Arylamine N-Acetyltransferase Is Required for Synthesis of Mycolic Acids and Complex Lipids in Mycobacterium bovis BCG and Represents a Novel Drug Target
牛分枝杆菌 BCG 中分枝杆菌酸和复合脂质的合成需要芳胺 N-乙酰转移酶，并且是一种新的药物靶点

Mycobacterium tuberculosis, the etiological agent of tuberculosis, is responsible for two to three million deaths per year, worldwide (1, 2). Chemotherapy is available for tuberculosis, but requires an extremely long, complex multiple therapy regimen to resolve infection (3). The length of this course leads to high rates of patient noncompliance, suspected to account for the increasing number of drug-resistant clinical isolates of M. tuberculosis now observed (4). Additional antituberculosis drugs are now urgently required both to treat organisms already resistant to existing therapeutics, and to limit the emergence of drug resistance by use with current treatment regimens to shorten the course of therapy.
结核分枝杆菌是结核病的病原体，每年导致全世界 2 至 300 万人死亡 (1, 2)。化疗可用于治疗结核病，但需要极长、复杂的多重治疗方案来解决感染 (3)。这个疗程的长度导致患者不依从的比例很高，这可能是目前观察到的结核分枝杆菌耐药临床分离株数量不断增加的原因(4)。现在迫切需要额外的抗结核药物来治疗已经对现有疗法产生耐药性的生物体，并通过与当前的治疗方案一起使用以缩短疗程来限制耐药性的出现。

M. tuberculosis belongs to the genus Mycobacterium, characterized by a unique cell wall rich in unusual glycolipids, polysaccharides, and lipids, including mycolic acids. Biosynthetic pathways of cell wall components have proved to be effective targets for several major antitubercular drugs, including the frontline drug isoniazid (INH), which inhibits mycolic acid biosynthesis. Mycolic acids are large, α alkyl β hydroxy fatty acids with the general structure of R₁-CH(OH)-CH(R₂)-COOH, where R₁ is a meromycolate chain typically containing 50–56 carbons and R₂ is a shorter α branch containing 22–26 carbons. They constitute the inner leaflet of the lipid bilayer of the mycobacterial cell wall and form an effective barrier to the penetration of antibiotics and chemotherapeutic agents (5). The effect of INH on cell wall and mycolic acid synthesis is long established (6–13), although there has been controversy over the molecular targets of INH. We recently reported arylamine N-acetyltransferase (NAT) of M. tuberculosis is a modifier of INH involved in mediating INH resistance (14,15).
结核分枝杆菌属于分枝杆菌属，其独特的细胞壁富含不寻常的糖脂、多糖和脂质，包括分枝菌酸。细胞壁成分的生物合成途径已被证明是几种主要抗结核药物的有效靶点，其中包括抑制霉菌酸生物合成的一线药物异烟肼（INH）。分枝菌酸是大的α烷基β羟基脂肪酸，其一般结构为R ₁ -CH(OH)-CH(R ₂ )-COOH，其中R ₁ 是包含 22-26 个碳的较短 α 支链。它们构成分枝杆菌细胞壁脂质双层的内层，并形成抗生素和化疗药物渗透的有效屏障 (5)。尽管 INH 的分子靶点存在争议，但 INH 对细胞壁和分枝菌酸合成的影响早已确定 (6-13)。我们最近报道了结核分枝杆菌的芳基胺 N-乙酰转移酶 (NAT) 是 INH 的修饰剂，参与介导 INH 耐药性 (14, 15)。

Genes encoding NAT are present in a range of bacterial genomes (15, 16). This observation first provoked intrigue because human NATs have long been identified as drug metabolizing enzymes (17). NAT represents one of the first examples of pharmacogenetic variation and its study revealed the role of acetyl-CoA as an acetyl donor. In particular, NAT2 in humans is known to be responsible for the inactivation of INH through acetylation (18–20). We have studied mycobacterial NATs in this laboratory as potential contributors to the variation in INH resistance among M. tuberculosis clinical isolates. It is now known that nat in M. tuberculosis is polymorphic (14). The expression product acetylates and inactivates INH in vitro, and it has been suggested that this activity and polymorphism might be a contributory factor to INH resistance (14, 15, 21). We know that nat is expressed in M. tuberculosis and Mycobacterium bovis BCG and the gene product is active (14). The genomes of M. tuberculosis (22) and M. bovis (23) have been sequenced. M. bovis is a member of the M. tuberculosis complex. M. bovis BCG is an attenuated M. bovis strain in use as a vaccine. The nat gene is maintained in M. bovis BCG and is identical in sequence to that of M. tuberculosis (14–16, 22, 23).
编码 NAT 的基因存在于一系列细菌基因组中 (15, 16)。这一观察结果首先引起了人们的兴趣，因为人类 NAT 长期以来一直被认为是药物代谢酶 (17)。 NAT 代表了药物遗传学变异的第一个例子，其研究揭示了乙酰辅酶 A 作为乙酰供体的作用。特别是，已知人类中的 NAT2 负责通过乙酰化作用使 INH 失活 (18–20)。我们在本实验室研究了分枝杆菌 NAT，将其视为结核分枝杆菌临床分离株 INH 耐药性变异的潜在因素。现在已知结核分枝杆菌中的 nat 是多态性的 (14)。表达产物在体外乙酰化并灭活 INH，有人认为这种活性和多态性可能是 INH 耐药性的一个促成因素 (14,15,21)。我们知道 nat 在结核分枝杆菌和牛分枝杆菌 BCG 中表达，并且基因产物具有活性 (14)。结核分枝杆菌 (22) 和牛分枝杆菌 (23) 的基因组已被测序。牛分枝杆菌是结核分枝杆菌复合群的成员。牛分枝杆菌 BCG 是用作疫苗的减毒牛分枝杆菌菌株。 nat 基因保留在牛分枝杆菌 BCG 中，其序列与结核分枝杆菌的序列相同 (14–16,22,23)。

