels by RIF in M. smegmatis may contribute towards its antituberculous activity.     

分枝杆菌RpsI序列差异对核糖体结构与功能的影响

核糖体结构存在动态调控,其变化与细菌发育、环境适应等过程密切相关。使用NCBI BLAST比对结核分枝杆菌(Mycobacterium tuberculosis)核糖体蛋白RpsI、RpmI和RpmJ与耻垢分枝杆菌(Mycobacterium smegmatis)相应蛋白的氨基酸序列,发现RpsI N端氨基酸序列存在较大差异。为了探究该N端序列差异对核糖体结构与功能的影响,将表达有结核分枝杆菌rpsI基因(rpsI_Rv)的质粒整合至耻垢分枝杆菌基因组中,并利用同源重组的方法敲除耻垢分枝杆菌rpsI基因,以此构建重组菌株。聚合酶链反应(polymerase chain reaction,PCR)结果表明该重组菌株构建成功。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)显示0.5 mmol/L异丙基-β-D-硫代半乳糖苷(IPTG)于16 ℃可诱导表达RpsI_Rv。用纯化的RpsI_Rv制备特异性多克隆抗体,其效价为 1 600 000。反转录PCR 和蛋白质印迹法(Western blot)显示rpsI_Rv在重组菌株中成功表达。测定重组菌株与空载对照菌株在不同温度下的生长曲线,该重组菌株在不同温度下的生长速率未发生改变。采用通用液体倍比稀释法测定作用于核糖体不同位点的5种抗生素最小抑菌浓度(MIC₉₀),重组菌株对阿米卡星(作用于核糖体小亚基A位点的抗生素)的敏感性升高,提示分枝杆菌RpsI序列差异导致核糖体小亚基A位点附近的结构发生改变,这为分枝杆菌核糖体结构与功能的机制研究提供了数据。     

Function of the cytochrome bc1-aa3 branch of the respiratory network in mycobacteria and network adaptation occurring in response to its disruption

The aerobic electron transport chain in Mycobacterium smegmatis can terminate in one of three possible terminal oxidase complexes. The structure and function of the electron transport pathway leading from the menaquinol-menaquinone pool to the cytochrome bc1 complex and terminating in the aa3-type cytochrome c oxidase was characterized. M. smegmatis strains with mutations in the bc1 complex and in subunit II of cyctochome c oxidase were found to be profoundly growth impaired, confirming the importance of this respiratory pathway for mycobacterial growth under aerobic conditions. Disruption of this pathway resulted in an adaptation of the respiratory network that is characterized by a marked up-regulation of cydAB, which encodes the bioenergetically less efficient and microaerobically induced cytochrome bd-type menaquinol oxidase that is required for the growth of M. smegmatis under O2-limiting conditions. Further insights into the adaptation of this organism to rerouting of the electron flux through the branch terminating in the bd-type oxidase were revealed by expression profiling of the bc1-deficient mutant strain using a partial-genome microarray of M. smegmatis that is enriched in essential genes. Although the expression profile was indicative of an increase in the reduced state of the respiratory chain, blockage of the bc1-aa3 pathway did not induce the sentinel genes of M. smegmatis that are induced by oxygen starvation and are regulated by the DosR two-component regulator.     

Glycine and alanine dehydrogenase activities are catalyzed by the same protein in Mycobacterium smegmatis: upregulation of both activities under microaerophilic adaptation

Microaerophilic adaptation has been described as one of the in vitro dormancy models for tuberculosis. Studies on Mycobacterium tuberculosis adapted to low oxygen levels showed an enhancement of glycine dehydrogenase (deaminating) activity. We studied the physiology of the fast-growing, nonpathogenic strain of Mycobacterium smegmatis ATCC 607 under low oxygen by shifting the actively growing M. smegmatis cells to static microaerophilic growth conditions. This shifting of M. smegmatis culture resulted in a similar phenomenon as seen with M. tuberculosis, i.e., elevated glycine dehydrogenase activity. Further purification of glycine dehydrogenase from M. smegmatis demonstrated glyoxylate amination, but failed to demonstrate glycine deamination, even in the purified fraction. Moreover, the purified protein showed pyruvate amination as well as L-alanine deamination activities. By activity staining, the protein band positive for glyoxylate amination demonstrated only pyruvate amination in the presence of NAD. Absence of glycine deamination activity strongly suggested that alanine dehydrogenase of M. smegmatis was responsible for glyoxylate amination in the cell lysate. This was further confirmed by demonstrating the similar level of upregulation of both glyoxylate and pyruvate amination activities in the cell lysate of the adapted culture.     

