,西南大学生命科学学院,三峡库区生态环境与生物资源省部共建国家重点实验室培育基地,现代生物医药研究所,重庆 400715Acid-resistant genes in Mycobacterium tuberculosis and the underlying regulatory network
Liyuan Zhang, Fujing Huang, Junqi Xu, Zhen Gong, Jianping Xie,Institute of Modern Biopharmaceuticals, State Key Laboratory Breeding Base of Eco-Environment and Bio-Resource of the Three Gorges Area, School of Life Sciences, Southwest University, Chongqing 400715, China第一联系人:
编委: 张天宇
收稿日期:2018-03-27修回日期:2018-05-8网络出版日期:2018-07-20
| 基金资助: |
Received:2018-03-27Revised:2018-05-8Online:2018-07-20
| Fund supported: |
作者简介 About authors
张沥元,本科,专业方向：生物专业E-mail:
黄芙静,本科,专业方向：生物专业E-mail:
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张沥元, 黄芙静, 许峻旗, 龚真, 谢建平. 结核分枝杆菌酸抗性基因及其调控网络. 遗传[J], 2018, 40(7): 546-560 doi:10.16288/j.yczz.18-077
Liyuan Zhang, Fujing Huang, Junqi Xu, Zhen Gong, Jianping Xie.
结核病(Tuberculosis, TB)现已超过艾滋病,成为全球第一大传染病。结核分枝杆菌(Mycobacterium tuberculosis, MTB)是结核病的致病菌。世界上1/3的人口为结核菌的潜伏感染者,每分钟至少两人死于结核病,2015和2016年每年新增1040万结核 病例,约130万人死于结核病[2]。多耐药性结核(multidrug-resistant tuberculosis, MDR-TB),甚至广泛耐药结核(extensively drug-resistant tuberculosis, XDR-TB)[3]的出现使结核病防控形势更加严峻[4, 5]。结核分枝杆菌能够长期在宿主细胞,尤其是巨噬细胞内持留[6],以抵抗或逃避宿主免疫攻击[7]和药物杀灭[8]。吞噬体酸化本来是巨噬细胞控制胞内致病菌的主要机制之一[9,10,11]。已有的研究显示结核分枝杆菌能够调控宿主细胞酸化进程,抵御吞噬体对自身的限制。因此,识别结核分枝杆菌应对酸性环境的基因及其调控网络有助于深化对结核分枝杆菌致病机理的认识,发现结核病防控的新干预节点。本文总结了结核分枝杆菌的3种酸抗性机制以及调控 通路。
1 结核分枝杆菌感染致病过程
结核分枝杆菌有效感染如图1A所示：(1) 微量结核分枝杆菌以气溶胶的形式感染上呼吸道,然后结核分枝杆菌进入下呼吸道的肺泡中。微量的结核分枝杆菌可以避免被宿主模式识别受体如Toll-like receptor (TLR)识别[12]。(2) 结核分枝杆菌表面的脂质如phthiocerol dimycocerosate (PDIM)可以干扰或者抵消被激活的巨噬细胞的杀灭功能[13, 14]。(3) 结核分枝杆菌产生的某些糖肽脂(glycopeptidolipid, GPL)可以刺激细胞产生趋化因子如chemokine (C-C motif) ligand 2 (CCL2),改变被招募来的免疫细胞的组分,进而抵御免疫细胞的杀伤作用。(4) 含有结核分枝杆菌的吞噬体的pH会降低。含有一般致病菌的吞噬体会与溶酶体融合,进而被溶酶体释放的酶类消灭。但结核分枝杆菌却会阻断吞噬体-溶酶体融合,并进一步酸化吞噬体[15],最终pH甚至可以达4.5[16]。被感染的巨噬细胞可能分泌某些尚未完全了解的分子,招募更多的免疫细胞,形成肉芽肿[17],限制其中结核分枝杆菌的生长和播散。当宿主免疫功能降低时,肉芽肿中坏死细胞可以作为结核分枝杆菌生长和繁殖所需的底物。随着活动性结核病患者的咳嗽而将结核分枝杆菌通过气溶胶形式排入空气,成为感染下一位宿主的源头[18]。图1
新窗口打开|下载原图ZIP|生成PPT图1结核分枝杆菌感染途径以及抗酸调控网络
A：结核分枝杆菌感染过程示意图。① 结核分枝杆菌进入巨噬细胞;② 巨噬细胞裂解释放出结核分枝杆菌;③ 结核分枝杆菌弥散在肺部;④结核分枝杆菌通过气溶胶形式传至下一个宿主。B：结核分枝杆菌抗酸通路示意图。① LysX通过转移赖氨酸残基抵御AMPs;② MarP活化蛋白RipA,剪切肽聚糖;③ ABC转运蛋白DrrA/DrrB减少质子进入细胞;④ PE/PPE家族蛋白降解游离脂肪酸等有害物质;⑤ 蛋白OmpATb参与调解胞内氮代谢释放铵根离子;⑥ 双组份系统PhoPR编码基因phoP/phoR调控aprABC以及whiB3的表达。PAMPs：pathogen-associated molecular pattern; PDIM: phthiocerol dimycocerosate; PGL: glycopeptidoliqid; AMP: anti-microbial peptides。
Fig. 1The infection and the acid resistance network of Mycobacterium tuberculosis
2 抗酸能力与结核分枝杆菌胞内存活、持留密切相关
抗酸能力是几乎所有生物都具备的特征,这在大肠杆菌等肠道微生物[19, 20]中研究比较多[21, 22]。抗酸能力与结核分枝杆菌的巨噬细胞内存活和持留密不可分[23]。结核分枝杆菌面临的酸性环境主要为：(1) 由于肉芽肿内缺氧环境而作为其中细胞能量主要来源的糖酵解产生的酸性物质更容易在肉芽肿中积累而形成局部的酸性环境。这使得从坏死的巨噬细胞中释放的结核分枝杆菌的生存和繁殖需要直接面对酸性环境;(2) 激活的巨噬细胞内吞噬体的酸化作用加强,其与溶酶体融合后形成的酸性环境直接影响其中的结核分枝杆菌。一般认为,酸性环境会导致包括结核分枝杆菌在内的生物的酸碱失衡,维持生命活动正常进行所需要的酶类失活,蛋白质被水解等,最终导致菌体死亡[24]。持留,指包括结核分枝杆菌在内的很多微生物的代谢非活跃状态,持留的微生物对绝大多数靶向代谢的药物不敏感[25, 26],因而持留结核分枝杆菌已经成为结核病治疗的三大难题之一[27]。结核分枝杆菌持留形成与其耐受或抵抗酸性环境的能力密不可分。一旦被巨噬细胞吞噬,结核分枝杆菌就面临着直接导致菌体死亡的酸性胁迫,为了在酸性环境中存活下来,调控许多抗酸机制以应对酸性胁迫,如通过调节体内的代谢水平,调控双组份系统来改变菌体的生长活跃状态,使其在酸性环境生存下来,而这样的生存伴随着菌体进入休眠状态,即其复制、基因表达及体内代谢活动大幅减弱,因此减少了其对大多数抗结核药物的敏感性,即进入持留状态。结核分枝杆菌抗酸的分子机制及其调控网络研究是结核病新防控措施的重要基础。
3 结核分枝杆菌的抗酸机制
为成功在巨噬细胞内存活下来,结核分枝杆菌会形成一个完整且复杂的酸性调控网络以应对胞内的酸性胁迫,其具体的调控机制主要包括以下几个方面(图1B)[28]。3.1 胞外酸性及其他有害物质的控制
结核分枝杆菌与抗酸相关的变化主要表现在：增加细胞壁的修复能力[29]、减少胞外酸性物质,以及酸性胁迫下毒性物质的降解能力增加[30,31,32]。3.1.1 细胞壁修复能力提高
宿主产生的攻击致病菌的抗菌肽(antimicrobial peptides, AMPs)的作用可以被结核分枝杆菌的赖氨酰tRNA合成酶(lysyl-tRNA synthetase, lysX)抵消[33]。lysX (128.208 kDa)是参与肽聚糖(peptidoglycan, PG)合成的膜蛋白,能够增加细胞表面的赖氨酸残基数量,降低抗菌肽对细菌表面的损伤[34]。有研究显示细菌存活率与lysX的表达水平成正相关[35]。
与肽聚糖相关的还有由结核分枝杆菌Rv3671c基因编码的跨膜丝氨酸蛋白酶(membrane-associated serine protease, MarP)。MarP (40.7124 kDa)位于结核分枝杆菌细胞周质[36]。MarP的蛋白水解活性与酸胁迫时结核分枝杆菌胞内pH维持有关。MarP可通过剪切活化Ras interacting protein (RipA)[36],RipA (49.8081 kDa)具有肽聚糖水解活性,使细胞壁中的肽聚糖可以正常水解,有利于细胞壁正常的修复过程,可能还参与维持细胞膜的稳定性[29]。
除此之外,Rv2136c编码的蛋白质对细胞壁的多种成分,如肽聚糖等的形成起关键作用。Rv2136c转座子突变菌株对十二烷基磺酸钠、亲脂性抗生素、高温、氧化压力和活性氮中间物敏感,在小鼠中减缓生长并且降低形成持留的能力[37]。低pH时,Rv2136c转座子突变菌株容易被环境中的Tween 80 和清蛋白刺激后释放的脂肪酸毒害[27]。使用磷酸钠缓冲液或其他表面活性剂,如泰洛沙泊,也称四丁酚醛,是烷基芳基聚醚醇型的非离子液体聚合物,可以有效增溶但不产生脂肪酸等有害物质,可以促进野生型结核分枝杆菌在pH为4.5环境下的存活事件,Rv2136c转座子突变菌株的存活率仍然降低[37]。但Rv2136c同源分子敲出的耻垢分枝杆菌与野生型对酸胁迫的表型相似[37]。Rv2136c的转座子插入突变可能会导致该基因编码的蛋白功能不完全或者没有蛋白产物,故Rv2136c转座子突变菌株的表型变化可能与插入导致的下游基因移码突变有关。
3.1.2 减少胞外酸性物质
基因drrA/drrB编码的是ABC超家族柔红霉素运输蛋白(Daunorubicin-transport ATP-binding protein ABC transporter) DrrA/DrrB。其中DrrA (35.8182 kDa)是ATP结合蛋白,DrrB (31.1089 kDa)是膜整合蛋白,两者结合在一起才具有外排泵活性。DrrA在膜上的定位取决于DrrB的表达情况,无DrrA时,DrrB对蛋白酶的水解非常敏感[38]。柔红霉素或者阿霉素促进结核分枝杆菌产生ATP,增加底物与转运蛋白结合,同时改变DrrA构象而更易结合ATP。DrrA/DrrB缺失或者活性降低导致结核分枝杆菌运输结核分枝杆菌醇二枝菌酸等复杂分子的能力下降,提示与毒力的密切关系[38]。酸性环境明显下调drrA/drrB基因的转录,该基因编码的产物属于ABC超家族,其对脂质的外排功能已经被大量报导过,在酸性条件下,脂质会水解成脂肪酸和甘油,对细胞产生毒害,推测结核分枝杆菌在酸性胁迫下,会通过一定的机制降低转运蛋白DrrA/DrrB的活性,降低脂质的外排量,从而减少胞外酸性物质,降低酸性环境对细胞的损伤。
3.1.3 降解有害物质
PE-PGRS (N-端功能域PE含有110个氨基酸,C-端PGRS功能域编码富含GC的多态性重复序列polymorphic GC-rich repeated sequence)和PPE (Pro- Pro-Glu)家族是分枝杆菌特异性蛋白家族,具有抗原性[39, 40]。PPEs和PEs可以活化T细胞[41,42,43]。PEs具有类似分子伴侣的功能。一些PGRS在C-端具有某些酶的活性结构域,如脂肪酶[44]。PPEs和PEs经常成对发挥作用。PE/PPE家族蛋白的分泌涉及VII型分泌系统[45, 46]。酸胁迫改变部分结核分枝杆菌的PE/PPE 家族蛋白的水平如PPE50和PE34[47, 48]。据此推测,该家族蛋白质可能与酸抗性有关,但具体分子机理仍有待深入研究。
3.2 通过调节代谢维持胞内酸碱平衡
自身的酸碱平衡对于结核分枝杆菌适应酸性环境至关重要。结核分枝杆菌Rv0899基因编码的外膜蛋白A (outer membrane protein A, OmpATb)可以在细胞膜上形成通道[49],但无孔蛋白活性[50, 51]。ompATb是相关酸碱调节基因的操纵子,这对结核分枝杆菌氨的快速分泌、酸碱快速中和及酸性条件下的生长至关重要。此外,ompATb的表达依赖于pH[51]：低pH上调ompATb的转录[52],同时逐步提高培养基pH值,这可能与体外培养时天冬酰胺是结核分枝杆菌的氮源分泌氨有关,但ompATb并不调控天冬酰胺的摄取,而在体内具体情况如何,仍有待研究[53]。3.3 通过双组份系统调控表达