An endogenous role for the N-acetylation activity of NAT has not been reported for any mycobacterial species and to this end we have generated an in-frame deletion of nat in M. bovis BCG. We examined the growth, cell morphology, and extractable cell wall lipid composition of the resulting knockout strain. The most important finding is that NAT is essential for normal mycolic acid synthesis in M. bovis BCG, suggesting that mycolic acid biosynthesis involves an as yet unidentified pathway involving NAT. In addition, loss of NAT activity resulted in increased intracellular killing of M. bovis BCG by macrophages. We were able to restore M. bovis BCG wild-type phenotype via functional complementation with M. tuberculosis nat, indicating that NAT has an endogenous role within mycobacteria. We propose that NAT, with its crucial role in mycolic acid biosynthesis, represents a novel antituberculosis drug target.
尚未报道任何分枝杆菌物种中 NAT N-乙酰化活性的内源性作用，为此，我们在牛分枝杆菌 BCG 中产生了 nat 的框内删除。我们检查了所得敲除菌株的生长、细胞形态和可提取的细胞壁脂质组成。最重要的发现是，NAT 对于牛分枝杆菌 BCG 中正常分枝菌酸合成至关重要，这表明分枝菌酸生物合成涉及一条尚未确定的涉及 NAT 的途径。此外，NAT 活性的丧失导致巨噬细胞对牛支原体 BCG 的细胞内杀伤增加。我们能够通过与结核分枝杆菌 nat 的功能互补来恢复牛分枝杆菌 BCG 野生型表型，这表明 NAT 在分枝杆菌中具有内源性作用。我们认为，NAT 在分枝菌酸生物合成中发挥着至关重要的作用，代表了一种新型的抗结核药物靶点。

M. bovis BCG Pasteur and genetically modified strains, including the Δnat complemented with nat in pACE1 (24), were cultured at 37°C in roller bottles with rotation at two revolutions per minute in Middlebrook 7H9 liquid medium containing 10% (vol/vol) albumin-dextrose-catalase (ADC; Difco) and 0.05% (vol/vol) Tween 80 (Sigma-Aldrich), and on Middlebrook 7H10 agar plus 10% (vol/vol) oleic acid–ADC (OADC; Difco), unless otherwise stated. Cultures were harvested from log phase at an OD₆₀₀ of 0.6–1.2. NAT activity in cell lysates was determined after HPLC analysis as previously described (14).
牛支原体 BCG 巴斯德和转基因菌株，包括 pACE1 中与 nat 互补的 Δnat (24)，在 37°C 的滚瓶中培养，在含有 10% (vol/vol) 的 Middlebrook 7H9 液体培养基中以每分钟两转的速度旋转) 白蛋白-葡萄糖-过氧化氢酶 (ADC; Difco) 和 0.05% (vol/vol) Tween 80 (Sigma-Aldrich)，以及 Middlebrook 7H10 琼脂加 10% (vol/vol) 油酸-ADC (OADC; Difco)，除非另有说明。培养物从对数期收获，OD ₆₀₀ 为 0.6–1.2。如前所述，在 HPLC 分析后测定细胞裂解物中的 NAT 活性 (14)。

M. bovis BCG Pasteur with an in-frame unmarked deletion of the nat open reading frame (ORF) was generated by homologous recombination, using plasmids and methods previously described (24, 25). The suicide construct comprised p2NIL, homology arms of ∼1 Kb flanking the M. bovis BCG nat ORF, and a selectable marker cassette from pGOAL19. Preparation and transformation of M. bovis BCG electrocompetent cells and selection of the knockout strain were performed exactly as described (25). For the resultant strain, the nat ORF (Rv3566c) and the five upstream ORFs (Rv3566A, Rv3567c, Rv3568c, Rv3569c, and Rv3570c) in the putative nat operon (16) were amplified using pFU DNA polymerase (Promega) with gene-specific primers (Rv3566c nat: 5′-GAC GAG GTC AGA ATG GCA AC-3′ and 5′-GGG GTT CGT TTG TTC GGA TA-3′; Rv3566A: 5′-GTGTCCGGCGCCGAT-3′ and 5′-TCAGATCCAGTGCCATGTTGC-3′; Rv3567c: 5′-ATGTCGGCTCAGATCGATCC-3′ and 5′-CTAGAGCCAGGTGTCCTGG-3′; Rv3568c: 5′-TGAGCATCCGGTCGCTG-3′ and 5′-CTAGCCGCGAGCGCCTAC-3′; Rv3569c: 5′-ATGACAGCTACCGAGGAATTG-3′ and 5′-TCATCTGCCACCTCCCAG-3′; Rv3570c: 5′-GTGACGTCCCATTCAACAGCG-3′ and 5′-TAGACCATGGTGTCGCCG-3′). DMSO at 6% (vol/vol) was added to all reactions. Mycobacterial cells were denatured at 95°C for 10 min as an extra cycle before the addition of the enzyme mix. The PCR cycle was repeated 30 times. The DNA sequences of the PCR products were confirmed by automated sequencing (Biochemistry Department, DNA Sequencing Facility, Oxford University).
使用之前描述的质粒和方法，通过同源重组生成具有读码框内未标记的 nat 开放阅读框 (ORF) 缺失的牛分枝杆菌 BCG 巴斯德 (24, 25)。自杀构建体包含 p2NIL、牛分枝杆菌 BCG nat ORF 侧翼约 1 Kb 的同源臂以及来自 pGOAL19 的选择性标记盒。牛支原体 BCG 电感受态细胞的制备和转化以及敲除菌株的选择完全按照所述进行（25）。对于所得菌株，使用带有基因特异性引物的 pFU DNA 聚合酶 (Promega) 扩增假定 nat 操纵子 (16) 中的 nat ORF (Rv3566c) 和五个上游 ORF（Rv3566A、Rv3567c、Rv3568c、Rv3569c 和 Rv3570c） (Rv3566c nat：5′-GAC GAG GTC AGA ATG GCA AC-3′和5′-GGG GTT CGT TTG TTC GGA TA-3′；Rv3566A：5′-GTGTCCGGCGCCGAT-3′和5′-TCAGATCCAGTGCCATGTTGC-3′； Rv3567c: 5'-ATGTCGGCTCAGATCGATCC-3' 和 5'-CTAGAGCCAGGTGTCCTGG-3'; Rv3568c: 5'-TGAGCATCCGGTCGCTG-3' 和 5'-CTAGCCGCGAGCGCCTAC-3'; Rv3569c: 5'-ATGACAGCTACCGAGGAATTG-3' 和 5'-TCATCTGCCACC TCCCAG -3'；Rv3570c：5'-GTGACGTCCCATTCAACAGCG-3'和5'-TAGACCATGGTGTCGCCG-3')。将 6%（体积/体积）的 DMSO 添加到所有反应中。在添加酶混合物之前，将分枝杆菌细胞在 95°C 下变性 10 分钟，作为额外循环。 PCR循环重复30次。 PCR产物的DNA序列通过自动测序（牛津大学生物化学系，DNA测序设施）进行确认。

