Mechanisms of pyrazinamide resistance in mycobacteria:: importance of lack of uptake in addition to lack of pyrazinamidase activity
Mechanisms of pyrazinamide resistance in mycobacteria:: importance of lack of uptake in addition to lack of pyrazinamidase activity
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DOI：
10.1099/13500872-145-6-1359
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发表时间：
1999-06-01
期刊：
影响因子：
2.8
通讯作者：
Daffé, M
中科院分区：
文献类型：
--
作者：
Raynaud, C;Lanéelle, MA;Daffé, M
已知分枝杆菌通过编码吡津酰胺酶(PZase)的基因突变而对抗结核药物吡津酰胺(PZA)产生抗药性。PZase是一种将PZA转化为吡津酸的酶，PZA被认为是PEA抗细菌的活性形式。已知存在对该药物产生抗药性的其他机制，但尚未充分调查。其中之一是未摄取前药物，这是本研究调查的一种可能性。研究了PEA在结核分枝杆菌体内的摄取机制，PEA是激活前药的必要步骤。在中性和酸性环境中，杆菌都能掺入[C-14]PZA，因为已知PEA活性在酸性ph值时最好。通过使用原载体(间氯苯肼，CCCP)、呋喃霉素、砷酸盐和低温，证明了依赖于ATP的转运系统参与了PZA的摄取。虽然结构相似的化合物烟酰胺抑制了PZA的转运系统，但其他结构相关的化合物，如吡嗪酸、异烟肼和胞嘧啶则没有。酸性条件也没有影响。基于脂质体中的扩散实验，发现PEA在膜双层中的扩散速度比甘油快，而结核分枝杆菌的孔蛋白样蛋白OmpATb在蛋白脂质体中的存在略微增加了药物的扩散。这一发现可以解释为什么细胞壁支原体疏水层不代表PEA扩散的限制步骤，这是通过使用结核分枝杆菌菌株及其同基因突变株的比较实验判断的，该突变株的共价连接支原体减少40%。比较不同分枝杆菌的PZase活性、PEA摄取率和敏感性。结核分枝杆菌是一种自然敏感的PEA物种，是唯一既表现出PZase活性又表现出PZA吸收的物种；与所研究的四种自然抗性物种没有观察到这种相关性。耻垢分枝杆菌具有功能性的PZase，但不摄取PZA；使用的PZase阴性的禽分枝杆菌则相反，其摄取PZA的情况与结核分枝杆菌相当。牛分枝杆菌BCC和Kansasii分枝杆菌既不显示PZase活性，也不摄取PZA。这些数据清楚地表明，PEA耐药的机制之一是菌株未能摄取药物，这表明分枝杆菌对PZA的敏感性需要功能PZase和PZA运输系统的存在。在所研究的菌株中，PZase和烟酰胺酶的出现与细胞位置之间没有相关性，这表明其中一种或两种酰胺都可以被其他分枝杆菌酰胺酶所降解。
Mycobacteria are known to acquire resistance to the antituberculous drug pyrazinamide (PZA) through mutations in the gene encoding pyrazinamidase (PZase), an enzyme that converts PZA into pyrazinoic acid, the presumed active form of PEA against bacteria. Additional mechanisms of resistance to the drug are known to exist but have not been fully investigated. Among these is the non-uptake of the pro-drug, a possibility investigated in the present study. The uptake mechanism of PEA, a requisite step for the activation of the pro-drug, was studied in Mycobacterium tuberculosis. The incorporation of [C-14]PZA by the bacilli was followed in both neutral and acidic environments since PEA activity is known to be optimal at acidic ph. By using a protonophore (carbonyl cyanide m-chlorophenylhydrazone; CCCP), valinomycin, arsenate and low temperature, it was shown that an ATP-dependent transport system is involved in the uptake of PZA. Whilst the structurally analogous compound nicotinamide inhibited the transport system of PZA, other structurally related compounds such as pyrazinoic acid, isoniazid and cytosine did not. Acidic conditions were also without effect. Based on diffusion experiments in liposomes, it was found that PEA diffuses rapidly through membrane bilayers, faster than glycerol, whilst the presence of OmpATb, the porin-like protein of M. tuberculosis, in proteoliposomes slightly increased the diffusion of the drug. This finding may explain why the cell wall mycolate hydrophobic layer does not represent the limiting step in the diffusion of PEA, as judged from comparative experiments using a M. tuberculosis strain and its isogenic mutant elaborating 40% less covarently linked mycolates. PZase activity, and PEA uptake and susceptibility in different mycobacterial species were compared. M. tuberculosis, a naturally PEA-susceptible species, was the only species that exhibited both PZase activity and PZA uptake; no such correlation was observed with the four naturally resistant species examined. Mycobacterium smegmatis possessed a functional PZase but did not take up PZA; the reverse was true for the PZase-negative strain of Mycobacterium avium used, with PZA uptake comparable to that of M. tuberculosis. Mycobacterium bovis BCC and Mycobacterium kansasii exhibited neither a PZase activity nor PZA uptake. These data clearly demonstrate that one of the mechanisms of resistance to PEA resides in the failure of strains to take up the drug, indicating that susceptibility to PZA in mycobacteria requires both the presence of a functional PZase and a PZA transport system. No correlation was observed between the occurrence and cellular location of PZase and of nicotinamidase in the strains examined, suggesting that one or both amides can be hydrolysed by other mycobacterial amidases.