巨噬细胞内的pH值约为5.2,这种酸性环境足以控制大多数病原微生物。但结核分枝杆菌却仍然能够在巨噬细胞这种酸性条件下存活。结核分枝杆菌可能具有感知吞噬体内pH并迅速调整其基因表达的能力。现在已知被酸和吞噬体调控的基因(Acid and Phagosome Regulated, apr) aprA (AprA: 7.90905 kDa)、aprB (AprB: 5.72264 kDa)和aprC (AprC: 30.7241 kDa)。该操纵子被双组份信号转导系统PhoPR调控：aprC的表达明显依赖于PhoPR的存在[54]。在aprABC的调控序列中可能有PhoP (27.5135 kDa)结合位点[55, 56]。推测依赖于aprABC的调控通路最终作用于一种目前未知的分子X,X的产生受phoP调控。缺失aprABC时,X的积累可能会影响其他基因的表达;当phoP缺失时,X不会形成,依赖aprABC的基因的表达也会被改变[57]。aprABC缺失的分枝杆菌的脂代谢[58]也会有缺陷。这提示脂质可能参与结核分枝杆菌酸抗性。PhoPR和Rv3416编码的胞内氧化还原感应分子WhiB3 (11.6122 kDa)在结核分枝杆菌酸抗性中发挥重要功能[59, 60]。低pH时,phoPR能直接激活whiB3的表达。whiB3调控包括cpsY在内的多个基因的表达,与结核分枝杆菌毒力和酸抗性有关[61]。whiB3及其上游基因Rv3415c的编码序列高度保守。两个基因间的非编码序列可分为两个区域：非保守区域1和相对保守区域2。结核分枝杆菌的启动子序列位于相对保守区域2,表明在分枝杆菌中转录起始保守。非编码区1的序列只在结核分枝杆菌和海分枝杆菌中保守,在耻垢分枝杆菌中并不保守,这和低pH下whiB3的表达水平差异正好一致[62]。这提示whiB3上游的非编码区1可能是调控转录起始的关键序列。结核分枝杆菌PhoP的DNA结合序列也位于whiB3上游的非编码区1。该DNA片段在结核分枝杆菌和海分枝杆菌中相同,但在耻垢分枝杆菌中则有差异。结核分枝杆菌PhoR (52.0164 kDa)的H259位点能发生磷酸化,磷酰基被转运到PhoP的D71位点。磷酸化与whiB3激活相关[61, 63]。该调控方式目前只在部分致病分枝杆菌中发现。
3.4 其他抗酸机制
结核分枝杆菌的谷氨酸脱羧酶、精氨酸脱羧酶、赖氨酸脱羧酶和鸟氨酸脱羧酶会通过消耗特定氨基酸的氢离子,最终生成胺类和CO2来提高胞内pH值[15]。谷氨酸脱羧酶主要是把谷氨酸脱羧生成γ-氨基丁酸(γ-aminobutyric acid, GABA)并转运到细胞外,从而消耗酸性环境下进入细胞内的质子,并将产物运到细胞外,阻止细胞内pH值过度降低[20, 64]。精氨酸脱羧酶会将精氨酸脱羧并消耗质子,最终生成胍丁胺和CO2,在精胺反向转运蛋白的作用下,精胺被运出细胞并同时交换新的精氨酸进入,再在精氨酸脱羧酶的作用下发生脱羧反应,如此循环将不断消耗细胞内的质子,从而维持酸性环境下胞内pH稳定。同样,赖氨酸脱羧酶和鸟氨酸脱羧酶也可以通过相应氨基酸脱羧维持胞内pH稳定。结核分枝杆菌的分子伴侣可在酸性条件下维持体内蛋白质稳定[65]。分子伴侣帮助新生蛋白质正确折叠,也可以促进错误折叠蛋白质的重新折叠,在酸抗性中也发挥重要作用。pH中性时,分子伴侣在结核分枝杆菌胞内主要以无活性的二聚体或多聚体形式存在,但当细菌处于极端酸性环境中,其变成有活性的形式,与其受体蛋白相结合,使受体蛋白免受酸的伤害或者帮助已经在酸性条件下沉淀的蛋白质恢复活性。一些非编码小RNA也参与酸抗性[66],小RNA可能主要调控一些关键的抗酸基因的表达。
关于结核分枝杆菌的酸抗性基因,研究还很少。上述基因很多是根据大肠杆菌的信息,通过比对获得。关于酸抗性,很多研究以大肠杆菌为模型进行。本实验室在大肠杆菌结果的基础上[67, 68],使用生物信息学方法,通过与结核分枝杆菌基因组进行比对,预测了结核分枝杆菌其他可能的酸抗性基因(表1),为后续在结核分枝杆菌中的验证提供线索。
Table 1
表1
表1 结核分枝杆菌潜在的酸抗性基因及其编码产物
Table 1
| 基因编号 | 编码产物功能 | 相关文献 |
|---|---|---|
| Rv0072 | 谷氨酰胺以及部分其他分子的跨膜运输 | [69] |
| Rv0247c | 参与富马酸盐和琥珀酸的相互转化(有氧呼吸),催化活性：琥珀酸+受体=延胡索酸 + 还原性受体 | [70] |
| Rv1032c | 双组份系统trcRS中的trcS,组氨酸激酶 | [71~74] |
| Rv1098c | 参与了三羧酸循环,延胡索酸脱氢酶,催化活性：(S)-苹果酸 = 延胡索酸 + 水 | [75, 76] |
| Rv1464 | 半胱氨酸脱硫酶(Cysteine desulfurase, Csd),催化硫和硒从L-半胱氨酸,L-胱氨酸, L-硒代半胱氨酸,和L-硒代胱氨酸的转移,产生L-丙氨酸 | [77~79] |
| Rv1558 | 功能未知 | [80] |
| Rv1811 | 可能的镁离子转运P型ATP酶C (Mg2+ transport P-type ATPase C,MgtC),催化活性： ATP + 水 + Mg2+(膜外) = ADP + 磷酸 + Mg2+(膜内) | [81~93] |
| Rv1854c | 可能的NADH脱氢酶(NADH dehydrogenase,Ndh),将电子从NADH转移到呼吸链 | [94~121] |
| Rv1979c | 可能的保守的透性酶,可能与氨基酸跨膜运输相关 | [122~127] |
| Rv2359 | 锌指调控蛋白(Zinc uptake regulation protein, Zur),负调控因子,以Zn2+作为辅因子 | [128~133] |
| Rv2710 | RNA聚合酶转录起始因子(RNA polymerase sigma factor B, SigB),可能控制生长稳定期 以及一般压力胁迫下的转运调控 | [134~143] |
| Rv2720 | 基因表达阻遏因子LexA,参与核苷酸切除修复和SOS反应 | [128, 144~150] |
| Rv2827c | 功能未知 | [151, 152] |
| Rv2995c | 可能的3-异丙基苹果酸脱氢酶(3-isopropylmalate dehydrogenase , LeuB)参与了亮氨酸生物合成 (第三步),催化活性：3-羧基-2-羟基-4-甲基戊酸乙酯+NAD+ = 3-羧基-4-甲基-2-戊酸乙酯+NADH | [153, 154] |
| 基因编号 | 编码产物功能 | 相关文献 |
| Rv0477 | 未知分泌蛋白 | |
| Rv2531c | 鸟氨酸/精氨酸/赖氨酸脱羧酶 | |
| Rv3253c | 带阳离子氨基酸以及一些底物分子的跨膜转运 | |
| Rv3432c | 谷氨酸盐脱羧酶(Glutamate decarboxylase, GadB),产生谷胱甘肽,催化活性： L-谷氨酸 = γ-氨基丁酸 + CO2 |
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4 问题与展望
结核分枝杆菌的抗酸系统是由多个分子组成的相互协调的网络。从对外界环境酸碱性的感应到细胞壁的变化,内部酸碱代谢平衡及相关基因表达的调控,最终形成对外界酸性环境高度耐受的状态,其中的每一个过程都涉及到复杂的调控。这个调控系统中部分基因已经明确,如细胞壁相关的跨膜丝氨酸蛋白酶MarP,酸碱代谢平衡调节相关的外膜蛋白A (OmpATb)。但是,对于结核分枝杆菌的耐酸基因的研究仍然颇少。借鉴研究比较深入的如大肠杆菌等肠道微生物耐受肠道酸性环境的基因[11]及其调控网络[10]有助于启发结核分枝杆菌的酸抗性机制及其持留机制。目前认为细胞壁相关基因与酸抗性密切相关,虽然已经识别了MarP等细胞壁成分维持相关基因,但环境pH值变化如何被分枝杆菌所感知并反馈到MarP等细胞壁相关基因的改变还不清楚。其他的研究也往往集中在鉴定酸抗性有关基因,将来需要深入研究鉴定酸抗性基因的作用分子机理,以及其相互作用网络。这将有助于更全面地揭示结核分枝杆菌持留机制。
致谢：
特别感谢李启明博士在图表处理方面的指导。参考文献 原文顺序
文献年度倒序
文中引用次数倒序
被引期刊影响因子
URL [本文引用: 1]
This publication provides the latest information and analysis about global tuberculosis (TB) data reported by 182 WHO member states (204 countries and territories) during 2012. Presented data include: (1) global burden of TB disease; (2) TB case notifications and treatment outcomes; (3) drug-resistant TB; (4) funding for TB care and control; (5) improvements in TB diagnostics; (6) TB/HIV co-inf...
URL [本文引用: 1]
Abstract The global problem of antimicrobial resistance is particularly pressing in developing countries, where the infectious disease burden is high and cost constraints prevent the widespread application of newer, more expensive agents. Gastrointestinal, respiratory, sexually transmitted, and nosocomial infections are leading causes of disease and death in the developing world, and management of all these conditions has been critically compromised by the appearance and rapid spread of resistance. In this first part of the review, we have summarised the present state of resistance in these infections from the available data. Even though surveillance of resistance in many developing countries is suboptimal, the general picture is one of accelerating rates of resistance spurred by antimicrobial misuse and shortfalls in infection control and public health. Reservoirs for resistance may be present in healthy human and animal populations. Considerable economic and health burdens emanate from bacterial resistance, and research is needed to accurately quantify the problem and propose and evaluate practicable solutions. In part II, to be published next month, we will review potential containment strategies that could address this burgeoning problem.