M. tuberculosis nat cloned into the Escherichia coli mycobacterial shuttle expression vector pACE1, under control of the inducible acetamidase promoter (15, 24), was used to complement M. bovis BCG Δnat. Preparation and transformation of electrocompetent M. bovis BCG cells with the construct were as previously described, with selection of transformants on 50 μg/ml 7H10 OADC agar-containing hygromycin. Cultures of M. bovis BCG Δnat complemented with nat in pACE1 were initially maintained in minimal medium induced with 2 mg/ml acetamide (15) and the log phase was reached after 7 d. For experiments in which growth characteristics were compared with other strains, the complemented strain was grown in liquid culture in 7H9 Middlebrook medium, under which conditions the acetamide promoter is known to drive basal level expression (26).
将结核分枝杆菌 nat 克隆到大肠杆菌分枝杆菌穿梭表达载体 pACE1 中，在诱导型乙酰胺酶启动子 (15, 24) 的控制下，用于补充牛分枝杆菌 BCG Δnat。使用构建体制备和转化电感受态牛分枝杆菌BCG细胞如前所述，在含有潮霉素的50μg/ml 7H10 OADC琼脂上选择转化体。在 pACE1 中用 nat 补充的牛分枝杆菌 BCG Δnat 培养物最初维持在用 2 mg/ml 乙酰胺诱导的基本培养基中 (15)，7 天后达到对数期。对于将生长特性与其他菌株进行比较的实验，互补菌株在 7H9 Middlebrook 培养基中的液体培养物中生长，已知在该条件下乙酰胺启动子可驱动基础水平表达 (26)。

We performed SDS-PAGE and Western blotting as previously described using rabbit antiserum raised against recombinant M. tuberculosis NAT as first antibody (21) at a dilution of 1:10,000.
我们使用针对重组结核分枝杆菌 NAT 的兔抗血清作为第一抗体（21）以 1:10,000 的稀释度进行 SDS-PAGE 和蛋白质印迹，如前所述。

Transmission electron microscopy (TEM) was performed as previously described (27) and viewed on a Philips EM410 transmission electron microscope. Digital images were taken using the Gatan multiscan Camera, model 791.
如前所述 (27) 进行透射电子显微镜 (TEM) 并在 Philips EM410 透射电子显微镜上观察。使用 Gatan 多重扫描相机（型号 791）拍摄数字图像。

Scanning electron microscopy (SEM) was performed on polylysine-coated sterile coverslips kept under PBS buffer within a 24-well plate. A drop of concentrated bacterial culture was placed on the surface, washed three times after 1 h with PBS, and then fixed with 3% glutaraldehyde and stained with 1% osmium tetroxide. Critical point drying was performed after a serial wash with 75, 85, and 95%, and twice in absolute alcohol and dry alcohol (kept under CuSO₄). The sample was coated with platinum vapor and observed by SEM (28).
在 24 孔板内保存在 PBS 缓冲液下的聚赖氨酸包被的无菌盖玻片上进行扫描电子显微镜 (SEM)。将一滴浓缩细菌培养物置于表面，1小时后用PBS洗涤3次，然后用3%戊二醛固定，并用1%四氧化锇染色。用75%、85%和95%的无水酒精和无水酒精（在CuSO ₄ 下保存）连续洗涤两次后进行临界点干燥。将样品涂上铂蒸气并通过SEM观察（28）。

100 ml roller cultures were harvested at mid-exponential phase (OD₆₀₀ = 1.0) for each strain. The CFU values for each culture were determined separately to confirm equivalence of biomass. The complex lipids and mycolic acids were extracted and the same proportion of each culture was loaded onto thin layer chromatography (TLC) plates as previously described (29) to allow direct visual comparison between the different strains. Identification of components was by comparison with authenticated standards.
每个菌株在指数中期 (OD ₆₀₀ = 1.0) 收获 100 ml 滚筒培养物。分别测定每种培养物的 CFU 值，以确认生物量的等效性。提取复合脂质和分枝菌酸，并将相同比例的每种培养物加载到薄层色谱（TLC）板上，如前所述（29），以允许不同菌株之间的直接视觉比较。通过与经过验证的标准进行比较来识别成分。

