Use of visual loop-mediated isotheral amplification of rimM sequence for rapid detection of Mycobacterium tuberculosis and Mycobacterium bovis

Mycobacterium tuberculosis and Mycobacterium bovis are pathogenic bacterial species in the genus Mycobacterium and the causative agents of most cases of tuberculosis (TB). Detection of M. tuberculosis and M. bovis using conventional culture- and biochemical-based assays is time-consuming and laborious. Therefore, a simple and sensitive method for rapid detection has been anxiously awaited. In the present study, a visual loop-mediated isothermal amplification (LAMP) assay was designed from the rimM (encoding 16S rRNA-processing protein) gene sequence and used to rapidly detect M. tuberculosis and M. bovis from clinical samples in South China. The visual LAMP reaction was performed by adding calcein and manganous ion, allowing the results to be read by simple visual observation of color change in a closed-tube system, and which takes less than 1 h at 65 °C. The assay correctly identified 84 M. tuberculosis isolates, 3 M. bovis strains and 1 M. bovis BCG samples, but did not detect 51 non-tuberculous mycobacteria (NTM) isolates and 8 other bacterial species. Sensitivity of this assay for detection of genomic DNA was 1 pg. Specific amplification was confirmed by the ladder-like pattern of gel electrophoresis and restriction enzyme HhaI digestion. The assay successfully detected M. tuberculosis and M. bovis not only in pure bacterial culture but also in clinical samples of sputum, pleural fluid and blood. The speed, specificity, sensitivity of the rimM LAMP, the lack of a need for expensive equipment, and the visual readout show great potential for clinical detection of M. tuberculosis and M. bovis.     

牛分枝杆菌Mb0950c的免疫特性及其血清学检测方法的建立

【目的】利用原核表达系统对牛分枝杆菌Mb0950c蛋白进行表达和纯化,通过小鼠模型评价其免疫原性,建立血清学间接ELISA方法用于牛结核病的临床检测。【方法】构建pET32a-Mb0950c原核表达质粒,并转化至BL21(DE3)中诱导蛋白的表达,对蛋白进行纯化。使用流式细胞术(flow cytometry,FCM)、ELISA等对该蛋白在小鼠中的免疫原性进行分析。建立基于Mb0950c的间接ELISA方法,评价该方法的临床检测潜力。【结果】SDS-PAGE和Western blotting结果显示,成功获得了可溶性Mb0950c蛋白,且具有良好免疫反应性;FCM结果显示,Mb0950c蛋白上调了T细胞表面CD69分子的表达。细胞因子和抗体结果表明,该蛋白能够诱导特异性的IFN-γ和IL-4的分泌,同时能诱导机体分泌特异性的抗体,且以IgG1型为主。建立了ELISA检测方法应用于牛结核临床检测,结果显示,该方法与牛结核外周血IFN-γ外释放试验和皮试试验结果的阳性符合率、阴性符合率和总符合率分别为65.7%、97.9%和72.4%。【结论】在原核表达系统中可溶性表达Mb0950c蛋白,...     

The ability of non-bacteriocin producing Streptococcus bovis strains to bind and transfer bovicin HC5 to other sensitive bacteria

Streptococcus bovis HC5 produces a broad spectrum lantibiotic (bovicin HC5), but S. bovis JB1 does not have antimicrobial activity. Preliminary experiments revealed an anomaly. When S. bovis JB1 cells were washed in stationary phase S. bovis HC5 cell-free culture supernatant, the S. bovis JB1 cells were subsequently able to inhibit hyper-ammonia producing ruminal bacteria (Clostridium sticklandii, Clostridium aminophilum and Peptostreptococcus anaerobius). Other non-bacteriocin producing S. bovis strains also had the ability to bind and transfer semi-purified bovicin HC5. Bovicin HC5 that was bound to S. bovis JB1 was much more resistant to Pronase E than cell-free bovicin HC5, but it could be inactivated if the incubation period was 24 h. Acidic NaCl treatment (100 mM, pH 2.0) liberates half of the bovicin HC5 from S. bovis HC5, but it did not prevent bovicin HC5 from binding to S. bovis JB1. Acidic NaCl liberated some bovicin HC5 from S. bovis JB1, but the decrease in activity was only 2-fold. Bovicin HC5 is a positively charged peptide, and the ability of S. bovis JB1 to bind bovicin HC5 could be inhibited by either calcium or magnesium (100 mM). Acidic NaCl-treated S. bovis JB1 cells were unable to accumulate potassium, but they were still able to bind bovicin HC5 and prevent potassium accumulation by untreated S. bovis JB1 cells. Based on these results, bovicin HC5 bound to S. bovis JB1 cells still acts as a pore-forming lantibiotic.     