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URLPMID:29219058 [本文引用: 1]
Objective: The objective of the present study was to develop targeted engineered nanoerythrosomes based intravenous formulation of antimalarial drug pyrimethamine. Material and methods: The nanoerythrosomes formulation was developed by sonication method and optimized for effective drug loading at variable drug concentration, surface morphology, viscosity and sedimentation volume. Results: The... [Show full abstract]
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URLPMID:29075866 [本文引用: 2]
Lactic acid bacteria (LAB) can take advantage of fermentable carbohydrates to produce lactic acid. They are proverbially applied in industry, agricultural production, animal husbandry, food enterprise, pharmaceutical engineering and some other important fields, which are closely related to human life. For performing the probiotic functions, LAB have to face the low pH environment of the gastrointestinal tract. Therefore, acid resistance of LAB is of great importance not only for their own growth, but also for fermentation and preparation of probiotic products. Recent research studies on acid resistance mechanisms of LAB are mainly focused on neutralization process, biofilm and cell density, proton pump, protection of macromolecules, pre-adaptation and cross-protection, and effect of solutes. In this context, biotechnological strategies such as synthetic biology, genome shuffling, high pressure homogenization and adaptive laboratory evolution were also used to improve the acid resistance of LAB to respond to constantly changing low pH environment.
URLPMID:24898062 [本文引用: 2]
Abstract As part of their life cycle, neutralophilic bacteria are often exposed to varying environmental stresses, among which fluctuations in pH are the most frequent. In particular, acid environments can be encountered in many situations from fermented food to the gastric compartment of the animal host. Herein, we review the current knowledge of the molecular mechanisms adopted by a range of Gram-positive and Gram-negative bacteria, mostly those affecting human health, for coping with acid stress. Because organic and inorganic acids have deleterious effects on the activity of the biological macromolecules to the point of significantly reducing growth and even threatening their viability, it is not unexpected that neutralophilic bacteria have evolved a number of different protective mechanisms, which provide them with an advantage in otherwise life-threatening conditions. The overall logic of these is to protect the cell from the deleterious effects of a harmful level of protons. Among the most favoured mechanisms are the pumping out of protons, production of ammonia and proton-consuming decarboxylation reactions, as well as modifications of the lipid content in the membrane. Several examples are provided to describe mechanisms adopted to sense the external acidic pH. Particular attention is paid to Escherichia coli extreme acid resistance mechanisms, the activity of which ensure survival and may be directly linked to virulence.
URL [本文引用: 1]
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结核分枝杆菌（Mycobacterium tuberculosis,MTB）是一种典型的胞内致病菌,巨噬细胞是MTB在体内的主要宿主细胞。巨噬细胞具有强大的吞噬功能,在机体固有免疫和适应性免疫中均发挥着重要作用,可有效保护宿主免受结核分枝杆菌的感染。MTB在与宿主巨噬细胞的长期相互作用过程中,逐渐形成多种逃避杀灭的有效策略,得以在宿主体内存活并增殖。该文从巨噬细胞抗MTB感染及MTB逃避巨噬细胞杀灭两个方面综述国内外的研究进展。
URL [本文引用: 1]
结核分枝杆菌（Mycobacterium tuberculosis,MTB）是一种典型的胞内致病菌,巨噬细胞是MTB在体内的主要宿主细胞。巨噬细胞具有强大的吞噬功能,在机体固有免疫和适应性免疫中均发挥着重要作用,可有效保护宿主免受结核分枝杆菌的感染。MTB在与宿主巨噬细胞的长期相互作用过程中,逐渐形成多种逃避杀灭的有效策略,得以在宿主体内存活并增殖。该文从巨噬细胞抗MTB感染及MTB逃避巨噬细胞杀灭两个方面综述国内外的研究进展。
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URLPMID:20346409 [本文引用: 1]
Metabolites, substrates and substrate impurities may be toxic to cells by damaging biological molecules, organelles, membranes or disrupting biological processes. Chemical stress is routinely encountered in bioprocessing to produce chemicals or fuels from renewable substrates, in whole-cell biocatalysis and bioremediation. Cells respond, adapt and may develop tolerance to chemicals by mechanisms only partially explored, especially for multiple simultaneous stresses. More is known about how cells respond to chemicals, but less about how to develop tolerant strains. Aiming to stimulate new metabolic engineering and synthetic-biology approaches for tolerant-strain development, this review takes a holistic, comparative and modular approach in bringing together the large literature on genes, programs, mechanisms, processes and molecules involved in chemical stress or imparting tolerance. These include stress proteins and transcription factors, efflux pumps, altered membrane composition, stress-adapted energy metabolism, chemical detoxification, and accumulation of small-molecule chaperons and compatible solutes. The modular organization (by chemicals, mechanism, organism, and methods used) imparts flexibility in exploring this complex literature, while comparative analyses point to hidden commonalities, such as an oxidative stress response underlying some solvent and carboxylic-acid stress. Successes involving one or a few genes, as well as global genomic approaches are reviewed with an eye to future developments that would engage novel genomic and systems-biology tools to create altered or semi-synthetic strains with superior tolerance characteristics for bioprocessing.
URLPMID:22995042 [本文引用: 2]
Abstract For successful colonization of the mammalian host, orally acquired bacteria must overcome the extreme acidic stress (pH65<652.5) encountered during transit through the host stomach. The glutamate-dependent acid resistance (GDAR) system is by far the most potent acid resistance system in commensal and pathogenic Escherichia coli, Shigella flexneri, Listeria monocytogenes and Lactococcus lactis. GDAR requires the activity of glutamate decarboxylase (GadB), an intracellular PLP-dependent enzyme which performs a proton-consuming decarboxylation reaction, and of the cognate antiporter (GadC), which performs the glutamatein /γ-aminobutyrateout (GABA) electrogenic antiport. Herein we review recent findings on the structural determinants responsible for pH-dependent intracellular activation of E.65coli GadB and GadC. A survey of genomes of bacteria (pathogenic and non-pathogenic), having in common the ability to colonize or to transit through the host gut, shows that the gadB and gadC genes frequently lie next or near each other. This gene arrangement is likely to be important to ensure timely co-regulation of the decarboxylase and the antiporter. Besides the involvement in acid resistance, GABA production and release were found to occur at very high levels in lactic acid bacteria originally isolated from traditionally fermented foods, supporting the evidence that GABA-enriched foods possess health-promoting properties. 08 2012 Blackwell Publishing Ltd.
URL [本文引用: 1]
依赖于谷氨酸脱羧酶的酸抗性系统对痢疾杆菌在宿主细胞内的生存和繁殖至关重要,hdeA、hdeB、yhiE和yhiF是其中几个重要的酸抗性基因。借助Red系统的重组功能,PCR扩增两翼与目的基因上下游同源的抗性基因片段,电击转化痢疾杆菌2457T,对筛选到的阳性转化子再导入编码FLP位点特异性重组酶的质粒pCP20以去除抗性基因。结果成功的敲除了hdeA、hdeB、yhiE和yhiF等4个酸抗性系统相关基因,为深入研究痢疾杆菌酸抗性基因的调控网络奠定了基础。
URL [本文引用: 1]
依赖于谷氨酸脱羧酶的酸抗性系统对痢疾杆菌在宿主细胞内的生存和繁殖至关重要,hdeA、hdeB、yhiE和yhiF是其中几个重要的酸抗性基因。借助Red系统的重组功能,PCR扩增两翼与目的基因上下游同源的抗性基因片段,电击转化痢疾杆菌2457T,对筛选到的阳性转化子再导入编码FLP位点特异性重组酶的质粒pCP20以去除抗性基因。结果成功的敲除了hdeA、hdeB、yhiE和yhiF等4个酸抗性系统相关基因,为深入研究痢疾杆菌酸抗性基因的调控网络奠定了基础。
Magsci [本文引用: 1]
胸膜肺炎放线杆菌(APP)是重要的猪呼吸道病原菌, 给世界养猪业造成严重的经济损失。信号标签突变(STM)技术是在宿主动物体内鉴定病原菌毒力因子的高通量方法。通过体外传代选育出APP血清1型和3型萘啶酸抗性菌株, 再以萘啶酸抗性菌株为受体菌, 以携带mini-Tn10的标签质粒(pLOF/TAG1-48)的E. coli CC118 λ pir或S17-1 λ pir为供体菌, 在或不在E. coli DH5α (pRK2073)的辅助下, 进行三亲本或两亲本接合, 通过抗性筛选、PCR和Southern杂交鉴定转座突变株。结果表明: 体外萘啶酸加压传代很容易选育出萘啶酸抗性APP菌株, 该抗性的产生与DNA促旋酶A亚基基因gyrA的突变有关。在APP与E. coli接合实验中, 两亲本接合比三亲本接合操作更简单, 效率也较高; APP不同菌株在接合和转座效率上存在很大差异, 血清1型菌株高于血清3型菌株, 3型标准菌株高于地方分离株JL03-R。本研究为APP STM突变体库的构建与毒力基因的鉴定奠定了基础。
Magsci [本文引用: 1]
胸膜肺炎放线杆菌(APP)是重要的猪呼吸道病原菌, 给世界养猪业造成严重的经济损失。信号标签突变(STM)技术是在宿主动物体内鉴定病原菌毒力因子的高通量方法。通过体外传代选育出APP血清1型和3型萘啶酸抗性菌株, 再以萘啶酸抗性菌株为受体菌, 以携带mini-Tn10的标签质粒(pLOF/TAG1-48)的E. coli CC118 λ pir或S17-1 λ pir为供体菌, 在或不在E. coli DH5α (pRK2073)的辅助下, 进行三亲本或两亲本接合, 通过抗性筛选、PCR和Southern杂交鉴定转座突变株。结果表明: 体外萘啶酸加压传代很容易选育出萘啶酸抗性APP菌株, 该抗性的产生与DNA促旋酶A亚基基因gyrA的突变有关。在APP与E. coli接合实验中, 两亲本接合比三亲本接合操作更简单, 效率也较高; APP不同菌株在接合和转座效率上存在很大差异, 血清1型菌株高于血清3型菌株, 3型标准菌株高于地方分离株JL03-R。本研究为APP STM突变体库的构建与毒力基因的鉴定奠定了基础。
URLMagsci [本文引用: 1]
<p>结核分枝杆菌能在宿主体内长期存活，很大一部分原因是能抵抗吞噬体的酸性环境。细菌一方面能抑制吞噬体与溶酶体融合，干扰吞噬体成熟、酸化过程；另一方面也能通过自身功能抵抗吞噬溶酶体内的酸性杀伤作用。本文主要介绍吞噬体的酸化过程及结核分枝杆菌耐酸机制的最新研究进展。</p>
URLMagsci [本文引用: 1]
<p>结核分枝杆菌能在宿主体内长期存活，很大一部分原因是能抵抗吞噬体的酸性环境。细菌一方面能抑制吞噬体与溶酶体融合，干扰吞噬体成熟、酸化过程；另一方面也能通过自身功能抵抗吞噬溶酶体内的酸性杀伤作用。本文主要介绍吞噬体的酸化过程及结核分枝杆菌耐酸机制的最新研究进展。</p>
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URLPMID:5698697 [本文引用: 1]
Targeting ofMycobacterium tuberculosis(MTB) latent antigens comprises a crucial strategy for the development of alternative tuberculosis (TB) vaccine(s) that protects against TB reactivation. Here, we generated a multistage DNA vaccine, A39, containing the early antigens Ag85A and Rv3425 as well as the latency-associated protein Rv2029c, which conferred protective immunity in a pre-exposure mouse model. Moreover, administration of the A39 vaccination after MTB exposure inhibited reactivation and resulted in significantly lower bacterial loads in the lungs and spleen of mice, compared to those in the control population. Subsequently, we investigated the effect of Rv2029c on innate immunity and characterized the molecular details of the interaction of this protein with the host via iTRAQ proteomic and biochemical assay analyses. Rv2029c activated macrophages, triggered the production of pro-inflammatory cytokines, and promoted toll-like receptor/mitogen-activated protein kinase (TLR/MAPK)-dependent macrophage apoptosis. Furthermore, Rv2029c treatment enhanced the ability ofMycobacterium bovisBacillus Calmette-Gu rin (BCG)-infected macrophages to present antigens to CD4+T cellsin vitro, which correlated with an increase in MHC-II expression. Lastly, Rv2029c-treated macrophages activated T cells, effectively polarized CD4+and CD8+T cells to secrete IFN- and IL-2, and specifically expanded a population of CD44highCD62LlowCD4+/CD8+effector/memory cells, indicating that Rv2029c, as a specific recall antigen, contributes to Th1 polarization in T cell immunity. These results suggest that Rv2029c and A39 comprise promising targets for the development of next-generation clinical TB therapeutic vaccines.