M. bovis BCG Pasteur and Δnat strains were grown to mid-exponential phase (OD₆₀₀ = 1.0) and serially diluted using fresh liquid medium (7H9 ADC Tween 80) and spotted at 10² cells/well in six-well plates with or without antibiotics in 5 ml 7H10 OADC agar. Growth was observed after 14 d of incubation at 37°C. Mid-exponential phase cultures were dispensed and grown in 7H9 Middlebrook medium containing ADC and INH from 0 to 0.3 μg/ml in 96-well plates until cultures without INH reached the late exponential phase (4 d). The growth rate was followed by measuring the turbidity at 600 nm. The relative growth rate is expressed as a fraction of the OD of the untreated wild-type cells (21).
将牛支原体 BCG 巴斯德和 Δnat 菌株生长至指数中期 (OD ₆₀₀ = 1.0)，并使用新鲜液体培养基 (7H9 ADC Tween 80) 进行系列稀释，并在 10 ² 六孔板中的细胞/孔，含或不含抗生素，含 5 ml 7H10 OADC 琼脂。 37℃培养14天后观察生长情况。将指数中期培养物分配并生长在96孔板中含有0至0.3μg/ml ADC和INH的7H9 Middlebrook培养基中，直到不含INH的培养物达到指数后期（4天）。通过测量 600 nm 处的浊度来跟踪生长速率。相对生长速率表示为未处理野生型细胞 OD 的分数 (21)。

Monolayers of the mouse macrophage cell line RAW 264.7 were grown in RPMI 1640 with 10% FBS and plated on coverslips in multiwell plates for 3–4 h before infection (30). Cells were washed three times with OPTIMEM and labeled with TOPRO3 (Molecular Probe) as previously described (31). Elicited macrophage were prepared as previously described (32). M. bovis BCG Pasteur or Δnat cells were harvested at mid-log phase, washed, and resuspended in PBS. One half of the mycobacterial cells were washed and resuspended in OPTIMEM for infection and killing assays, and the remainder were labeled with 500 μg/ml FITC in NaHCO₃, pH 8.5. For opsonization, mycobacteria were incubated at 37°C for 30 min with human serum (4:1 vol/vol), and used immediately. All solutions contained 0.01% Tween 80 (Sigma-Aldrich).
小鼠巨噬细胞系 RAW 264.7 的单层在含有 10% FBS 的 RPMI 1640 中生长，并在感染前铺在多孔板的盖玻片上 3-4 小时 (30)。细胞用 OPTIMEM 洗涤 3 次，并用 TOPRO3（分子探针）标记，如前所述 (31)。如前所述制备引出的巨噬细胞（32）。在对数中期收获牛支原体 BCG 巴斯德或 Δnat 细胞，洗涤并重悬于 PBS 中。将一半的分枝杆菌细胞洗涤并重悬于 OPTIMEM 中进行感染和杀灭测定，其余细胞用 NaHCO ₃ （pH 8.5）中的 500 μg/ml FITC 进行标记。对于调理作用，将分枝杆菌与人血清（4:1 体积/体积）在 37°C 下孵育 30 分钟，并立即使用。所有溶液均含有 0.01% Tween 80 (Sigma-Aldrich)。

Bacteria were added to macrophages in a ratio of 10:1 for fluorimetry, FACS^® analysis, and microscopy. Cultures were incubated for 2 h at 37°C, washed three times in PBS, and fixed with 4% paraformaldehyde. Cells were either stained with ZN (Tb-color kit; Bund Deutscher Hebammen Laboratory) for light microscopy or with TOPRO 3 for 1 h at room temperature after permeabilization with 1% Triton X-100 (31) and washing, before FACS^® analysis. For FACS^® and fluorimetry, cells were recovered by scraping. Protein was measured using the Bradford Colorimetric Assay (Sigma-Aldrich).
将细菌以 10:1 的比例添加到巨噬细胞中，用于荧光测定、FACS ^® 分析和显微镜检查。培养物在37°C孵育2小时，用PBS洗涤3次，并用4%多聚甲醛固定。在 FACS ^® 分析。对于 FACS ^® 和荧光测定法，通过刮擦回收细胞。使用布拉德福德比色测定法（Sigma-Aldrich）测量蛋白质。

After infection with M. bovis BCG Pasteur and the corresponding Δnat mutant in 16-well plates for 2 h, macrophage were washed thoroughly with OPTIMEM with 0.01% Tween, incubated at 37°C for 2 h, 3 d, or 7 d, and then washed with PBS with 0.01% Tween 80. After discarding the medium, cells were lysed in distilled water at room temperature for 10 min, and dilutions were incubated on Middlebrook 7H10 OADC agar at 37°C for 28 d to determine the CFUs.
在16孔板中用牛分枝杆菌BCG巴斯德和相应的Δnat突变体感染2小时后，用含0.01%吐温的OPTIMEM彻底洗涤巨噬细胞，在37°C下孵育2小时、3天或7天，并且然后用含 0.01% Tween 80 的 PBS 清洗。弃去培养基后，室温下在蒸馏水中裂解细胞 10 分钟，并将稀释液在 Middlebrook 7H10 OADC 琼脂上于 37°C 孵育 28 d，以确定 CFU。