结核分枝杆菌PhoP系统研究进展

Pho P与Pho R组成的Pho PR是结核分枝杆菌重要的双组分调节系统。Pho P作为应答调节子调节基因的表达,这些基因参与细胞壁脂质合成,并对结核分枝杆菌毒力有重要调控作用。本文综述了Pho P的结构、性质以及相关的结核分枝杆菌疫苗研发情况,并提出了未来可能的研究趋势。     

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...     

Physical and functional interaction between d-ribokinase and topoisomerase I has opposite effects on their respective activity in Mycobacterium smegmatis and Mycobacterium tuberculosis

d-ribose is an essential component of multiple important biological molecules and must first be phosphorylated by ribokinase before entering metabolic pathways. However, the function and regulation of ribokinases in Mycobacterium tuberculosis, the causative agent of tuberculosis, and its related species are largely unknown. In this study, we have characterized the activities of two putative ribokinases, Rv2436 and Ms4585, from M. tuberculosis and Mycobacterium smegmatis, respectively. The mycobacterial topoisomerase I (TopA) was found to physically interact with its ribokinase both in vitro and in vivo. By creating two ribokinase mutants that showed defective interactions with TopA, we further showed that the interaction between ribokinase and TopA had opposite effects on their respective function. While the interaction between the two proteins inhibited the ability of TopA to relax supercoiled DNA, it stimulated ribokinase activity. A cross-regulation assay revealed that the interaction between the two proteins was conserved in the two mycobacterial species. Thus, we uncovered an interplay between ribokinase and topoisomerase I in mycobacteria, which implies the existence of a novel regulatory strategy for efficient utilization of d-ribose in M. tuberculosis that may be useful in stressful environments with restricted access to nutrients.     

The novel protein BboRhop68 is expressed by intraerythrocytic stages of Babesia bovis

The apical complex of intracellular hemoparasites contains organelles like micronemes and rhoptries, specialized structures required for adherence and invasion of host cells. Several molecules discharged from rhoptries have been identified from Plasmodium spp., but only a single rhoptry associated protein-1 (RAP-1) has been characterized from Babesia bovis. In silico search of the B. bovis genome allowed to identifying a sequence homologous to the gene that encodes a P. falciparum rhoptry protein PfRhop148. The intron-less 1830 bp novel gene, predicted a 68 kDa protein, and it was highly conserved among different B. bovis strains and isolates. The deducted protein from the B. bovis T2Bo strain, named BboRhop68, showed two putative transmembrane domains, at least seven B-cell epitopes, and a well conserved DUF501 super family domain. The bborhop68 gene was amplified, analyzed and compared among different B. bovis strains and isolates showing overall high sequence conservation. A fragment of bborhop68 was expressed as a recombinant fusion protein (rBboRhop68). The mice anti-rBboRhop68 serum identified the novel protein in intraerythrocytic trophozoites and merozoites by WB and ELISA, but not in free merozoites. Sera from naturally and experimentally infected bovines also recognized BboRhop68, suggesting that it is expressed and immunogenic during B. bovis infection. Fluorescence microscopy analysis using anti-rBboRhop68 antibodies showed a rod structure associated to trophozoites and merozoites infected erythrocytes, but this pattern of reactivity was not observed in free merozoites. The BboRhop68 was also not detected in ELISA based on solubilized merozoites. Thus, at least three independent lines of evidence support differential expression of BboRhop68 in intraerythrocytic stages of B. bovis and its possible functional role immediately after B. bovis erythrocyte invasion. The results of this work suggest that BboRhop68 could be considered as a novel additional target for developing improved methods to control bovine babesiosis.     

Easy differentiation of Mycobacterium mucogenicum from other species of the Mycobacterium fortuitum complex by thin-layer and gas chromatography of fatty esters and alcohols

The mycolate pattern of a recently recognized mycobacterial pathogen, Mycobacterium mucogenicum (formerly Mycobacterium chelonae-like organism), was established for the first time. The reference strains, together with 31 environmental and clinical isolates belonging to this species, were examined for their mycolate composition by thin-layer chromatography. All strains tested exhibited the same mycolate profile. Mycolates were identified as belonging to the type without additional oxygenated chemical groups (mycolate I) and the type with a dicarboxylic group (mycolate VI); the identification of the latter was reinforced by the presence of 2-octadecanol, as seen by gas-liquid capillary chromatography. This mycolate profile permits the clear differentiation of M. mucogenicum from other related species, as members of the Mycobacterium fortuitum complex. This fact is especially important because strains of M. mucogenicum are very difficult to differentiate from other species of the M. fortuitum complex by means of conventional biochemical tests. Moreover, the characteristic mycolate profile exhibited by the strains of M. mucogenicum supports the recent proposal which considers them as members of a new species.     