URLPMID:11826478 [本文引用: 1]
The obligate intracellularism of Mycobacterium leprae may be attributable to the effects of mutations in major metabolic areas due to a genome capable of encoding only about 1600 proteins. Yet cell wall biosynthesis capability remains relatively intact and comparisons with the genome of Mycobacterium tuberculosis provide insights into the genetic basis of a minimal mycobacterial cell wall.
URLPMID:29461881 [本文引用: 2]
Abstract Tuberculosis (TB) is a major bacterial infectious disease worldwide that is predominantly caused by Mycobacterium tuberculosis (Mtb). The comorbidity of multiple drug-resistant TB strains with HIV and diabetes is widespread. In the presence of these diseases, host immunity is weakened, allowing the recovery of dormant bacilli and leading to recurrent TB infection. As an important component of the host innate and adaptive immune responses, macrophage autophagy plays a significant role in protecting the host against TB. However, dormant bacilli can escape from autophagosomes and/or suppress autophagy, thus surviving within the host for an extended period of time, although the underlying mechanism remains elusive. Heat shock protein 16.3 (Hsp16.3, HspX, Rv2031c, and Acr) is one of the immunodominant proteins expressed during latent TB infection (LTBI). It may help maintain the protein stability and long-term viability of Mtb by inhibiting macrophage autophagy, resulting in LBTI. In this review, we discuss how dormant bacilli escape from autophagosomes, and we focus on the role of Hsp16.3 in regulating macrophage autophagy in LTBI so as to provide a firm basis for further studies. Hsp16.3 may represent a potential biomarker of LTBI and novel pharmacological target for anti-tubercular drugs.
URLPMID:17652161 [本文引用: 1]
Mycobacterium tuberculosis evades the innate antimicrobial defenses of macrophages by inhibiting the maturation of its phagosome to a bactericidal phagolysosome. Despite intense studies of the mycobacterial phagosome, the mechanism of mycobacterial persistence dependent on prolonged phagosomal retention of the coat protein coronin-1 is still unclear. The present study demonstrated that several mycobacterial proteins traffic intracellularly in M. bovis BCG-infected cells and that one of them, with an apparent subunit size of M(r) 50,000, actively retains coronin-1 on the phagosomal membrane. This protein was initially termed coronin-interacting protein (CIP)50 and was shown to be also expressed by M. tuberculosis but not by the non-pathogenic species M. smegmatis. Cell-free system experiments using a GST-coronin-1 construct showed that binding of CIP50 to coronin-1 required cholesterol. Thereafter, mass spectrometry sequencing identified mycobacterial lipoamide dehydrogenase C (LpdC) as a coronin-1 binding protein. M. smegmatis over-expressing Mtb LpdC protein acquired the capacity to maintain coronin-1 on the phagosomal membrane and this prolonged its survival within the macrophage. Importantly, IFNgamma-induced phagolysosome fusion in cells infected with BCG resulted in the dissociation of the LpdC-coronin-1 complex by a mechanism dependent, at least in part, on IFNgamma-induced LRG-47 expression. These findings provide further support for the relevance of the LpdC-coronin-1 interaction in phagosome maturation arrest.
URLPMID:3642297 [本文引用: 2]
The transmembrane serine protease MarP is important for pH homeostasis in Mycobacterium tuberculosis (Mtb). Previous structural studies revealed that MarP contains a chymotrypsin fold and a disulfide bond that stabilizes the protease active site in the substrate-bound conformation. Here, we determined that MarP is located in the Mtb periplasm and showed that this localization is essential for function. Using the recombinant protease domain of MarP, we identified its substrate specificity using two independent assays: positional-scanning synthetic combinatorial library profiling and multiplex substrate profiling by mass spectrometry. These methods revealed that MarP prefers bulky residues at P4, tryptophan or leucine at P2, arginine or hydrophobic residues at P1, and alanine or asparagine at P1'. Guided by these data, we designed fluorogenic peptide substrates and characterized the kinetic properties of MarP. Finally, we tested the impact of mutating MarP cysteine residues on the peptidolytic activity of recombinant MarP and its ability to complement phenotypes of Mtb Delta MarP. Taken together, our studies provide insight into the enzymatic properties of MarP, its substrate preference, and the importance of its transmembrane helices and disulfide bond.
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URLPMID:17374885 [本文引用: 1]
The PE and PPE proteins of Mycobacterium tuberculosis form a source of antigenic variation among different strains of M. tuberculosis. One of the PE-PGRS proteins, Rv1818c, plays a role in the pathogenesis of mycobacterial infection and specifically influences host-cell responses to tuberculosis infection. Although little is known about these two classes of protein, an immunoinformatics approach has indicated the possibility of their participation in eliciting a major histocompatibility complex (MHC class 1-mediated immune response against tuberculosis, as peptides derived from Rv1818c are predicted to bind to MHC class I molecules with high affinity. In the present work, a DNA vaccine was constructed encoding the full-length Rv1818c protein of M. tuberculosis and its immunogenicity was analysed in BALB/c mice. Immunization with Rv1818c DNA induced a strong CD8(+) cytotoxic lymphocyte and Th1-type response, with high levels of gamma interferon and low levels of interleukin-4. Two nonameric peptides (Peptides (6-14) and Peptide(385-393)) from Rv1818c were identified by their ability to induce the production of IFN-gamma by CD8(+) T cells in mice immunized with Rv1818c DNA. An epitope-specific response was demonstrated by the lysis of peptide-pulsed antigen-presenting cells, release of cytotoxic granules and IFN-gamma production. These peptides bound with high affinity to MHC H-2K(d) and showed low dissociation rates of peptide-MHC complexes. These results could form the basis for testing the identified T-cell epitopes of PE_PGRS proteins in the induction of protective immunity against M. tuberculosis challenge in the mouse model.
URL [本文引用: 1]
Records of milk production and pedigree information of Mahdasht dairy herd from 1994 to 1998 were used to survey the factors that influence economic traits and evaluated environmental factors and genetic parameters. Respectively. The total milk yield, peak production, days in milk, days open and calving interval were 6782±1006(kg), 32±6(kg), 290±31 (day), 121±52 (day) and 390±46 (day), respectively. The total milk yield, days in milk and calving interval were affected by days open (P<0.05). The best days open was 80 to 110 days. The effects of year and month of calving were significant on days open (P<0.05). The Heritability of days open was 0.06±0.03. The additive genetic correlation of days open with total milk yield, peak production, day in milk and calving interval were 0.19, 0.1, 0.13 and 0.87 and phenotypic correlation were 0.08, 0.3, 0.1 and 0.66, respectively.
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URLPMID:16803587 [本文引用: 2]
Mycobacteria are characterized by an unusual cell wall that controls nutrient and small hydrophilic compound permeability. Porin-like proteins are necessary to ensure the transport of molecules into the cell. Here, we investigated the pore-forming properties of OmpATb, a porin from Mycobacterium tuberculosis, in lipid bilayers. Multi-channel experiments showed an asymmetric behaviour with channel closures at negative critical voltages (Vc) and a strong decrease in Vc at acidic pH. Single-channel experiments gave conductance values of about 850 +/- 80 pS in 1 M KCl and displayed a weak cationic selectivity in 4-8 pH range. The production and characterization of a series of truncated OmpATb proteins, showed that the central domain (OmpATb(73-220)) was sufficient to induce the ion channel properties of the native protein in lipid bilayers, i.e. asymmetric insertion, pH-dependent voltage closure, cationic selectivity and similar conductance values in 1 M KCl. Western blot analysis suggests that the presence of OmpATb is only restricted to certain pathogenic species. Therefore, the propensity of channels of native OmpATb to close at low pH may represent an intrinsic property allowing pathogenic mycobacteria to adapt and survive to mildly acidic conditions, such as those encountered within the macrophage phagosome.
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URLPMID:25536998 [本文引用: 1]
The regulatory mechanisms that control the ESX-1 secretion system, a key player in the pathogenesis of Mycobacterium tuberculosis, have not been fully elucidated. However, factors that regulate the ESX-1 substrate EspA usually affect ESX-1 function. Previous studies showed that espA is directly regulated by the nucleoid-associated protein EspR and the two-component system (TCS) MprAB. The PhoPR TCS also activates espA, but the direct target of PhoP was unknown. In this report, we reveal that EspR is directly regulated by MprA and PhoP-Rv, but not by PhoP-Ra. PhoP-Rv and MprA binding sites in the espR promoter were determined by gel-shift and DNase I footprinting assays, which identified a PhoP-protected region centred approximately 205 bp before the espR start codon and that encompasses MprA Region-1, one of two MprA-protected regions. MprA Region-2 is located approximately 60 bp downstream of MprA Region-1 and overlaps a known EspR binding site. Nucleotides essential for the binding of PhoP and/or MprA were identified through site-directed DNA mutagenesis. Our studies also indicate that MprA Region-2, but not MprA Region-1/PhoP region, is required for the full expression of espR. Recombinant strains carrying mutations at MprA Region-2 exhibited lower transcription levels for espR, espA and espD, and had reduced EspR and EspA levels in cell lysates. These findings indicate that EspR may mediate the regulatory effect of PhoPR and MprAB, and provide more insight into the mechanisms underlying ESX-1 control.
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URLPMID:3535892 [本文引用: 1]
The mechanism by which the antitubercular drug isoxyl (ISO) inhibits mycolic acid biosynthesis has not yet been reported. We found that point mutations in either the HadA or HadC component of the type II fatty acid synthase (FAS-II) are associated with increased levels of resistance to ISO in Mycobacterium tuberculosis. Overexpression of the HadAB, HadBC, or HadABC heterocomplex also produced high-level resistance. These results show that the FAS- II dehydratases are involved in ISO resistance.
URLPMID:26637353 [本文引用: 1]
Abstract The ability of Mycobacterium tuberculosis to resist intraphagosomal stresses, such as oxygen radicals and low pH, is critical for its persistence. Here, we show that a cytoplasmic redox sensor, WhiB3, and the major M. tuberculosis thiol, mycothiol (MSH), are required to resist acidic stress during infection. WhiB3 regulates the expression of genes involved in lipid anabolism, secretion, and redox metabolism, in response to acidic pH. Furthermore, inactivation of the MSH pathway subverted the expression of whiB3 along with other pH-specific genes in M. tuberculosis. Using a genetic biosensor of mycothiol redox potential (EMSH), we demonstrated that a modest decrease in phagosomal pH is sufficient to generate redox heterogeneity in EMSH of the M. tuberculosis population in a WhiB3-dependent manner. Data indicate that M. tuberculosis needs low pH as a signal to alter cytoplasmic EMSH, which activates WhiB3-mediated gene expression and acid resistance. Importantly, WhiB3 regulates intraphagosomal pH by down-regulating the expression of innate immune genes and blocking phagosomal maturation. We show that this block in phagosomal maturation is in part due to WhiB3-dependent production of polyketide lipids. Consistent with these observations, Mtb0200whiB3 displayed intramacrophage survival defect, which can be rescued bypharmacological inhibition of phagosomal acidification. Last, Mtb0200whiB3 displayed marked attenuation in the lungs of guinea pigs. Altogether, our study revealed an intimate link between vacuolar acidification, redox physiology, and virulence in M. tuberculosis and discovered WhiB3 as crucial mediator of phagosomal maturation arrest and acid resistance in M. tuberculosis. 0008 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
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URLPMID:23530220 [本文引用: 1]
Abstract To successfully colonize the human gut, enteric bacteria must activate acid resistance systems to survive the extreme acidity (pH 1.5-3.5) of the stomach. The antiporter AdiC is the master orchestrator of the arginine-dependent system. Upon acid shock, it imports extracellular arginine (Arg) into the cytoplasm, providing the substrate for arginine decarboxylases, which consume a cellular proton ending up in a C-H bond of the decarboxylated product agmatine (Agm(2+)). Agm(2+) and the "virtual" proton it carries are exported via AdiC subsequently. It is widely accepted that AdiC counters intracellular acidification by continuously pumping out virtual protons. However, in the gastric environment, Arg is present in two carboxyl-protonation forms, Arg(+) and Arg(2+). Virtual proton pumping can only be achieved by Arg(+)/Agm(2+) exchange, whereas Arg(2+)/Agm(2+) exchange would produce no net proton movement. This study experimentally asks which exchange AdiC catalyzes, an issue previously unapproachable due to the absence of a reconstituted system mimicking the situation of bacteria in the stomach. Here, using an oriented liposome system able to hold a three-unit pH gradient, we demonstrate that Arg/Agm exchange by AdiC is strongly electrogenic with positive charge moved outward, and thus that AdiC mainly mediates Arg(+)/Agm(2+) exchange to support effective virtual proton pumping. Further experiments reveal a mechanistic surprise--that AdiC selects Arg(+) against Arg(2+) on the basis of gross valence, rather than by local scrutiny of protonation states of the carboxyl group, as had been suggested by Arg-bound AdiC crystal structures.