M. bovis BCG with an in-frame unmarked deletion of the nat gene (M. bovis BCG Δnat) was generated using homologous recombination (25). Deletion of nat in M. bovis BCG Δnat was confirmed by PCR, Southern blotting, and sequencing of the vestigial nat sequence. We also confirmed the integrity of the adjacent genes by sequencing and PCR. The effect of deleting nat was confirmed by NAT activity, which fell from 17.2 pmoles INH acetylated/min/mg protein to an undetectable level in lysates of the M. bovis BCG Δnat strain. Western blotting using an antibody that specifically recognizes NAT in cell lysates of M. tuberculosis and M. bovis BCG (fig. 1 D) showed NAT to be clearly present in the parental strain, but we do not detect NAT protein in the lysate of M. bovis BCG Δnat. We introduced the E. coli mycobacterial shuttle expression vector pACE1 (24) containing M. tuberculosis nat (14) into M. bovis BCG Δnat to generate a complemented M. bovis BCG Δnat strain. The level of expression of NAT in the complemented strain as determined by Western blotting appears to be similar to the wild-type strain (Fig. 1 D).
使用同源重组生成具有读码框内未标记 nat 基因缺失的牛支原体 BCG (牛支原体 BCG Δnat) (25)。通过 PCR、Southern 印迹和残留 nat 序列测序证实了牛分枝杆菌 BCG Δnat 中 nat 的缺失。我们还通过测序和 PCR 确认了邻近基因的完整性。 NAT 活性证实了删除 nat 的效果，在牛支原体 BCG Δnat 菌株的裂解物中，NAT 活性从 17.2 pmoles INH 乙酰化/min/mg 蛋白降至不可检测的水平。使用特异性识别结核分枝杆菌和牛分枝杆菌 BCG 细胞裂解物中 NAT 的抗体进行蛋白质印迹分析（图 1 D），结果显示 NAT 清楚地存在于亲本菌株中，但我们没有在结核分枝杆菌裂解物中检测到 NAT 蛋白。牛 BCG Δnat。我们将含有结核分枝杆菌 nat (14) 的大肠杆菌分枝杆菌穿梭表达载体 pACE1 (24) 引入牛分枝杆菌 BCG Δnat，以生成互补的牛分枝杆菌 BCG Δnat 菌株。通过蛋白质印迹测定，互补菌株中 NAT 的表达水平似乎与野生型菌株相似（图 1D）。

The growth of M. bovis BCG is slower when nat is deleted. This is observed both on solid agar and in liquid cultures. On agar, colonies in which the nat gene is deleted appear at day 28, as opposed to day 21 for the parental strain (Fig. 1 A). Growth curves in liquid culture reveal that the strain with the nat gene deleted has an increased lag phase (Fig. 1 C), whereas its exponential growth rate mirrors that of the parental strain. In the complemented strain, the growth is restored to that of the wild-type, consistent with the restoration of NAT protein.
当 nat 被删除时，牛支原体 BCG 的生长较慢。这在固体琼脂和液体培养物中都观察到。在琼脂上，nat 基因被删除的集落出现在第 28 天，而亲本菌株则出现在第 21 天（图 1 A）。液体培养中的生长曲线显示，删除 nat 基因的菌株具有更长的滞后期（图 1 C），而其指数生长速率则与亲本菌株相同。在补充菌株中，生长恢复至野生型，与 NAT 蛋白的恢复一致。

We observed, initially by eye, that colonies of M. bovis BCG Δnat appear smaller than their parental strain counterparts (Fig. 1, A and B). This was confirmed by light microscopy; the knockout colonies appear smaller. We attempted to quantify this morphological change using SEM and TEM. From SEM, the individual cells of M. bovis BCG Δnat are significantly (P < 0.05) smaller (Fig. 2 C) than those of the parental cells (M. bovis BCG, total area 5.47 ± 0.84 μ²; M. bovis BCG Δnat, total area 3.97 ± 0.65; average ± SD, n = 40).
我们最初通过肉眼观察到，牛支原体 BCG Δnat 的菌落看起来比其亲本菌株对应物小（图 1、A 和 B）。光学显微镜证实了这一点；敲除的菌落显得更小。我们尝试使用 SEM 和 TEM 量化这种形态变化。从SEM观察，牛分枝杆菌BCG Δnat的单个细胞明显（P < 0.05）小于亲本细胞（牛分枝杆菌BCG，总面积5.47 ± 0.84 μ ²

TEM also reveals the much smoother surface of the M. bovis BCG Δnat cells compared with those of the parental strain (Fig. 2, A and B). Moreover, it is clear from the scanning electron micrographs that the M. bovis BCG Δnat strain lacks cord formation, which is clearly visible in the parental cells (Fig. 2 C).
TEM 还显示，与亲本菌株相比，牛支原体 BCG Δnat 细胞的表面更加光滑（图 2，A 和 B）。此外，从扫描电子显微照片中可以清楚地看出，牛分枝杆菌BCG Δnat菌株缺乏索状形成，这在亲本细胞中清晰可见（图2C）。