禽分枝杆菌对豚鼠的致病性分析

【背景】我国禽型结核菌素(avian tuberculin)的制造用菌株为CVCC 68201、CVCC 68202和CVCC 68203株,但目前仍未明确这3株菌的生物学特性及对豚鼠致病性的情况。【目的】探究禽分枝杆菌(Mycobacterium avium)的生物学特性及对动物机体的致病性,为禽结核病和牛结核病的防控工作提供技术支撑。【方法】对3株禽分枝杆菌基因组进行鉴定分析及核酸相似度分析;用3株禽分枝杆菌分别感染豚鼠,观察感染后的临床症状、病理学变化、体重增重情况分析、皮内变态反应结果、脏器系数变化等,进而分析3株禽分枝杆菌对豚鼠的致病力。【结果】种型鉴定和进化分析结果表明,CVCC 68201、CVCC 68202和CVCC 68203均为禽分枝杆菌,基因组与Mycobacterium avium subsp. avium FDAARGOS_1608最为相近;在感染前期、中期、后期对3株禽分枝杆菌感染豚鼠的体重增重情况分析发现,感染禽分枝杆菌影响豚鼠增重,主要表现为生长迟缓,感染第5周时,CVCC 68201、CVCC 68202组豚鼠的平均体重明显轻于未感染组;皮内变态反应试验结果显示,感染CVCC 68201组豚鼠的皮肤红肿面积明显大于其他2个感染组,CVCC 68201可引起机体更为强烈的迟发型变态反应;3株禽分枝杆菌感染后,豚鼠脾脏和肺脏存在不同程度的肿大与出血,其中感染CVCC 68201豚鼠的肺脏系数与未感染组相比差异显著(P<0.01);病理学观察结果显示,豚鼠肺脏可见不同程度病变,其中CVCC 68201组更为严重,表现为肿大和轻微出血。各感染组豚鼠肺脏和脾脏组织切片抗酸染色均可见红色的分枝杆菌散在浸润。【结论】3株禽分枝杆菌对豚鼠均有一定程度的致病性,可引发局部病变。本研究为禽分枝杆菌的制备和鉴定提供依据,也为牛结核病的鉴别诊断方法研究提供参考。     

Visualization of Mycobacterium avium in Crohn's tissue by oil-immersion microscopy

Studies seeking Mycobacterium avium subsp. paratuberculosis in Crohn's disease by PCR have generated inconsistent findings. As an alternative, microscopy offers a number of advantages, including direct visualization of organisms in tissue. Experimental infections have demonstrated that M. avium organisms can be seen by both acid-fast staining and species-specific in situ hybridization, but because they are smaller than M. tuberculosis, oil-immersion microscopy (×1000 magnification) is needed. We performed a blinded search for M. avium in paraffin-embedded surgical resections from Crohn's and control subjects at two centres. Specimens were coded and subjected to acid-fast staining and ribosomal RNA in situ hybridization for M. avium rRNA. Agreement between these two methods was good (42/52 patients, κ = 0.60) and similar results were observed for patients from two centers. Together, both methods provided positive results in 10 of 17 Crohn's subjects (59%, 95% CI: 36–78), contrasting with only 5 of 35 control subjects (Odds ratio for Crohn's vs. controls = 8.6, p = 0.002). M. avium organisms had an intracellular localization within inflammatory lesions, but were often observed as lone organisms outside of granulomas. Using two assays in two settings, presence of M. avium organisms was strongly associated with Crohn's disease.     

用小鼠模型分析可溶性表达结核分枝杆菌Ag85A的免疫原性

【目的】可溶性表达结核分枝杆菌Ag85A蛋白,并评价其免疫原性。【方法】利用冷休克表达质粒和含有伴侣质粒的大肠杆菌对Ag85A蛋白进行可溶性原核表达,并进行纯化与鉴定,通过C57BL/6小鼠模型对Ag85A蛋白的免疫原性,包括诱导机体特异性体液免疫应答和细胞免疫应答水平进行分析。【结果】重组菌诱导后裂解上清中检测到可溶性Ag85A蛋白的表达,经过亲和层析纯化收获了纯度在90%以上的Ag85A蛋白,Western blot鉴定显示其具有较好的免疫反应性。Ag85A蛋白免疫小鼠后,血清中可以检测到高水平的Ig G抗体效价,其中Ig G2b水平要高于Ig G1。通过特异性多肽、蛋白刺激脾脏和腹股沟淋巴结细胞可分泌高水平的IFN-γ、TNF-α等Th1型细胞因子。【结论】实现了Ag85A蛋白的可溶性表达,免疫特性评价显示Ag85A蛋白可诱导机体产生强烈的特异性体液免疫应答及Th1型的细胞免疫应答,从而为其进一步免疫学功能的研究奠定了重要基础。     