URLPMID:22459131 [本文引用: 1]
Abstract The extremely acidic environment of the mammalian stomach not only serves to facilitate food digestion but also acts as a natural barrier against infections of food-borne pathogens. Many pathogenic bacteria, such as enterohemorrhagic Escherichia coli, can breach this host defense and cause severe diseases. These pathogens have evolved multiple intricate strategies to overcome the bactericidal activity of acids. In particular, recent studies have uncovered the central roles of two periplasmic chaperones, HdeA and HdeB, in protecting enteric bacteria from extremely acidic conditions. Here, we review recent advances in the understanding of the acid resistance mechanisms of Gram-negative bacteria and focus on the mechanisms of HdeA and HdeB in preventing acid-induced protein aggregation and facilitating protein refolding following pH neutralization. Copyright 2012 Elsevier Ltd. All rights reserved.
URLPMID:24795365 [本文引用: 1]
Glucose confers acid resistance on exponentially growing bacteria by repressing formation of the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex and consequently activating acid resistance genes. Therefore, in a glucose-rich growth environment, bacteria are capable of resisting acidic stresses due to low levels of cAMP-CRP. Here we reveal a second mechanism for glucose-conferred acid resistance. We show that glucose induces acid resistance in exponentially growing bacteria through pyruvate, the glycolysis product. Pyruvate and/or the downstream metabolites induce expression of the small noncoding RNA (sncRNA) Spot42, and the sncRNA, in turn, activates expression of the master regulator of acid resistance, RpoS. In contrast to glucose, pyruvate has little effect on levels of the cAMP-CRP complex and does not require the complex for its effects on acid resistance. Another important difference between glucose and pyruvate is that pyruvate can be produced by bacteria. This means that bacteria have the potential to protect themselves from acidic stresses by controlling glucose-derived generation of pyruvate, pyruvate-acetate efflux, or reversion from acetate to pyruvate. We tested this possibility by shutting down pyruvate-acetate efflux and found that the resulting accumulation of pyruvate elevated acid resistance. Many sugars can be broken into glucose, and the subsequent glycolysis generates pyruvate. Therefore, pyruvate-associated acid resistance is not confined to glucose-grown bacteria but is functional in bacteria grown on various sugars.
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URLPMID:3191967
Abstract Onset of the adaptive immune response in mice infected with Mycobacterium tuberculosis is accompanied by slowing of bacterial replication and establishment of a chronic infection. Stabilization of bacterial numbers during the chronic phase of infection is dependent on the activity of the gamma interferon (IFN-0206)-inducible nitric oxide synthase (NOS2). Previously, we described a differential signature-tagged mutagenesis screen designed to identify M. tuberculosis "counterimmune" mechanisms and reported the isolation of three mutants in the H37Rv strain background containing transposon insertions in the rv0072, rv0405, and rv2958c genes. These mutants were impaired for replication and virulence in NOS2(-/-) mice but were growth-proficient and virulent in IFN-0206(-/-) mice, suggesting that the disrupted genes were required for bacterial resistance to an IFN-0206-dependent immune mechanism other than NOS2. Here, we report that the attenuation of these strains is attributable to an underlying transposon-independent deficiency in biosynthesis of phthiocerol dimycocerosate (PDIM), a cell wall lipid that is required for full virulence in mice. We performed whole-genome resequencing of a PDIM-deficient clone and identified a spontaneous point mutation in the putative polyketide synthase PpsD that results in a G44C amino acid substitution. We demonstrate by complementation with the wild-type ppsD gene and reversion of the ppsD gene to the wild-type sequence that the ppsD(G44C) point mutation is responsible for PDIM deficiency, virulence attenuation in NOS2(-/-) and wild-type C57BL/6 mice, and a growth advantage in vitro in liquid culture. We conclude that PDIM biosynthesis is required for M. tuberculosis resistance to an IFN-0206-mediated immune response that is independent of NOS2.
URLPMID:4477654
Bacterial pathogens adapt to changing environments within their hosts, and the signaling molecule adenosine 3', 5'-cyclic monophosphate (cAMP) facilitates this process. In this study, we characterized in vivo DNA binding and gene regulation by the cAMP-responsive protein CRP in M. bovis BCG as a model for tuberculosis (TB)-complex bacteria. Chromatin immunoprecipitation followed by deep-sequencing (ChIP-seq) showed that CRP associates with 900 DNA binding regions, most of which occur within genes. The most highly enriched binding region was upstream of a putative copper transporter gene (ctpB), and crp-deleted bacteria showed increased sensitivity to copper toxicity. Detailed mutational analysis of four CRP binding sites upstream of the virulence-associated Rv0249c-Rv0247c succinate dehydrogenase genes demonstrated that CRP directly regulates Rv0249c-Rv0247c expression from two promoters, one of which requires sequences intragenic to Rv0250c for maximum expression. The high percentage of intragenic CRP binding sites and our demonstration that these intragenic DNA sequences significantly contribute to biologically relevant gene expression greatly expand the genome space that must be considered for gene regulatory analyses in mycobacteria. These findings also have practical implications for an important bacterial pathogen in which identification of mutations that affect expression of drug target-related genes is widely used for rapid drug resistance screening.
URLPMID:26082118
Abstract Two-component regulatory systems (2CRSs) are widely used by bacteria to sense and respond to environmental stimuli with coordinated changes in gene expression. Systems are normally comprised of a sensory kinase protein that activates a transcriptional regulator by phosphorylation. Mycobacteria have few 2CRSs, but they are of key importance for bacterial survival and play important roles in pathogenicity. Mycobacterium tuberculosis has 12 paired two-component regulatory systems (which include a system with two regulators and one sensor, and a split sensor system), as well as four orphan regulators. Several systems are involved in virulence, and disruption of different systems leads to attenuation or hypervirulence. PhoPR plays a major role in regulating cell wall composition, and its inactivation results in sufficient attenuation of M. tuberculosis that deletion strains are live vaccine candidates. MprAB controls the stress response and is required for persistent infections. SenX3-RegX3 is required for control of aerobic respiration and phosphate uptake, and PrrAB is required for adaptation to intracellular infection. MtrAB is an essential system that controls DNA replication and cell division. The remaining systems (KdpDE, NarL, TrcRS, TcrXY, TcrA, PdtaRS, and four orphan regulators) are less well understood. The structure and binding motifs for several regulators have been characterized, revealing variations in function and operation. The sensors are less well characterized, and stimuli for many remain to be confirmed. This chapter reviews our current understanding of the role of two-component systems in mycobacteria, in particular M. tuberculosis.
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The -propeller gene Rv1057 of is activated by envelope stress and was first characterized as a regulatory target of the TrcRS two-component system (TCS). Rv1057 expression is repressed by TrcRS, and the Rv1057 proximal promoter contains a TrcR binding site. In this study, we determined that Rv1057 is also directly regulated by MprAB, a TCS associated with envelope stress. Multiple potential MprA binding sites (MprA boxes) were identified in the 1 kb intergenic region upstream of Rv1057, and four sites were shown to bind MprA. Although MprA boxes were found in the proximal promoter, analyses suggest that MprA and TrcR do not compete for binding in this region. An MprAB-dependent, detergent-inducible transcriptional start point for Rv1057 was identified downstream of the MprA boxes, and a second TrcR binding site and small ORF of the 13E12 family were discovered in the distal promoter. MprAB was required for activation of Rv1057 during growth in macrophages and under detergent stress, and promoter constructs suggest the entire intergenic region is utilized during MprAB-dependent activation of Rv1057. These findings indicate that Rv1057 has an extensive and complex promoter, and provide evidence for coordinated regulation of stress response genes by TCSs.
URLPMID:28803470
Abstract Native mass spectrometry is an ideal technique to investigate the effects of DMSO on protein structure and interac-tions. Given the frequent use of DMSO in biochemical and biophysical assays, it is desirable to understand the influence of DMSO concentration on the dissociation or unfolding behavior of proteins. In this study, the effects of DMSO on the structure and interac-tions of avidin and Mycobacterium tuberculosis (Mtb) CYP142A1 were assessed through collision-induced dissociation (CID) and collision-induced unfolding (CIU) as monitored by nanoelectrospray ionization-ion mobility-mass spectrometry (nESI-IM-MS). DMSO concentrations higher than 4% (v/v) destabilize the avidin tetramer towards dissociation and unfolding, via both its effects on charge state distribution (CSD) as well as at the level of individual charge states. In contrast, DMSO both protects against heme loss and increases the stability of CYP142A1 towards unfolding even up to 40% DMSO. Tandem MS/MS experiments showed that DMSO could modify the dissociation pathway of CYP142A1, while CIU revealed the protective effect of the heme group on the structure of CYP142A1.
URLPMID:26786650
Despite the known association of several genes to clinical Isoniazid (INH) resistance, its molecular basis remains unknown in 6516% of clinical isolates ofMycobacterium tuberculosis(M.02tb). While screening a set ofMycobacterium smegmatis(M.02smegmatis) transposon mutants with altered colony morphology for differential susceptibility to INH, we found six resistant mutants and mapped their transposon insertion sites. The disrupted genes in six INH resistant mutants were homologs ofM.tb ctaE, rplY, tatA, csdandtatBwith one insertion mapping to the promoter region ofM.02smegmatis ctaE. MIC measurements indicated a wide spectrum of INH resistance in these mutants, with complementation analyses of four selected mutants with the cognateM.02smegmatisgenes and theirM.02tbhomologs confirming the association of the disrupted genes with INH resistance. Our discovery of novel genes associated with INH resistance could lead to the identification of novel INH resistance mechanisms and possibly new diagnostic modalities as well.
URLPMID:24478415
Abstract Lymph node enlargement is a common medical problem, and in a large number of patients, the causes of lymphadenopathy remain undiagnosed. We report a thorough microbiological analysis of 1,688 lymph node biopsy specimens collected in our bartonellosis reference center. We studied lymph node biopsy samples from patients with suspected regional infectious lymph node enlargement from January 2008 to December 2012. To evaluate a useful strategy for the diagnosis of infectious lymphadenitis, specimens were cultured and subjected to molecular assays. Histologic analysis was done when possible. A total of 642 (38%) biopsy specimens were infected with a bacterial agent, and quantitative PCR (qPCR) was significantly better than 16S rRNA gene PCR (rrs) for the detection of Bartonella henselae (P = 0.05), Mycobacterium tuberculosis (P = 0.05), and Mycobacterium avium (P = 0.007). Molecular assays were significantly better than bacterial cultures for the diagnosis of Francisella tularensis (P = 0.017) but were less effective for detecting M. tuberculosis (P = 0.004) and M. avium (P = 0.001). Histologic analysis was done for 412 lymph nodes, and 20% of these were compatible with an infectious lymphadenitis, whereas a neoplasm was found in 29% of these lymph nodes. M. tuberculosis was detected significantly more in female than in male patients (P = 0.01), and patients with cat scratch disease (CSD) were younger than patients with M. tuberculosis, Tropheryma whipplei, and F. tularensis. Negative rrs PCR does not exclude the diagnosis of infectious lymphadenitis. Histologic analysis of lymph node biopsy specimens is critical, as a diagnosis of infectious lymphadenitis does not preclude other concurrent diseases.