The effects of deleting nat on the morphology of the individual cells suggest that the cell wall might be altered. Therefore, we compared the lipid composition of the parental and M. bovis BCG Δnat cells at the same stage of the growth cycle. These comparisons show a distinct difference in the total lipid composition of the M. bovis BCG Δnat strain (Fig. 3, A–C). The mutant appeared to have very reduced quantities of a number of complex lipids and glycolipids, which were identified in the wild-type as phthiocerol dimycocerosate (PDIM), menaquinone (MK), glucose monomycolate (GMM), and trehalose dimycolate or cord factor (CF) using two-dimensional TLC (Fig. 3, A and B). We further analyzed the delipidated cell walls to compare bound mycolic acids (Fig. 3 C). There appears to be very little mycolic acid present within M. bovis BCG Δnat, either in extractable lipids or bound to the cell wall (Fig. 3, A and B). However, the presence of normal mycolic acid production and complex lipid patterns is restored on complementation with M. tuberculosis nat. The cultures used for mycolic acid analyses were plated out to ensure no contamination by nonmycobacterial organisms had occurred. It is these cultures that are illustrated in Fig. 1 A. We see no visible difference between the Δnat and parental strain among other extractable components of the cell wall, particularly the phospholipids (Fig. 3 E), although the amount of extractable fatty acid is slightly diminished in the M. bovis BCG Δnat strain (Fig. 3 D).
删除 nat 对单个细胞形态的影响表明细胞壁可能发生改变。因此，我们比较了亲本和牛支原体 BCG Δnat 细胞在生长周期同一阶段的脂质组成。这些比较显示牛支原体 BCG Δnat 菌株的总脂质组成存在明显差异（图 3，A-C）。该突变体的多种复合脂质和糖脂的数量似乎非常减少，这些复合脂质和糖脂在野生型中被鉴定为硫醇二霉菌酸酯（PDIM）、甲基萘醌（MK）、葡萄糖单霉菌酸酯（GMM）和海藻糖二霉菌酸酯或索状因子。 CF）使用二维薄层色谱（图 3，A 和 B）。我们进一步分析了脱脂的细胞壁以比较结合的分枝菌酸（图3C）。牛分枝杆菌 BCG Δnat 中似乎存在很少的分枝菌酸，无论是在可提取的脂质中还是与细胞壁结合（图 3，A 和 B）。然而，正常的分枝菌酸产生和复杂的脂质模式的存在在与结核分枝杆菌 nat 的补充后得以恢复。将用于分枝杆菌酸分析的培养物铺板以确保没有发生非分枝杆菌生物体的污染。这些培养物如图 1 A 所示。我们发现 Δnat 菌株和亲本菌株在细胞壁的其他可提取成分中没有明显差异，特别是磷脂（图 3 E），尽管可提取脂肪酸的量不同。在牛分枝杆菌 BCG Δnat 菌株中略有减弱（图 3D）。

When unopsonized M. bovis BCG or M. bovis BCG Δnat bacilli are incubated with the mouse macrophage cell line (RAW 264.7), we observed no difference in cellular uptake (Fig. 4, A and B) into macrophages. Experiments with elicited macrophages showed the same results. However, intracellular M. bovis BCG Δnat bacilli are killed after 3 d, whereas the wild-type strain is not (Fig. 4 C). Opsonization does not appear to alter the marked difference in intracellular killing, although after opsonization there is a fivefold increase in the number of M. bovis BCG cells taken up for both the M. bovis BCG and M. bovis BCG Δnat bacilli.
当未调理的牛支原体 BCG 或牛支原体 BCG Δnat 杆菌与小鼠巨噬细胞系 (RAW 264.7) 一起孵育时，我们观察到巨噬细胞的细胞摄取没有差异（图 4，A 和 B）。用引发的巨噬细胞进行的实验显示了相同的结果。然而，细胞内牛支原体 BCG Δnat 杆菌在 3 天后被杀死，而野生型菌株则不然（图 4 C）。调理作用似乎不会改变细胞内杀伤的显着差异，尽管在调理作用后，牛分枝杆菌BCG和牛分枝杆菌BCG Δnat杆菌所吸收的牛分枝杆菌BCG细胞数量增加了五倍。

We reasoned that the change in the composition of the cell wall of M. bovis BCG Δnat strain would result in greater accessibility of antibiotics. We show this is the case using the antibiotics hygromycin and gentamycin. These antibiotics are approximately one order of magnitude more effective in the strains with the nat gene deleted (Table I). Studies with β lactam antibiotics show that deletion of nat increases susceptibility only marginally.
我们推断，牛支原体 BCG Δnat 菌株细胞壁组成的变化将导致抗生素更容易获得。我们使用抗生素潮霉素和庆大霉素来证明这种情况。这些抗生素在 nat 基因缺失的菌株中的效果大约高一个数量级（表 I）。 β 内酰胺类抗生素的研究表明，删除 nat 仅略微增加敏感性。

The M. bovis BCG Δnat strain is twofold more sensitive to INH than the parental strain of M. bovis BCG Pasteur (Fig. 5). The growth of the Δnat strain was inhibited at a twofold lower INH concentration than the wild-type, whereas the complemented strain was indistinguishable from the wild-type in its INH sensitivity.
牛分枝杆菌 BCG Δnat 菌株对 INH 的敏感性是牛分枝杆菌 BCG 巴斯德亲本菌株的两倍（图 5）。 Δnat 菌株的生长在比野生型低两倍的 INH 浓度下受到抑制，而补充菌株的 INH 敏感性与野生型没有区别。