结核分枝杆菌Rv0081基因编码蛋白的生物信息学分析

运用生物信息学分析软件预测结核分枝杆菌（Mycobacterium tuberculosis, Mtb）Rv0081蛋白的生物学特征及筛选潜在的优势抗原表位。 从NCBI数据库获取Mtb Rv0081蛋白的氨基酸序列，利用生物信息学分析软件ProtParam、ProtScale及TMPRED分析Rv0081蛋白的理化性质及亲疏水性；TMHMM、SignalP-5.0 Server预测蛋白的跨膜区及信号肽；NetNGlyc-1.0 Server、NetPhos 3.1 Server分别预测蛋白的糖基化位点及磷酸化位点；STRING预测能与Rv0081相互作用的蛋白；分别运用SOPMA、SWISS-MODEL预测蛋白的二、三级结构；综合运用softberry、WoLF PSORT预测蛋白的亚细胞定位；运用DNAStar预测蛋白的B细胞抗原表位；综合运用SYFPEITHI、NetCTL 1.2 Server、Net MHC pan 4.1 server预测蛋白的CTL细胞抗原表位；综合运用SYFPEITHI、Net MHCII pan 4.0 server预测蛋白的Th细胞抗原表位。 结果表明，Rv0081蛋白由114个氨基酸组成，相对分子质量为12 356.32，亚细胞定位于细胞质中，为稳定的疏水性蛋白，无跨膜区和信号肽，含有1个糖基化位点及9个磷酸化位点；二级结构主要由α-螺旋和无规则卷曲构成，结构较松散；与hycE、hycP、Rv0088、Rv0083、hycD、hycQ、Rv0082、devR、Rv0080及Rv0079蛋白存在相互作用关系；综合分析各软件预测结果筛选出6个优势B细胞抗原表位、6个优势CTL细胞抗原表位及7个优势Th细胞抗原表位。Mtb Rv0081蛋白具有较多潜在的候选B、T细胞抗原表位，可作为研发新型结核疫苗的候选抗原。     

Role of the mitogen-activated protein kinase pathway in the differential response of bovine monocytes to Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium

We compared the kinetics of activation and antimicrobial activities of MAPK-p38 and MAPK-ERK in bovine monocytes infected with Mycobacterium avium subsp. paratuberculosis (MAP) and Mycobacterium avium subsp. avium (Maa). Monocytes were incubated with MAP or Maa organisms with or without a specific inhibitor of the MAPK-p38 pathway (SB203580), and MAPK phosphorylation and antimicrobial functions of monocytes were evaluated. At early time points MAPK-p38 phosphorylation was greater in MAP-infected bovine monocytes than in Maa-infected monocytes. At later time points MAPK-p38 phosphorylation by both organisms was similar. MAPKp38 phosphorylation in MAP-infected monocytes was similar to negative control cells, whereas in Maa-infected this activation remained greater than negative control cells. Increase phosphorylation MAPK-ERK was similar at all time points for both organisms. Bovine monocytes had minimal capacity to kill MAP organisms, to acidify MAP-containing phagosomes, or to form phagolysosome. Alternatively, bovine monocytes were able to kill Maa organisms. Addition of SB203580 to monocyte cultures increased phagosome acidification, phagolysosome formation, and killing of MAP and Maa organisms. Taken together these data indicate that early transient activation of MAPK-p38 in bovine mononuclear phagocytes by MAP organisms may be a key mechanism involved in the capacity of MAP to survive in bovine monocytes.     

耻垢分枝杆菌感染人脐带间充质干细胞模型的建立及其特征

【背景】结核分枝杆菌(Mycobacterium tuberculosis, Mtb)休眠菌形成被认为是潜伏结核感染(latent tuberculosis infection, LTBI)的主要原因,但目前缺乏体内和体外模型进行机制研究。新近研究表明Mtb可感染间充质干细胞(mesenchymal stem cells, MSC)并以休眠状态在细胞中长期存活。然而Mtb感染细胞模型存在周期长和生物安全要求高等问题,需要探索可用的MSC感染细胞模型用于Mtb休眠机制的研究。【目的】建立快速生长型耻垢分枝杆菌(Mycobacteriumsmegmatis,Ms)感染人脐带间充质干细胞(human umbilical cord MSC, hUCMSC)的细胞模型并研究其特征。【方法】取分离好的hUCMSC,流式细胞术鉴定其表面标志性抗原;以Ms菌株感染hUCMSC,DiI标记细胞膜,荧光显微镜下观察细胞吞噬作用;平板法计数Ms胞内存活率;油红O染色观察细胞脂滴形成;鬼笔环肽荧光染色观察细胞骨架变化;实时荧光定量PCR(realtimequantitativePCR,RT-qPCR)检测Ms...     