URLPMID:4469704
Abstract Pathogenic bacteria have developed strategies to adapt to host environment and resist host immune response. Several intracellular bacterial pathogens, including Salmonella enterica and Mycobacterium tuberculosis, share the horizontally-acquired MgtC virulence factor that is important for multiplication inside macrophages. MgtC is also found in pathogenic Pseudomonas species. Here we investigate for the first time the role of MgtC in the virulence of an extracellular pathogen, Pseudomonas aeruginosa. A P. aeruginosa mgtC mutant is attenuated in the systemic infection model of zebrafish embryos, and strikingly, the attenuated phenotype is dependent on the presence of macrophages. In ex vivo experiments, the P. aeruginosa mgtC mutant is more sensitive to macrophage killing than the wild-type strain. However, wild-type and mutant strains behave similarly toward macrophage killing when macrophages are treated with an inhibitor of the vacuolar proton ATPase. Importantly, P. aeruginosa mgtC gene expression is strongly induced within macrophages and phagosome acidification contributes to an optimal expression of the gene. Thus, our results support the implication of a macrophage intracellular stage during P. aeruginosa acute infection and suggest that Pseudomonas MgtC requires phagosome acidification to play its intracellular role. Moreover, we demonstrate that P. aeruginosa MgtC is required for optimal growth in Mg2+ deprived medium, a property shared by MgtC factors from intracellular pathogens and, under Mg2+ limitation, P. aeruginosa MgtC prevents biofilm formation. We propose that MgtC shares a similar function in intracellular and extracellular pathogens, which contributes to macrophage resistance and fine-tune adaptation to host immune response in relation to the different bacterial lifestyles. In addition, the phenotypes observed with the mgtC mutant in infection models can be mimicked in wild-type P. aeruginosa strain by producing a MgtC antagonistic peptide, thus highlighting MgtC as a promising new target for anti-virulence strategies.
URLPMID:25545682
Abstract MgtC is a virulence factor involved in intramacrophage growth that has been reported in several intracellular pathogens, including Mycobacterium tuberculosis and Salmonella enterica serovar Typhimurium. MgtC participates also in adaptation to Mg2+ deprivation. Herein, we have constructed a mgtC mutant in Mycobacterium marinum to further investigate the role of MgtC in mycobacteria. We show that the M. marinum mgtC gene (Mma mgtC) is strongly induced upon Mg2+ deprivation and is required for optimal growth in Mg2+-deprived medium. The behaviour of the Mma mgtC mutant has been investigated in the Danio rerio infection model using a transgenic reporter zebrafish line that specifically labels neutrophils. Although the mgtC mutant is not attenuated in the zebrafish embryo model based on survival curves, our results indicate that phagocytosis by neutrophils is enhanced with the mgtC mutant compared to the wild-type strain following subcutaneous injection. Increased phagocytosis of the mutant strain is also observed ex vivo with the murine J774 macrophage cell line. On the other hand, no difference was found between the mgtC mutant and the wild-type strain in bacterial adhesion to macrophages and in the internalization into epithelial cells. Unlike the role reported for MgtC in other intracellular pathogens, Mma MgtC does not contribute significantly to intramacrophage replication. Taken together, these results indicate an unanticipated function of Mma MgtC at early step of infection within phagocytic cells. Hence, our results indicate that although the MgtC function is conserved among pathogens regarding adaptation to Mg2+ deprivation, its role towards phagocytic cells can differ, possibly in relation with the specific pathogen's lifestyles.
URLPMID:39225975
Background Different polymorphisms have been described as markers to classify the lineages of the Mycobacterium tuberculosis complex. The analysis of nine single nucleotide polymorphisms (SNPs) was used to describe seven SNPs cluster groups (SCGs). We attempted to classify those strains that could not been categorized into lineages by the genotyping methods used in the routine testing. Results The M. tuberculosis complex isolates collected in 2010 in our region were analysed. A new method based on multiplex-PCRs and pyrosequencing to analyse these SNPs was designed. For the pyrosequencing assay nine SNPs that defined the seven SCGs were selected from the literature: 1977, 74092, 105139, 232574, 311613, 913274, 2460626, 3352929 and gyrA95. In addition, SNPs in katG463, mgtC182, Ag85C103 and RDRio deletion were detected. Conclusions This work has permitted to achieve a better classification of Aragonian strains into SCGs and in some cases, to assign strains to its certain lineage. Besides, the description of a new pattern shared by two isolates ???SCG-6c??? reinforces the interest of SNPs to follow the evolution of M. tuberculosis complex.
URLPMID:3736803
A Salmonella virulence protein targets and inhibits Salmonella’s own F1Fo ATP synthase to help maintain physiological ATP levels, enabling its survival within acidified host phagosomes.
URLPMID:22984256
MgtC is a virulence factor of unknown function important for survival inside macrophages in several intracellular bacterial pathogens, including Mycobacterium tuberculosis. It is also involved in adaptation to Mg(2+) deprivation, but previous work suggested that MgtC is not a Mg(2+) transporter. In this study, we demonstrated that the amount of the M. tuberculosis MgtC protein is not significantly increased by Mg(2+) deprivation. Members of the MgtC protein family share a conserved membrane N-terminal domain and a more divergent cytoplasmic C-terminal domain. To get insights into MgtC functional and structural organization, we have determined the nuclear magnetic resonance (NMR) structure of the C-terminal domain of M. tuberculosis MgtC. This structure is not affected by the Mg(2+) concentration, indicating that it does not bind Mg(2+). The structure of the C-terminal domain forms a βαββαβ fold found in small molecule binding domains called ACT domains. However, the M. tuberculosis MgtC ACT domain differs from canonical ACT domains because it appears to lack the ability to dimerize and to bind small molecules. We have shown, using a bacterial two-hybrid system, that the M. tuberculosis MgtC protein can dimerize and that the C-terminal domain somehow facilitates this dimerization. Taken together, these results indicate that M. tuberculosis MgtC does not have an intrinsic function related to Mg(2+) uptake or binding but could act as a regulatory factor based on protein-protein interaction that could be facilitated by its ACT domain.
URLPMID:20624486
Abstract We developed a new multiplexed-PCR assay to accurately classify Mycobacterium tuberculosis complex (MTC) isolates at the sublineage level by single nucleotide polymorphisms (SNPs). This method relies on 7 SNPs located in different genes of the MTC strains (recC, rec0, recR, ligB, ligC, alkA, and mgtC). Most of these genes are involved in replication, repair and recombination (3R) functions of M. tuberculosis strains, four of the mutations are synonymous, and thus neutral. Genes were chosen as a first empirical approach to assess the congruence between spoligotyping-based phylogeographical classification and SNP typing. This scheme efficiently classifies most of MTC phylogeographical groups: (1) confirming and identifying new sublineage-specific SNPs, (2) unraveling phylogenetical relationships between spoligotyping-defined MTC sublineages, (3) appropriately assigning sublineages to some spoligotypes and reassigning sublineages to other mis-labeled spoligotype signatures. This study opens the way to a more meaningful taxonomic, evolutionary and epidemiological classification. It also allows evaluation of spoligotype-signature significance towards a more comprehensive understanding of the evolutionary mechanisms of the clustered regularly interspaced short palindromic repeat (CRISPR) locus in MTC. Copyright 2010 Elsevier B.V. All rights reserved.
URLPMID:14708580
MgtC is a virulence factor required for intramacrophage survival and growth in low Mg2+ medium in two pathogens that are not phylogenetically related, Salmonella typhimurium and Mycobacterium tuberculosis. In S. typhimurium, mgtC is carried by the SPI-3 pathogenicity island and hybridization studies have suggested that the distribution of mgtC among enterobacteria is limited. In the present study, we searched for the presence of mgtC-like sequences in eubacterial genomes. Analyses of MgtC-like proteins phylogeny and mgtC-like chromosomal context support the hypothesis that mgtC has been acquired by horizontal gene transfer repeatedly throughout bacterial evolution. In addition, the phylogenetic analysis revealed the existence of a subgroup of proteins, that includes the S. typhimurium and M. tuberculosis MgtC proteins, as well as MgtC-related proteins from other pathogens that are able to survive in macrophages, B. melitensis and Y. pestis. We propose that MgtC has a similar function in all these distantly related pathogens, most likely providing the ability to grow in a low Mg2+ environment.
URLPMID:28754952
Abstract Bacteria can proliferate perpetually without ageing, but they also face conditions where they must persist. Mycobacteria can survive for a long period. This state appears during mycobacterial diseases such as tuberculosis and leprosy, which are chronic and develop after long-term persistent infections. However, the fundamental mechanisms of the long-term living of mycobacteria are unknown. Every Mycobacterium species expresses Mycobacterial DNA-binding protein 1 (MDP1), a histone-like nucleoid associated protein. Mycobacterium smegmatis is a saprophytic fast grower and used as a model of mycobacterial persistence, since it shares the characteristics of the long-term survival observed in pathogenic mycobacteria. Here we show that MDP1-deficient M. smegmatis dies more rapidly than the parental strain after entering stationary phase. Proteomic analyses revealed 21 upregulated proteins with more than 3-fold in MDP1-deficient strain, including DnaA, a replication initiator, NDH, a NADH dehydrogenase that catalyzes downhill electron transfer, Fas1, a critical fatty acid synthase, and antioxidants such as AhpC and KatG. Biochemical analyses showed elevated levels of DNA and ATP syntheses, a decreased NADH/NAD + ratio, and a loss of resistance to oxidative stress in the MDP1-knockout strain. This study suggests the importance of MDP1-dependent simultaneous control of the cellular functions in the long-term survival of mycobacteria.
URLPMID:28472892
Abstract Introduction: Tuberculosis (TB) is highly dangerous due to the development of resistance to first-line drugs. Moreover, Mycobacterium tuberculosis (Mtb) has also developed resistance to newly approved antitubercular drug bedaquiline. This necessitates the search for drugs acting on newer molecular targets. The energy metabolism of mycobacteria is the prime focus for the discovery of novel antitubercular drugs. Targeting type-2 NADH dehydrogenase (NDH-2) involved in the production of respiratory ATP could, therefore, be effective in treating the disease. Areas Covered: This review describes the energetics of mycobacteria and the role of NDH-2 in ATP synthesis. Special attention has been given for genetic and chemical validation of NDH-2 as a molecular target. The reported kinetics and crystal structures of NDH-2 have been given in detail for better understanding of the enzyme. Expert Opinion: NDH-2 is an essential enzyme for ATP synthesis and has a potential role in dormancy and persistence of Mtb. The human counterpart lacks this enzyme and hence NDH-2 inhibitors could have more clinical importance. Phenothiazines are potent inhibitor of NDH-2 and are effective against both drug-susceptible and drug-resistant Mtb. Thus, it is highly desirable to optimize this phenothiazine class of compounds for the development of next generation anti-TB drugs.