VapC toxins from Mycobacterium tuberculosis are ribonucleases that differentially inhibit growth and are neutralized by cognate VapB antitoxins

The chromosome of Mycobacterium tuberculosis (Mtb) encodes forty seven toxin-antitoxin modules belonging to the VapBC family. The role of these modules in the physiology of Mtb and the function(s) served by their expansion are unknown. We investigated ten vapBC modules from Mtb and the single vapBC from M. smegmatis. Of the Mtb vapCs assessed, only Rv0549c, Rv0595c, Rv2549c and Rv2829c were toxic when expressed from a tetracycline-regulated promoter in M. smegmatis. The same genes displayed toxicity when conditionally expressed in Mtb. Toxicity of Rv2549c in M. smegmatis correlated with the level of protein expressed, suggesting that the VapC level must exceed a threshold for toxicity to be observed. In addition, the level of Rv2456 protein induced in M. smegmatis was markedly lower than Rv2549c, which may account for the lack of toxicity of this and other VapCs scored as 'non-toxic'. The growth inhibitory effects of toxic VapCs were neutralized by expression of the cognate VapB as part of a vapBC operon or from a different chromosomal locus, while that of non-cognate antitoxins did not. These results demonstrated a specificity of interaction between VapCs and their cognate VapBs, a finding corroborated by yeast two-hybrid analyses. Deletion of selected vapC or vapBC genes did not affect mycobacterial growth in vitro, but rendered the organisms more susceptible to growth inhibition following toxic VapC expression. However, toxicity of 'non-toxic' VapCs was not unveiled in deletion mutant strains, even when the mutation eliminated the corresponding cognate VapB, presumably due to insufficient levels of VapC protein. Together with the ribonuclease (RNase) activity demonstrated for Rv0065 and Rv0617--VapC proteins with similarity to Rv0549c and Rv3320c, respectively--these results suggest that the VapBC family potentially provides an abundant source of RNase activity in Mtb, which may profoundly impact the physiology of the organism.     

异烟肼影响重组耻垢分支杆菌Rv1776c基因表达的实验研究

目的 研究经典抗结核药物异烟肼对重组耻垢分支杆菌生长增殖及其Rv1776c基因表达的影响。方法 将重组菌MS-Rv1776c接种于LB培养基培养作为对照组,实验组给予异烟肼药物处理,不同时间点取菌液测量A600值,根据所测A值绘制增殖曲线;提取菌液DNA以RT-PCR方法检测异烟肼对Rv1776c基因表达的影响;免疫印迹法SDS-PAGE及Western blot检测异烟肼对Rv1776c蛋白表达的影响。结果 异烟肼对两组重组耻垢分枝杆菌增殖无显著影响（P>0.05）;异烟肼可抑制Rv1776c基因的表达（P<0.05）;SDS-PAGE及Western blot检测发现异烟肼可显著降低ERv1776c蛋白的表达（P<0.05）。结论 异烟肼对重组耻垢分支杆菌的增殖无影响,但可抑制结核分枝杆菌Rv1776c基因及其表达的蛋白。该结果对研究结核菌从休眠菌到复苏初期及活跃期的药物预防提供了实验依据。     

NHEJ protects mycobacteria in stationary phase against the harmful effects of desiccation

The physiological role of the non-homologous end-joining (NHEJ) pathway in the repair of DNA double-strand breaks (DSBs) was examined in Mycobacterium smegmatis using DNA repair mutants (DeltarecA, Deltaku, DeltaligD, Deltaku/ligD, DeltarecA/ku/ligD). Wild-type and mutant strains were exposed to a range of doses of ionizing radiation at specific points in their life-cycle. NHEJ-mutant strains (Deltaku, DeltaligD, Deltaku/ligD) were significantly more sensitive to ionizing radiation (IR) during stationary phase than wild-type M. smegmatis. However, there was little difference in IR sensitivity between NHEJ-mutant and wild-type strains in logarithmic phase. Similarly, NHEJ-mutant strains were more sensitive to prolonged desiccation than wild-type M. smegmatis. A DeltarecA mutant strain was more sensitive to desiccation and IR during both stationary and especially in logarithmic phase, compared to wild-type strain, but it was significantly less sensitive to IR than the DeltarecA/ku/ligD triple mutant during stationary phase. These data suggest that NHEJ and homologous recombination are the preferred DSB repair pathways employed by M. smegmatis during stationary and logarithmic phases, respectively.