副结核分枝杆菌感染的免疫应答与检测方法研究进展北大核心CSCD

副结核分枝杆菌常引起感染牛的产奶量下降、持续性消瘦、顽固性下痢甚至死亡,给畜牧业带来了巨大的经济损失。牛通常在幼年期经口感染该菌并具有一个较长的亚临床期,后期才表现出临床症状,感染前期以细胞免疫为主并伴随着间歇排菌,经过一个2到5年的亚临床期后体液免疫应答增强,同时排菌量明显增加。目前对牛副结核病常用的检测方法有病原学检测方法,基于细胞免疫反应和基于体液免疫反应的检测方法。由于不同方法的反应原理不同,加之副结核分枝杆菌感染动物的特定免疫应答规律,在某一时间内各方法之间敏感性差异较大。本文简要阐述了牛副结核分枝杆菌的传播途径以及牛感染后的免疫应答特点,对牛副结核病的常见诊断方法进行了综述。     

Detection of streptomycin resistance in Mycobacterium tuberculosis clinical isolates from China as determined by denaturing HPLC analysis and DNA sequencing

China is regarded by the World Health Organization as a major hot-spot region for Mycobacterium tuberculosis infection. Streptomycin has been deployed in China for over 50 years and is still widely used for tuberculosis treatment. We have developed a denaturing HPLC (DHPLC) method for detecting various gene mutations conferring drug resistance in M. tuberculosis. The present study focused on rpsL and rrs mutation analysis. Two hundred and fifteen M. tuberculosis clinical isolates (115 proved to be streptomycin-resistant and 100 susceptible by a routine proportional method) from China were tested to determine the streptomycin minimal inhibitory concentration (MIC), and subjected to DHPLC and concurrent DNA sequencing to determine rpsL and rrs mutations. The results showed that 85.2% (98/115) of streptomycin-resistant isolates harbored rpsL or rrs mutation, while rpsL mutation (76.5%, 88/115) dominated. MIC of 98 mutated isolates revealed no close correlation between mutation types and levels of streptomycin resistance. No mutation was found in any of the susceptible isolates. The DHPLC results were completely consistent with those of sequencing. The DHPLC method devised in this study can be regarded as a useful and powerful tool for detection of streptomycin resistance. This is the first report to describe DHPLC analysis of mutations in the rpsL and rrs genes of M. tuberculosis in a large number of clinical isolates.     

相对定量分析异烟肼、链霉素单耐药与多耐药结核分枝杆菌临床分离株的蛋白质组差异

【目的】探讨异烟肼(isoniazid,INH)、链霉素(streptomycin,SM)单耐药结核分枝杆菌(Mycobacterium tuberculosis,MTB)与INH/SM多耐药MTB蛋白质组差异。【方法】应用i TRAQ结合Nano LC-MS/MS定量蛋白质组学技术,分析临床分离INH、SM或INH/SM耐药MTB与H37Rv标准株间均表达差异蛋白;并以INH/SM耐药MTB与H37Rv比值为对照,相对定量分析单耐药与多耐药MTB蛋白表达差异倍数;运用DAVID 6.7分析差异蛋白生物功能;STITCH 5.0分析差异蛋白与INH和SM相互作用。【结果】与H37Rv标准株比较,58个蛋白在INH、SM耐药与INH/SM耐药MTB间均有表达差异,共同差异蛋白生物功能主要为氧化还原酶活性和转移酶活性;主要参与丙酸代谢信号通路。共同差异蛋白中,与INH/SM耐药MTB比较,Rv2986c和Rv1908c在INH、SM耐药MTB均表达上调1.25倍;Rv3133c和Rv0577则均表达下调0.7倍;生物信息学预测发现以上4种蛋白可直接或间接与INH、SM进行相互作用。【结论】INH、SM单耐药和INH/SM多耐药MTB蛋白表达谱有较大差异,蛋白Rv2986c、Rv1908c、Rv3133c和Rv0577表达水平及相互作用可能与INH和SM耐药有关。