Cytological and transcript analyses reveal fat and lazy persister-like bacilli in tuberculous sputum.
Cytological and transcript analyses reveal fat and lazy persister-like bacilli in tuberculous sputum.
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DOI：
10.1371/journal.pmed.0050075
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发表时间：
2008-04-01
期刊：
影响因子：
15.8
通讯作者：
Barer, Michael R.
中科院分区：
文献类型：
--
作者：
Garton, Natalie J.;Waddell, Simon J.;Sherratt, Anna L.;Lee, Su-Min;Smith, Rebecca J.;Senner, Claire;Hinds, Jason;Rajakumar, Kumar;Adegbola, Richard A.;Besra, Gurdyal S.;Butcher, Philip D.;Barer, Michael R.
结核痰提供了必须通过化疗消除的杆菌样本，并且可能会继续传播感染。初步观察发现，结核分枝杆菌细胞在痰中含有三酰甘油脂体，但在体外生长时则不含三酰甘油脂体，这促使我们研究这种现象的程度及其生理基础。通过尼罗红和金胺联合染色，研究了来自英国和冈比亚的显微镜检查阳性痰样本中脂体阳性分枝杆菌的含量。所有样本均含有脂质体阳性群体，占抗酸杆菌的 3% 至 86%。最近发现，当分枝杆菌进入体外非复制持久性时，三酰甘油合酶由分枝杆菌表达，这促使我们研究这种状态是否也与脂质体形成相关。我们发现，当置于诱导非复制持久性的实验室条件下时，两种结核分枝杆菌菌株的脂质体水平与痰中发现的水平相当。我们通过将生长和非复制持久性培养物的结核分枝杆菌转录组与直接从痰样本中获得的转录组进行比较，进一步研究了这些生理学发现。尽管传统上认为痰液中含有活跃生长的结核杆菌，但我们的转录本分析反驳了这些细胞占主导地位的假设。相反，它们通过揭示可明显归因于缓慢复制或非复制分枝杆菌的转录特征来强化脂体分析的结果。最后，脂质体计数与诊断液体培养物中的阳性时间高度相关（R2 = 0.64，p < 0.03），从而在该细胞学特征与潜在非复制群体的大小之间建立了直接联系。由于非复制型结核杆菌能够耐受抗生素的杀灭作用并且能够抵抗多种应激，因此鉴定痰中这种类似持久性结核杆菌群体为研究对化疗的反应和结核病的传播提供了令人兴奋且易于处理的新机会。 Michael Barer 及其同事通过研究肺结核患者的痰液，检测到含有脂质体的分枝杆菌。将这种细胞学特征与缓慢生长的表型联系起来的分析揭示了持久性。每年，近 900 万人患上结核病（一种通常发生在肺部的传染性感染），约 200 万人死于该病。结核病是由结核分枝杆菌引起的，当结核病患者咳嗽或打喷嚏时，这种细菌会通过空气中的飞沫传播。结核病的症状包括持续咳嗽、体重减轻和盗汗。诊断测试包括胸部 X 光检查、结核菌素皮试和痰液分析。对于最后一项测试，收集痰液样本（咳嗽时从肺部带出的粘液和其他物质），然后带到实验室，细菌学家使用特殊染色剂（结核病阳性痰液中含有“抗酸杆菌”）寻找结核分枝杆菌，并尝试从样本中培养细菌。服用几种强效抗生素几个月即可治愈结核病。完成这种治疗对于确保体内所有结核分枝杆菌被杀死并防止耐药菌的出现非常重要。人们正在付出巨大的努力来减轻全球结核病的负担，但由于多种原因，迄今为止收效甚微。成功的一个障碍是结核分枝杆菌从一个人传播到另一个人的效率。人们对细菌生命周期的这一部分知之甚少。如果科学家能够更多地了解结核分枝杆菌在人与人之间的传播，他们可能会找到新的治疗和预防目标。因此，在这项研究中，研究人员检查了结核病阳性痰样本中的抗酸杆菌，以获取结核分枝杆菌在传播给新人时的快照，并询问这些杆菌的特征与实验室中生长的结核分枝杆菌的特征有何不同。研究人员在英国和冈比亚的结核病患者接受任何治疗之前收集了他们的痰样本，并寻找含有“脂质体”的抗酸杆菌的存在。这些小结构含有一种称为三酰甘油的脂肪。当结核分枝杆菌暴露于感染过程中存在的几种压力（例如，氧气减少或缺氧）时，它会积累三酰甘油，研究人员表明，这种脂肪的存在可能有助于细菌在传播过程中生存并建立新的感染。他们发现所有样本都含有一些脂质体阳性抗酸杆菌。接下来，研究人员发现，在实验室低氧条件下生长的结核分枝杆菌也会积累脂质体，这种条件会诱导细菌进入一种称为“非复制持久”（NRP）状态的抗生素耐受状态。这一结果表明痰中的脂体阳性抗酸杆菌可能处于NRP状态。为了验证这个想法，研究人员比较了结核分枝杆菌生长培养物、维持在 NRP 状态的结核分枝杆菌以及几个痰样本中的抗酸杆菌产生的 mRNA 模式（产生蛋白质的模板；mRNA 的模式称为转录组，并给出了细胞在给定条件下产生哪些蛋白质的概念）。痰样本的转录组显示许多 NRP 状态下产生的蛋白质。最后，研究人员表明，随着痰中脂体阳性抗酸杆菌比例的增加，从痰样本中培养结核分枝杆菌所需的时间也会增加，就像人们怀疑脂体的存在是否意味着非生长细胞一样。人们普遍认为，从结核病患者收集的痰液中的抗酸杆菌正在快速复制从肺部感染区域释放的结核分枝杆菌。通过在两个地理位置检查的所有痰样本中鉴定出含有脂质体且处于类 NRP 状态的细菌群体，这项研究强烈挑战了这一假设。研究人员认为，这一细菌群体的特征可能有助于它们在结核分枝杆菌在人际传播过程中遇到的不利条件下生存，并可能部分解释为什么完全清除结核分枝杆菌需要延长抗生素治疗。为了确定这些发现的临床意义，未来的研究需要检查抗生素治疗是否会影响患者痰中脂质体阳性结核分枝杆菌的频率，以及该测量值与传染性或对治疗的临床反应之间是否存在任何关系。请通过此摘要的在线版本访问这些网站：http://dx.doi.org/10.1371/journal.pmed.0050075。 MedlinePlus 百科全书包含有关结核病和痰培养的页面（英文和西班牙文） 美国国家过敏和传染病研究所提供有关结核病各个方面的信息 美国疾病控制和预防中心结核病消除部门提供了有关结核病的若干情况说明书和其他信息资源 世界卫生组织提供了有关减轻全球结核病负担的努力的信息
Tuberculous sputum provides a sample of bacilli that must be eliminated by chemotherapy and that may go on to transmit infection. A preliminary observation that Mycobacterium tuberculosis cells contain triacylglycerol lipid bodies in sputum, but not when growing in vitro, led us to investigate the extent of this phenomenon and its physiological basis. Microscopy-positive sputum samples from the UK and The Gambia were investigated for their content of lipid body–positive mycobacteria by combined Nile red and auramine staining. All samples contained a lipid body–positive population varying from 3% to 86% of the acid-fast bacilli present. The recent finding that triacylglycerol synthase is expressed by mycobacteria when they enter in vitro nonreplicating persistence led us to investigate whether this state was also associated