URLPMID:28304162
A High-throughput screen (HTS) was undertaken against the respiratory chain dehydrogenase component, NADH:menaquinone oxidoreductase (Ndh) of Mycobacterium tuberculosis (Mtb). 11,000 compounds were selected for the HTS based on the known phenothiazine Ndh inhibitors, trifluoperazine and thioridazine. Combined HTS (11,000 compounds) and in-house screening of a limited number of quinolones (50 compounds) identified ~100 hits and four distinct chemotypes, the most promising of which contained the quinolone core. Subsequent Mtb screening of the complete in-house quinolone library (350 compounds) identified a further ~90 hits across three quinolone sub-templates. Quinolones containing the amine based side chain were selected as the pharmacophore for further modification, resulting in metabolically stable quinolones effective against multi drug resistant (MDR) Mtb. The lead compound, MTC420 displays acceptable anti-tuberculosis activity (Mtb IC50 =525 nM, Mtb Wayne IC50 = 76 nM and MDR Mtb patient isolates IC50...
URLPMID:27450017
We spoligotyped and screened 1490 clinicalMycobacterium tuberculosiscomplex strains from Northern and Greater Accra regions of Ghana against INH and RIF using the microplate alamar blue phenotypic assay. Specific drug resistance associated genetic elements of drug resistant strains were analyzed for mutations. A total of 111 (7.5%), 10 (0.7%) and 40 (2.6%) were mono-resistant to INH, RIF, and MDR, respectively. We found the Ghana spoligotype to be associated with drug resistance (INH: 22.1%; p02=020.0000, RIF: 6.2%; p02=020.0103, MDR: 4.6%; p02=020.0240) as compared to the Cameroon spoligotype (INH: 6.7%, RIF: 2.4%, MDR: 1.6%). The propensity for an isolate to harbourkatGS315T mutation was higher inM. tuberculosis(75.8%) thanMycobacterium africanum(51.7%) (p02=020.0000) whereas the opposite was true forinhApromutations; MAF (48.3%) compared to MTBSS (26.7%) (p02=020.0419). We identified possible novel compensatory INH resistance mutations ininhA(G204D) andahpCpro(-88G/A and -142G/A) and a novelndhmutation K32R. We detected two possiblerpoCmutations (G332R and V483G), which occurred independently withrpoBS450L, respectively. The study provides the first evidence that associate the Ghana spoligotype with DR-TB and calls for further genome analyses for proper classification of this spoligotype and to explore for fitness implications and mechanisms underlying this observation.
URLPMID:27365187
Abstract Objectives: Quinolinequinones (QQ) have been shown to inhibit the growth of mycobacterial species, but their mode(s) of action and molecular target(s) remain unknown. To facilitate further development of QQ as antimycobacterial drugs, we investigated the molecular mechanism and target of QQ in mycobacteria. Methods: Cell viability of Mycobacterium tuberculosis and Mycobacterium bovis bacillus Calmette u rin was determined in the presence of QQ8c, a representative QQ compound, and isoniazid, a frontline antitubercular drug. The effect of QQ8c on mycobacterial energetics was studied using inverted membrane vesicles. NADH oxidation and formation of reactive oxygen species (ROS) were measured in the presence and absence of KCN. Generation of ROS was measured via oxygen consumption in an oxygen electrode. The effects of QQ8c were compared with the antimycobacterial drug clofazimine in side-by-side experiments. Results: QQ8c challenge resulted in complete sterilization of cultures with no refractory resistant population observed. QQ8c stimulated NADH oxidation by type II NADH dehydrogenase (NDH-2) and oxygen consumption in inverted membrane vesicles. Large quantities of ROS were produced in the presence of QQ8. Even when oxygen consumption was blocked with KCN, activation of NDH-2 by QQ8c occurred suggesting QQ8c was redox cycling. Conclusions: QQ8c targets NDH-2 of the mycobacterial respiratory chain leading to activation of NADH oxidation and generating bactericidal levels of ROS in a manner similar to, but more effectively than, the antimycobacterial drug clofazimine. Our results validate respiratory-generated ROS as an avenue for antimycobacterial drug development.
URLPMID:26369965
Ethionamide (ETH) is an antibiotic used for the treatment of multidrug-resistant (MDR) tuberculosis (TB) (MDR-TB), and its use may be limited with the emergence of resistance in the Mycobacterium tuberculosis population. ETH resistance in M. tuberculosis is phenomenon independent or cross related when accompanied with isoniazid (INH) resistance. In most cases, resistance to INH and ETH is explained by mutations in the inhA promoter and in the following genes: katG, ethA, ethR, mshA, ndh, and inhA. We sequenced the above genes in 64 M. tuberculosis isolates (n = 57 ETH-resistant MDR-TB isolates; n = 3 ETH-susceptible MDR-TB isolates; and n = 4 fully susceptible isolates). Each isolate was tested for susceptibility to first- and second-line drugs using the agar proportion method. Mutations were observed in ETH-resistant MDR-TB isolates at the following rates: 100% in katG, 72% in ethA, 45.6% in mshA, 8.7% in ndh, and 33.3% in inhA or its promoter. Of the three ETH-susceptible MDR-TB isolates, all showed mutations in katG; one had a mutation in ethA, and another, in mshA and inhA. Finally, of the four fully susceptible isolates, two showed no detectable mutation in the studied genes, and two had mutations in mshA gene unrelated to the resistance. Mutations not previously reported were found in the ethA, mshA, katG, and ndh genes. The concordance between the phenotypic susceptibility testing to INH and ETH and the sequencing was 1 and 0.45, respectively. Among isolates exhibiting INH resistance, the high frequency of independent resistance and cross-resistance with ETH in the M. tuberculosis isolates suggests the need to confirm the susceptibility to ETH before considering it in the treatment of patients with MDR-TB.
URLPMID:19486070
Nucleotide sequences of genes conferring isoniazid resistance ( katG, inhA, oxyR–ahpC and ndh ) and ethionamide resistance ( ethA ) in 160 drug-resistant Mycobacterium tuberculosis clinical isolates from Thailand were analysed. Mutations in the katG gene were found in 129 isolates, predominantly at codon 315, which was mutated in 127 isolates. Twenty-two isolates had mutations in the inhA promoter and coding region. Mutations in the oxyR–ahpC intergenic region and in ndh were detected in four and one isolate(s), respectively. Of 24 ethionamide-resistant isolates, 13 had mutations in the ethA gene. However, these mutations were dispersed along the entire gene, with no codon predominating significantly.
URLPMID:24900541
NDH-2 is an essential respiratory enzyme in Mycobacterium tuberculosis (Mtb), which plays an important role in the physiology of Mtb. Herein, we present a target-based effort to identify a new structural class of inhibitors for NDH-2. High-throughput screening of the AstraZeneca corporate collection resulted in the identification of quinolinyl pyrimidines as the most promising class of NDH-2 inhibitors. Structure ctivity relationship studies showed improved enzyme inhibition (IC50) against the NDH-2 target, which in turn translated into cellular activity against Mtb. Thus, the compounds in this class show a good correlation between enzyme inhibition and cellular potency. Furthermore, early ADME profiling of the best compounds showed promising results and highlighted the quinolinyl pyrimidine class as a potential lead for further development.
URLPMID:23228936
Objectives: Phenothiazines have been shown to exhibit in vitro and in vivo activity against Mycobacterium tuberculosis (Mtb) and multidrug-resistant Mtb. They are predicted to target the genetically validated respiratory chain component type II NADH:quinone oxidoreductase (Ndh). Using a set of compounds containing the phenothiazine pharmacophore, we have (i) investigated whether chemical validation data support the molecular target and (ii) evaluated pharmacophore tractability for further drug development.Methods: Recombinant Mtb Ndh was generated and its functionality confirmed by steady-state kinetics. Pharmacodynamic profiling of the phenothiazines, including antitubercular efficacy in aerobic and O-2-limited conditions, time-kill assays and isobole analyses against first-line antituberculars, was performed. Potential mitochondrial toxicity was assessed in a modified HepG2 cell-line assay and against bovine cytochrome bc(1).Results: Steady-state kinetic analyses revealed a substrate preference for coenzyme Q(2) and an inability to utilize NADPH. A positive correlation between recombinant Ndh inhibition and kill of aerobically cultured Mtb was observed, whilst enhanced potency was demonstrated in a hypoxic model. Time-kill studies revealed the phenothiazines to be bactericidal whilst isobolograms exposed antagonism with isoniazid, indicative of intracellular NADH/NAD(+) couple perturbation. At therapeutic levels, phenothiazine-mediated toxicity was appreciable; however, specific mitochondrial targeting was excluded.Conclusions: Data generated support the hypothesis that Ndh is the molecular target of phenothiazines. The favourable pharmacodynamic properties of the phenothiazines are consistent with a target product profile that includes activity against dormant/persistent bacilli, rapid bactericidal activity and activity against drug-resistant Mtb by a previously unexploited mode of action. These properties warrant further medicinal chemistry to improve potency and safety.
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URLPMID:25150092
Tuberculosis (TB) is a difficult to treat disease caused by the bacterium Mycobacterium tuberculosis. The need for improved therapies is required to kill different M. tuberculosis populations present during infection and to kill drug resistant strains. Protein complexes associated with energy generation, required for the survival of all M. tuberculosis populations, have shown promise as targets for novel therapies (e.g., phenothiazines that target type II NADH dehydrogenase (NDH-2) in the electron transport chain). However, the low efficacy of these compounds and their off-target effects has made the development of phenothiazines as a therapeutic agent for TB limited. This study reports that a series of alkyltriphenylphosphonium (alkylTPP) cations, a known intracellular delivery functionality, improves the localization and effective concentration of phenothiazines at the mycobacterial membrane. AlkylTPP cations were shown to accumulate at biological membranes in a range of bacteria and lipophilicity was revealed as an important feature of the structure unction relationship. Incorporation of the alkylTPP cationic function significantly increased the concentration and potency of a series of phenothiazine derivatives at the mycobacterial membrane (the site of NDH-2), where the lead compound 3a showed inhibition of M. tuberculosis growth at 0.5 g/mL. Compound 3a was shown to act in a similar manner to that previously published for other active phenothiazines by targeting energetic processes (i.e., NADH oxidation and oxygen consumption), occurring in the mycobacterial membrane. This shows the enormous potential of alkylTPP cations to improve the delivery and therefore efficacy of bioactive agents targeting oxidative phosphorylation in the mycobacterial membrane.
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URLPMID:24709059
61Two new type 2 NADH dehydrogenases from S. aureus are purified and characterized.61A new protocol is developed to more closely mimic the protein's native environment.61Phenothiazines inhibit both enzymes, though with different potencies.61Phenothiazines are shown to be also uncouplers of oxidative phosphorylation.
URLPMID:26786219
Coupling an appropriate reporter to the promoter of gene of can make the reporter expression respiration sensitive and thereby reliably detect both active and dormant populations of the reporter strain.
URLPMID:28320727
Abstract Clofazimine has been repurposed for the treatment of tuberculosis, especially for multidrug-resistant tuberculosis (MDR-TB). To test the susceptibility to clofazimine of Mycobacterium tuberculosis clinical isolates, MICs of clofazimine were determined using the microplate alamarBlue assay (MABA) method for 80 drug-resistant isolates and 10 drug-susceptible isolates for comparison. For five clofazimine-resistant strains isolated from previously treated pre-extensively drug-resistant TB (pre-XDR-TB) and XDR-TB patients without prior exposure to clofazimine or bedaquiline, clofazimine MICs were 0909061.2 0204g/ml. Four isolates with cross-resistance to bedaquiline had Rv0678 mutations. The other isolate with no resistance to bedaquiline had an Rv1979c mutation. This study adds to a recent study showing that 6.3% of MDR-TB patients without prior clofazimine or bedaquiline exposure harbored isolates with Rv0678 mutations, which raises concern that preexisting resistance to these drugs may be associated with prior TB treatment. Furthermore, we propose a tentative breakpoint of 1.2 0204g/ml for clofazimine resistance using the MABA method. More-widespread surveillance and individualized testing for clofazimine and bedaquiline resistance, together with assessment of their clinical usage, especially among previously treated and MDR-TB patients, are warranted. Copyright 0008 2017 American Society for Microbiology.