To investigate the endogenous role of NAT in mycobacteria, we deleted the nat gene from M. bovis BCG Pasteur and observed phenotypic changes on (a) growth, (b) ultrastructure and cell morphology, (c) cell wall lipid composition, and (d) intracellular killing of M. bovis BCG Pasteur by mouse macrophages. Deletion of nat affects growth of M. bovis BCG Pasteur on plates and retards the growth in liquid culture by increasing the duration of the lag phase. It has been demonstrated that the nat gene in certain clinical isolates of M. tuberculosis (from amongst a related group of isolates that share a particular IS1160 genotype and are known as family 28) harbor a mutation (33) in the nat gene that renders the NAT protein less active (14). Observations on the growth of these clinical isolates have repeatedly demonstrated that they grow exceptionally slowly on agar and in liquid culture. Although the members of family 28 harbor other mutations, these data indicate that NAT activity may influence growth of M. tuberculosis as well as of M. bovis BCG. We have recently generated a strain of M. tuberculosis H37Rv in which the nat gene has been deleted and have observed that the effect on colony morphology mirrors our results on the effect of deleting the nat gene in M. bovis BCG.
为了研究 NAT 在分枝杆菌中的内源性作用，我们从牛分枝杆菌 BCG 巴斯德中删除了 nat 基因，并观察了 (a) 生长、(b) 超微结构和细胞形态、(c) 细胞壁脂质组成和 (d) 的表型变化。 ) 小鼠巨噬细胞对牛分枝杆菌 BCG 巴斯德的细胞内杀伤。 nat 的删除会影响牛支原体 BCG 巴斯德在平板上的生长，并通过增加滞后期的持续时间来延迟液体培养物中的生长。已经证明，结核分枝杆菌的某些临床分离株（来自共享特定 IS1160 基因型并被称为家族 28 的一组相关分离株）的 nat 基因在 nat 基因中存在突变 (33)，从而导致NAT 蛋白活性较低 (14)。对这些临床分离株生长的观察多次表明它们在琼脂和液体培养物中生长异常缓慢。尽管家族 28 的成员存在其他突变，但这些数据表明 NAT 活性可能影响结核分枝杆菌和牛分枝杆菌 BCG 的生长。我们最近生成了结核分枝杆菌 H37Rv 菌株，其中 nat 基因已被删除，并观察到对菌落形态的影响反映了我们对牛分枝杆菌 BCG 中删除 nat 基因的影响的结果。

The ultrastructure of M. bovis BCG Pasteur is affected when nat is deleted and the cell wall is greatly diminished as observed in TEM. Biochemical analysis of the cell wall lipids clearly shows that the M. bovis BCG Δnat mutant has less mycolic acids and other mycolic acid derivatives like GMM and CF as well as other complex lipids, such as PDIM and MK. Consistent with the lack of CF, we have found cord formation is absent in M. bovis BCG Δnat mutant from ultrastructural analysis by SEM. Studies of these cell wall components of mycobacteria are also consistent with the biochemical findings. PDIM, which is found only in pathogenic mycobacteria, is required for growth in the lungs of mice and is associated with virulence (34). Mycobacterial GMM is a target of the human immune response to mycobacteria (35) and CF is able to stimulate innate, early adaptive, and both humoral and cellular adaptive immunity (36), and induces prolonged mycobacterial survival (37). MK is a component of the respiratory chain of mycobacteria. The effects of environmental conditions on the structure and function of the respiratory chain are beginning to be understood (38). MK appears to be essential (39) and its level in the Δnat strain might be below the level of detection in the experiments presented here. We have found that the changes in mycolates and associated complex lipids from the cell wall is associated with increased sensitivity of mycobacteria to intracellular killing by mouse macrophages, regardless of opsonization. Dilapidation of M. tuberculosis similarly affects survival in bone marrow–derived macrophages (40). It is not certain how a decrease in the content of complex lipids contributes to the observed phenotype or whether the pattern of lipid changes is a primary defect or a secondary effect as a result of interference of normal mycolic acid synthesis, for example through shedding. It is possible that NAT has a role in maintaining homeostasis of acetyl-CoA, a central metabolite in lipid synthesis and hence its lack would affect different synthetic pathways. In this context, it has been observed that a truncation mutant of NAT that catalyses hydrolysis of acetyl-CoA is toxic to E. coli (41). The effects of deleting the nat gene on lipid biosynthesis in M. bovis BCG is not universal. Although mycolic acids and their methyl esters are greatly affected, fatty acids are only diminished slightly. These data suggest that NAT may play a role in extension of the chain length of fatty acids before esterification. The biochemical mechanisms of extension of fatty acyl chains is dependent on chain length (11) and in this context it might be significant that in preliminary experiments, we have found NAT associated with the cell membrane fraction under certain growth conditions.
当 nat 被删除时，牛支原体 BCG 巴斯德的超微结构会受到影响，并且如 TEM 中观察到的细胞壁大大减少。细胞壁脂质的生化分析清楚地表明，牛分枝杆菌 BCG Δnat 突变体具有较少的分枝菌酸和其他分枝菌酸衍生物（如 GMM 和 CF）以及其他复杂脂质（如 PDIM 和 MK）。与 CF 的缺乏一致，我们通过 SEM 的超微结构分析发现牛分枝杆菌 BCG Δnat 突变体中不存在索状形成。对分枝杆菌这些细胞壁成分的研究也与生化结果一致。 PDIM 仅存在于致病性分枝杆菌中，是小鼠肺部生长所必需的，并且与毒力相关 (34)。分枝杆菌 GMM 是人类对分枝杆菌免疫反应的目标 (35)，而 CF 能够刺激先天性、早期适应性以及体液和细胞适应性免疫 (36)，并诱导分枝杆菌存活时间延长 (37)。 MK 是分枝杆菌呼吸链的组成部分。人们开始了解环境条件对呼吸链结构和功能的影响（38）。 MK 似乎是必需的 (39)，其在 Δnat 菌株中的水平可能低于此处介绍的实验中的检测水平。我们发现，无论调理作用如何，细胞壁分枝杆菌和相关复合脂质的变化与分枝杆菌对小鼠巨噬细胞细胞内杀伤的敏感性增加有关。结核分枝杆菌的损坏同样会影响骨髓来源的巨噬细胞的存活率（40）。 目前尚不清楚复合脂质含量的减少如何影响观察到的表型，也不确定脂质变化的模式是主要缺陷还是由于干扰正常分枝菌酸合成（例如通过脱落）而产生的次要影响。 NAT 可能在维持乙酰辅酶 A 的稳态中发挥作用，乙酰辅酶A是脂质合成的中心代谢物，因此它的缺乏会影响不同的合成途径。在这种情况下，据观察，催化乙酰辅酶 A 水解的 NAT 截短突变体对大肠杆菌有毒 (41)。删除 nat 基因对牛分枝杆菌 BCG 脂质生物合成的影响并不普遍。尽管分枝菌酸及其甲酯受到很大影响，但脂肪酸仅略有减少。这些数据表明，NAT 可能在酯化前延长脂肪酸链长方面发挥作用。脂肪酰链延伸的生化机制取决于链长度 (11)，在这种情况下，在初步实验中，我们发现 NAT 在某些生长条件下与细胞膜部分相关，这一点可能很重要。