with lipid body formation. We found that, when placed in laboratory conditions inducing nonreplicating persistence, two M. tuberculosis strains had lipid body levels comparable to those found in sputum. We investigated these physiological findings further by comparing the M. tuberculosis transcriptome of growing and nonreplicating persistence cultures with that obtained directly from sputum samples. Although sputum has traditionally been thought to contain actively growing tubercle bacilli, our transcript analyses refute the hypothesis that these cells predominate. Rather, they reinforce the results of the lipid body analyses by revealing transcriptional signatures that can be clearly attributed to slowly replicating or nonreplicating mycobacteria. Finally, the lipid body count was highly correlated (R2 = 0.64, p < 0.03) with time to positivity in diagnostic liquid cultures, thereby establishing a direct link between this cytological feature and the size of a potential nonreplicating population. As nonreplicating tubercle bacilli are tolerant to the cidal action of antibiotics and resistant to multiple stresses, identification of this persister-like population of tubercle bacilli in sputum presents exciting and tractable new opportunities to investigate both responses to chemotherapy and the transmission of tuberculosis. Studying sputum from humans with pulmonary tuberculosis, Michael Barer and colleagues detect mycobacteria containing lipid bodies. Analyses linking this cytological feature to a slow-growing phenotype sheds light on persistence. Every year, nearly nine million people develop tuberculosis—a contagious infection usually of the lungs—and about two million people die from the disease. Tuberculosis is caused by Mycobacterium tuberculosis, bacteria that are spread in airborne droplets when people with the disease cough or sneeze. The symptoms of tuberculosis include a persistent cough, weight loss, and night sweats. Diagnostic tests include chest X-rays, the tuberculin skin test, and sputum analysis. For the last of these tests, a sample of sputum (mucus and other matter brought up from the lungs by coughing) is collected and then taken to a laboratory where bacteriologists look for M. tuberculosis using special stains—tuberculosis-positive sputum contains “acid-fast bacilli”—and also try to grow bacteria from the sample. Tuberculosis can be cured by taking several powerful antibiotics for several months. It is very important that this treatment is completed to ensure that all the M. tuberculosis bacteria in the body are killed and to prevent the emergence of drug-resistant bacteria. Strenuous efforts are being made to reduce the global burden of tuberculosis but with limited success so far for many reasons. One barrier to success is the efficiency with which M. tuberculosis spreads from one person to another. Very little is known about this part of the bacteria's life cycle. If scientists could understand more about the transmission of M. tuberculosis between people, they might identify new therapeutic and preventative targets. In the study, therefore, the researchers examine the acid-fast bacilli in tuberculosis-positive sputum samples to get a snapshot of M. tuberculosis at the point of its transmission to a new person and ask how the characteristics of these bacilli compare with those of M. tuberculosis growing in the laboratory. The researchers collected sputum samples from patients with tuberculosis in the UK and The Gambia before they received any treatment, and looked for the presence of acid-fast bacilli containing “lipid bodies.” These small structures contain a fat called triacylglycerol. M. tuberculosis accumulates triacylglycerol when it is exposed to several stresses present during infection (for example, reduced oxygen or hypoxia) and the researchers suggest that the presence of this fat may help the bacteria survive during transmission and establish a new infection. They found that all the samples contained some lipid body–positive acid-fast bacilli. Next, the researchers showed that M. tuberculosis grown in the laboratory under hypoxic conditions, which induce the bacteria to enter an antibiotic-tolerant condition called a “nonreplicating persistent” (NRP) state, also accumulated lipid bodies. This result suggests that the lipid body–positive acid-fast bacilli in sputum might be in an NRP state. To test this idea, the researchers compared the pattern of mRNAs (the templates from which proteins are produced; the pattern of mRNAs is called the transcriptome and gives an idea of which proteins a cell is making under given conditions) made by growing cultures of M. tuberculosis, by M. tuberculosis maintained in the NRP state, and by the acid-fast bacilli in several sputum samples. The transcriptome of the sputum sample revealed production of many proteins made in the NRP state. Finally, the researchers showed that the time needed to grow M. tuberculosis from sputum samples increased as the proportion of lipid body–positive acid-fast bacilli in the sputum increased, just as one would suspect if the presence of lipid bodies signifies nongrowing cells. It has been generally assumed that the acid-fast bacilli in sputum collected from patients with tuberculosis are rapidly replicating M. tuberculosis released from infected areas of the lungs. By identifying a population of bacteria that contain lipid bodies and that are in an NRP-like state in all the samples of sputum examined from two geographical sites, this study strongly challenges this assumption. The characteristics of this population of bacteria, the researchers suggest, might help them survive the adverse conditions that M. tuberculosis encounters during transmission between people and might partly explain why complete clearance of M. tuberculosis requires extended treatment with antibiotics. To establish the clinical significance of these findings, future studies will need to examine whether antibiotic treatment affects the frequency of lipid body–positive M. tuberculosis bacteria in patients' sputum and whether there is any relationship between this measurement and infectiousness, or clinical response to treatment. Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0050075. The MedlinePlus encyclopedia contains pages on tuberculosis and on sputum culture (in English and Spanish) The US National Institute of Allergy and Infectious Diseases provides information on all aspects of tuberculosis The US Centers for Disease Control and Prevention Division of Tuberculosis Elimination provides several fact sheets and other information resources about tuberculosis The World Health Organization provides information on efforts to reduce the global burden of tuberculosis
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为什么要治愈结核病需要长期治疗？
影响因子：
15.8
作者：
Connolly, Lynn E.;Edelstein, Paul H.;Ramakrishnan, Lalita
通讯作者：
Ramakrishnan, Lalita
影响因子：
3.2
作者：
Beste, D. J. V.;Laing, E.;McFadden, J. J.
通讯作者：
McFadden, J. J.
影响因子：
3.2
作者：
Daniel, J;Deb, C;Kolattukudy, PE
通讯作者：
Kolattukudy, PE
影响因子：
4.2
作者：
Alvarez, HM;Silva, RA;Steinbüchel, A
通讯作者：
Steinbüchel, A
影响因子：
3.2
作者：
Gomez, JE;McKinney, JD
通讯作者：
McKinney, JD