Through mutant selection on agar containing pyrazinoic acid (POA), the bioactive form of the prodrug pyrazinamide (PZA), we recently showed that missense mutations in the aspartate decarboxylase PanD and the unfoldase ClpC1, and loss-of-function mutation of polyketide synthases Mas and PpsA-E involved in phthiocerol dimycocerosate synthesis, cause resistance to POA and PZA in Mycobacterium tuberculosis. Here we first asked whether these in vitro-selected POA/PZA-resistant mutants are attenuated in vivo, to potentially explain the lack of evidence of these mutations among PZA-resistant clinical isolates. Infection of mice with panD, clpC1, and mas/ppsA-E mutants showed that whereas growth of clpC1 and mas/ppsA-E mutants was attenuated, the panD mutant grew as well as the wild-type. To determine whether these resistance mechanisms can emerge within the host, mice infected with wild-type M. tuberculosis were treated with POA, and POA-resistant colonies were confirmed for PZA and POA resistance. Genome sequencing revealed that 82 and 18% of the strains contained missense mutations in panD and clpC1, respectively. Consistent with their lower fitness and POA resistance level, independent mas/ppsA-E mutants were not found. In conclusion, we show that the POA/PZA resistance mechanisms due to panD and clpC1 missense mutations are recapitulated in vivo. Whereas the representative clpC1 mutant was attenuated for growth in the mouse infection model, providing a possible explanation for their absence among clinical isolates, the growth kinetics of the representative panD mutant was unaffected. Why POA/PZA resistance-conferring panD mutations are observed in POA-treated mice but not yet among clinical strains isolated from PZA-treated patients remains to be determined.

中文翻译：

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体内选择耐吡嗪酸的结核分枝杆菌菌株在天冬氨酸脱羧酶PanD和解折叠酶ClpC1中怀有突变。

通过在含有吡嗪酸（POA），前药吡嗪酰胺（PZA）的生物活性形式的琼脂上进行突变选择，我们最近显示了天冬氨酸脱羧酶PanD和解折叠酶ClpC1的错义突变以及聚酮化合物合酶Mas的功能丧失突变PphA-E和PpsA-E参与二硫代木蜡二椰油酸酯的合成，在结核分枝杆菌中引起对POA和PZA的抗性。在这里，我们首先询问这些体外选择的POA / PZA耐药突变体是否在体内减弱，以潜在地解释在PZA耐药临床分离株中缺乏这些突变的证据。与小鼠的感染PAND，clpC1，和MAS / PPSA-E突变体显示，而生长clpC1和mas / ppsA-E突变体减毒，panD突变体与野生型一样生长。为了确定这些抗性机制是否能在宿主内出现，用POA处理感染了野生型结核分枝杆菌的小鼠，并确认POA抗性菌落具有PZA和POA抗性。基因组测序，发现该菌株的82和18％含有错义突变在PAND和clpC1，分别。与其较低的适应性和POA抗性水平一致，未发现独立的mas / ppsA-E突变体。总之，我们证明了POA / PZA耐药机制由于PAND和clpC1在体内概括了错义突变。尽管代表性的clpC1突变体在小鼠感染模型中的生长被减弱，为临床分离株中的缺失提供了可能的解释，但是代表性的panD突变体的生长动力学并未受到影响。为什么在POA治疗的小鼠中观察到赋予POA / PZA耐药性的panD突变，但在从PZA治疗的患者中分离出的临床菌株中尚未观察到尚待确定。