INH is a substrate of NAT and hence INH is indisputably a NAT ligand (42). We have obtained a three-dimensional crystallographic structure of NAT with INH bound (unpublished data). The precise target of INH in mycobacteria leading to inhibition of mycolic acid synthesis has been the subject of continued debate (43, 44). Studies that we have performed with pure proteins suggest that KatG and NAT compete for INH, supporting the interpretation that NAT acts to control the amount of active INH available and hence modulates INH sensitivity. The interest in mycolic acids stems from their exclusivity to mycobacteria, making these biosynthetic pathways obvious targets for antimycobacterial drugs. Routes leading to mycolic acid biosynthesis are not fully established (45, 46), although studies with a viable strain of Mycobacterium smegmatis defective in mycolate biosynthesis (5) as well as Mycobacterium aurum treated with INH (47) suggest that mycobacterial cells can survive with severely reduced mycolic acid content of the cell wall. A reduction in mycolates makes these organisms more permeable and is in agreement with the results presented here on increased sensitivity to antibiotics in the Δnat strain of M. bovis BCG Pasteur.
INH 是 NAT 的底物，因此 INH 无疑是 NAT 配体 (42)。我们获得了具有INH结合的NAT的三维晶体结构（未发表的数据）。 INH 在分枝杆菌中的精确靶标导致分枝杆菌酸合成的抑制一直是争论的主题 (43, 44)。我们用纯蛋白质进行的研究表明，KatG 和 NAT 竞争 INH，这支持了这样的解释：NAT 的作用是控制可用的活性 INH 的量，从而调节 INH 敏感性。对分枝杆菌酸的兴趣源于其对分枝杆菌的排他性，使得这些生物合成途径成为抗分枝杆菌药物的明显靶标。导致分枝菌酸生物合成的途径尚未完全确定 (45, 46)，尽管对分枝菌酸生物合成缺陷的耻垢分枝杆菌活菌株的研究 (5) 以及用异烟肼处理的金黄色分枝杆菌 (47) 表明，分枝杆菌细胞可以在细胞壁分枝菌酸含量严重降低。霉菌酸盐的减少使得这些生物体更具渗透性，并且与本文中关于牛分枝杆菌 BCG 巴斯德的 Δnat 菌株对抗生素敏感性增加的结果一致。

Although INH is a substrate for M. tuberculosis NAT (14), NAT is unlikely to be an additional target for INH. Nevertheless, a reduction in mycolic acid as well as decreasing the effectiveness of the cell wall as a barrier to protein extrusion (47), mirrors the ultrastructural effects we have observed on deleting the nat gene. These data, together with the increased intracellular killing by macrophages of M. bovis BCG Pasteur Δnat, indicate that specifically targeting the NAT protein may serve both to increase the effectiveness of combination therapy by an order of magnitude and shorten the treatment time for active infection through inhibiting cell wall biosynthesis.
尽管 INH 是结核分枝杆菌 NAT 的底物 (14)，但 NAT 不太可能成为 INH 的其他靶标。然而，分枝菌酸的减少以及细胞壁作为蛋白质挤出屏障的有效性的降低（47），反映了我们在删除 nat 基因时观察到的超微结构效应。这些数据，加上牛分枝杆菌 BCG Pasteur Δnat 巨噬细胞增加的细胞内杀伤作用，表明特异性靶向 NAT 蛋白可能既可以将联合治疗的有效性提高一个数量级，又可以通过以下方式缩短活动性感染的治疗时间：抑制细胞壁生物合成。

To target the NAT protein, we have developed a high throughput assay (48) and are using this with combinatorial chemistry and modeling on the NAT 3-D structure (42) to generate compounds to test both on mycobacterial growth in culture and mycobacterial killing in macrophages.
为了靶向 NAT 蛋白，我们开发了一种高通量测定法 (48)，并将其与组合化学和 NAT 3-D 结构建模 (42) 结合使用，以生成化合物来测试培养物中分枝杆菌的生长和杀灭分枝杆菌的情况。巨噬细胞。

We thank Mimi Mo and Drs. F. Pompeo, J. Harris, and P. Deepalakshmi for assistance.
我们感谢咪咪莫和博士。 F. Pompeo、J. Harris 和 P. Deepalakshmi 寻求帮助。

S. Bhakta is a Wellcome Travelling Research Fellow and G.S. Besra is a Lister Institute-Jenner Research Fellow. We acknowledge support from the Wellcome Trust and Medical Research Council, Biotechnology and Biological Sciences Research Council, and GlaxoSmithKline for a studentship for A.M. Upton.
S. Bhakta 是 Wellcome 旅行研究员，G.S. Besra 是 Lister Institute-Jenner 研究员。我们感谢威康信托和医学研究委员会、生物技术和生物科学研究委员会以及葛兰素史克公司对 A.M. 学生奖学金的支持。厄普顿。