URLPMID:21184855
The ongoing evolution of BCG after its introduction in 1921 resulted in strains that differ genetically and phenotypically. Based on a genomic deletion (Region of Difference 2 or RD2) that occurred between 1927 and 1931, BCG strains can be sub-classified by the presence or absence of RD2. The existence of other mutations that distinguish BCG strains precludes simple comparison of RD2-positive and RD2-negative BCG strains to determine the importance, if any, of RD2 for vaccine protection. In this study, we have compared the RD2-containing BCG Russia, BCG Pasteur (which is a natural mutant for RD2), and BCG Pasteur complemented with RD2-genes Rv1979c-Rv1982 through various in vitro and in vivo assays of immunogenicity and protection. We determined that the presence of RD2 did not affect vaccine persistence, but lead to increased immunogenicity, as measured by ELISpot. Additionally, T-cells from animals immunized with BCG Russia and BCG Pasteur::Rv1979c-82 were more effective at killing Mycobacterium tuberculosis in macrophages than T-cells from animals immunized with BCG Pasteur. In a mouse vaccine-challenge model, the presence of RD2 had no effect on pulmonary TB, as measured by M. tuberculosis burden and degree of histopathology, at 4, 8 or 12 weeks post-infection. The presence of RD2 was however associated with decreased dissemination of M. tuberculosis to the spleen. Together, our data demonstrated that the loss of RD2 resulted in decreased immunogenicity but did not affect protection against pulmonary TB, indicating a dissociation between these phenotypes associated with BCG vaccination.
URLPMID:12438379
An independent review for the British government has concluded that the development of a cattle vaccine against Mycobacterium bovis holds the best long-term prospects for tuberculosis control in British herds. The development of complementary diagnostic tests to differentiate between vaccinated and infected animals is necessary to allow the continuation of test-and-slaughter-based control policies alongside vaccination. Vaccination with M. bovis bacillus Calmette-Gu rin (BCG), the only available vaccine, results in tuberculin purified protein derivative sensitivity and has shown varying vaccine efficacies in cattle. Thus, identification of more-specific reagents to distinguish between vaccination and infection, as well as the identification of subunit vaccine candidates for improved tuberculosis vaccines, is a research priority. In the present study, we applied comparative genomics to identify M. bovis-Mycobacterium tuberculosis antigens whose genes had been deleted in BCG Pasteur. In total, 13 open reading frames (ORFs) from the RD1, RD2, and RD14 regions of the M. tuberculosis genome were selected. Pools of overlapping peptides spanning these ORFs were tested in M. bovis-infected (n = 22), BCG-vaccinated (n = 6), and unvaccinated (n = 10) control cattle. All were recognized in infected cattle, with responder frequencies varying between 16 and 86%. In particular, eight highly immunogenic antigens were identified whose potentials as diagnostic reagents or as subunit vaccines warrant further study (Rv1983, Rv1986, Rv3872, Rv3873, Rv3878, Rv3879c, Rv1979c, and Rv1769).
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This paper presents an overview of the role of image processing in Intelligent Transportation Systems (ITS). The authors suggest areas for improvement, including resolution and dynamic range in imaging devices. They also recommend using depth maps from laser radar or microwave radar as input to image processing. Typical on-board applications include detection of lane markers, obstacles, and proximity vehicles. Traffic signal and sign recognition, road surface monitoring, and driver monitoring are also included. Roadside applications include traffic surveillance, incident/accident detection, license number recognition, and parking lot surveillance.
URLPMID:28993339
Abstract Emergence and spread of drug resistant Mycobacterium tuberculosis strains threatens our ability the possibility to treat this disease in the future. Even if two new antitubercular drugs were recently introduced, there is still the need to design new molecules whose mechanism of action could reduce treatment length. We showed that two alternative sigma factors of M. tuberculosis (SigE and SigB) have a major role in determining the level of basal resistance to several drugs and the amount of persisters surviving long drug treatment. We also demonstrate that ethambutol, a bacteriostatic drug is highly bactericidal for M. tuberculosis mutants missing either SigE or SigB. We suggest that developing molecules able to interfere with their activity could not only reduce M. tuberculosis virulence in vivo , but also boost the effect of other drugs by increasing its sensitivity and reducing the number of persisters able to escape killing.
URLPMID:4596819
We have used RNASeq and qRT-PCR to study mRNA levels for all σ-factors in differentMycobacterium marinumstrains under various growth and stress conditions. We also studied their levels inM.marinumfrom infected fish and mosquito larvae. The annotated σ-factors were expressed and transcripts varied in relation to growth and stress conditions. Some were highly abundant such assigA,sigB,sigC,sigD,sigEandsigHwhile others were not. The σ-factor mRNA profiles were similar after heat stress, during infection of fish and mosquito larvae. The similarity also applies to some of the known heat shock genes such as the α-crystallin gene. Therefore, it seems probable that the physiological state ofM.marinumis similar when exposed to these different conditions. Moreover, the mosquito larvae data suggest that this is the state that the fish encounter when infected, at least with respect to σ-factor mRNA levels. Comparative genomic analysis of σ-factor gene localizations in threeM.marinumstrains andMycobacterium tuberculosisH37Rv revealed chromosomal rearrangements that changed the localization of especiallysigA,sigB,sigD,sigE,sigFandsigJafter the divergence of these two species. This may explain the variation in species-specific expression upon exposure to different growth conditions.
URLPMID:25833257
The resuscitation-promoting factors of Mycobacterium tuberculosis are hydrolytic enzymes, which are required for resuscitation of dormant cells. RpfB, a peptidoglycan remodelling enzyme similar to the lytic transglycosylase of Escherichia coli, is required for reactivation of M. tuberculosis from chronic infection in vivo, underscoring the need to understand its transcriptional regulation. Here, we identified the transcriptional and translational start points of rpfB, and suggested from rpf promoter-driven GFP expression and in vitro transcription assays that its transcription possibly occurs in a SigB-dependent manner. We further demonstrated that rpfB transcription is regulated by MtrA the response regulator of the essential two-component system MtrAB. Association of MtrA with the rpfB promoter region in vivo was confirmed by chromatin immunoprecipitation analysis. Electrophoretic mobility shift assays (EMSAs) revealed a loose direct repeat sequence associated with MtrA binding. Binding of MtrA was enhanced upon phosphorylation. MtrA could be pulled down from lysates of M. tuberculosis using a biotinylated DNA fragment encompassing the MtrA-binding site on the rpfB promoter, confirming that MtrA binds to the rpfB promoter. Enhanced GFP fluorescence driven by the rpfB promoter, upon deletion of the MtrA-binding site, and repression of rpfB expression, upon overexpression of MtrA, suggested that MtrA functions as a repressor of rpfB transcription. This was corroborated by EMSAs showing diminished association of RNA polymerase (RNAP) with the rpfB promoter in the presence of MtrA. In vitro transcription assays confirmed that MtrA inhibits RNAP-driven rpfB transcription.
URLPMID:4176334
RbpA, a transcriptional activator that is essential for Mycobacterium tuberculosis replication and survival during antibiotic treatment, binds to RNA polymerase (RNAP) in the absence of promoter DNA. It has been hypothesized that RbpA stimulates housekeeping gene expression by promoting assembly of the σ(A) subunit with core RNAP. Here, using a purified in vitro transcription system of M. tuberculosis, we show that RbpA functions in a promoter-dependent manner as a companion of RNAP essential for promoter DNA unwinding and formation of the catalytically active open promoter complex (RPo). Screening for RbpA activity using a full panel of the M. tuberculosis σ subunits demonstrated that RbpA targets σ(A) and stress-response σ(B), but not the alternative σ subunits from the groups 3 and 4. In contrast to σ(A), the σ(B) subunit activity displayed stringent dependency upon RbpA. These results suggest that RbpA-dependent control of RPo formation provides a mechanism for tuning gene expression during the switch between different physiological states, and in the stress response.
URLPMID:21517741
Use of the old antipsychotic phenothiazine thioridazine (THZ) for therapy of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB) infection is now being seriously considered. It is reported that THZ primarily acts on enzymes involved in fatty acid metabolism and membrane proteins, particularly efflux pumps, as well as oxidoreductases and proteins involved in aerobic respiration that overlap with a number of conventional antituberculous drugs. It targets the products of the Rv3160c-Rv3161c operon, which are perhaps required for the detoxification of THZ, members of the sigma factor SigB regulon that play a crucial role in protecting the pathogen against cell envelope damage, and Rv2745c, a transcription factor that regulates ATP-dependent proteolysis. Some of these genes have been shown to be essential for the survival or persistence of Mycobacterium tuberculosis in the infected host. Since THZ targets multiple pathways, including those involved in cell wall processes and respiratory chain components, it may serve as a model for multi-target drug development, as well as constitute a highly potent addition to a combination of antituberculous drug regimens. The discussion of some of the patents relevant to thioridazine to combat tuberculosis is also included in the present manuscript. <br/> <br/> <br/>
URLPMID:24405327
Abstract Mycobacterium tuberculosis (Mtb) is an intracellular human parasite that causes tuberculosis (TB). The parasite is capable of surviving under stress conditions. The gene expression in Mtb is regulated by sigma factor family of proteins. The SigF protein belongs to the sigma factor family, expressed during stationary and growth phase, 14 genes are directly regulated by SigF and has a role in the expression of the principal sigma factor SigB as well. The interacting partner Usfx, the anti SigF protein, controls the regulation of SigF. The structures of SigF and Usfx were evaluated using comparative modelling techniques and validated. The active sites of the two proteins were identified. The protein-protein interaction studies between SigF and Usfx reveal His53, Phe226 and Asp227 residues of SigF protein to be involved in binding with Arg108, Arg130 and Glu140 amino acids of Usfx. The present study focuses on identification of important residues involved in binding of SigF protein with Usfx, which are essential in the inhibition of transcription initiation and survival of Mtb.
URLPMID:28024526
Mycobacterium tuberculosis, the causative agent of tuberculosis-one of the most devastating infectious diseases, is a successful intracellular pathogen capable of surviving diverse stresses. Unveiling the molecular mechanisms governing this superior adaptation will inspire better control measures against tuberculosis. To define the role of Rv2788, a manganese-dependent transcriptional repressor, M.smegmatis was used as the host strain for heterologous expression Rv2788. Rv2788 can significantly change the colony morphology and fatty acids and permeability of cell wall, enhance the growth of the recombinants and resistance to diverse stresses, such as hydrogen peroxide (H60O60), diamide exposure, surface stress, acidic condition, multiple antibiotics treatment including chloramphenicol, vancomycin and amikacin. The dysregulation of the target genes of Rv2788, such as whiB1 and lexA , might underpin such phenotypes. The results implicate important roles of Rv2788 in the survival of Mycobacterium under stresses, and might represent ideal novel antibiotics target candidate.
URLPMID:3381160
The DNA damage response is crucial for bacterial survival. The transcriptional repressor LexA is a key component of the SOS response, the main mechanism for the regulation of DNA repair genes in many bacteria. In contrast, in mycobacteria gene induction by DNA damage is carried out by two mechanisms; a relatively small number of genes are thought to be regulated by LexA, and a larger number by an alternate, independent mechanism. In this study we have used ChIP-seq analysis to identify 25 in vivo LexA-binding sites, including nine regulating genes not previously known to be part of this regulon. Some of these binding sites were found to be internal to the predicted open reading frame of the gene they are thought to regulate; experimental analysis has confirmed that these LexA-binding sites regulate the expression of the expected genes, and transcriptional start site analysis has found that their apparent relative location is due to misannotation of these genes. We have also identified novel binding sites for LexA in the promoters of genes that show no apparent DNA damage induction, show positive regulation by LexA, and those encoding small RNAs.
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URLPMID:16880548
Abstract The hypothetical protein Rv2827c from Mycobacterium tuberculosis was cloned and heterologously expressed in Escherichia coli. It was purified using affinity and size-exclusion chromatographic techniques and then crystallized. Preliminary X-ray diffraction data analysis suggests the presence of two translationally related molecules in the asymmetric unit of the orthorhombic crystals.
