Overexpression of Adenylyl Cyclase Encoded by the Mycobacterium tuberculosis Rv2212 Gene Confers Improved Fitness, Accelerated Recovery from Dormancy and Enhanced Virulence in Mice.
Overexpression of Adenylyl Cyclase Encoded by the Mycobacterium tuberculosis Rv2212 Gene Confers Improved Fitness, Accelerated Recovery from Dormancy and Enhanced Virulence in Mice.
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DOI：
10.3389/fcimb.2017.00370
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发表时间：
2017
影响因子：
5.7
通讯作者：
Kaprelyants AS
中科院分区：
文献类型：
--
作者：
Shleeva MO;Kondratieva TK;Demina GR;Rubakova EI;Goncharenko AV;Apt AS;Kaprelyants AS
早些时候，我们证明了MSMEG_4279基因编码的腺苷环化酶(AC)在耻垢分枝杆菌休眠状态下的复苏和生长中起着关键作用，该基因的过表达导致细胞内cAMP浓度的增加，并阻止耻垢分枝杆菌在伴随着中等酸化的延长的静止期从活跃生长向休眠转变。我们推测，cAMP的主要产生者结核分枝杆菌(Mtb)的同源Rv2212基因在这些条件下支持休眠细菌的活跃状态和重新激活，发挥着类似的生理作用。为了验证这一假设，我们建立了高表达Rv2212的Mtb菌株，并将其体外和体内生长特性与对照菌株进行了比较。在体外，AC过表达的pMindRv2212菌株在液体培养基中生长更快，形成CFU的能力延长，并显著延迟甚至阻止了向休眠的过渡。AC过表达的细胞更容易从休眠中恢复。在体内，AC高表达细菌在感染小鼠的肺和脾中显示出比对照菌株显著更高的生长率(毒力)，并且与后者不同的是，在感染8个月前，耐结核病菌株中的小鼠死亡。即使在没有选择潮霉素B的情况下，所有的pMindRv2212 CFU在体内生长过程中都保留了Rv2212插入片段，这有力地表明AC的过度表达对细菌有利。综上所述，我们的结果表明，cAMP在体外支持在不利条件下保持结核分枝杆菌细胞的活力，并在体内支持其毒力。
Earlier we demonstrated that the adenylyl cyclase (AC) encoded by the MSMEG_4279 gene plays a key role in the resuscitation and growth of dormant Mycobacterium smegmatis and that overexpression of this gene leads to an increase in intracellular cAMP concentration and prevents the transition of M. smegmatis from active growth to dormancy in an extended stationary phase accompanied by medium acidification. We surmised that the homologous Rv2212 gene of M. tuberculosis (Mtb), the main cAMP producer, plays similar physiological roles by supporting, under these conditions, the active state and reactivation of dormant bacteria. To test this hypothesis, we established Mtb strain overexpressing Rv2212 and compared its in vitro and in vivo growth characteristics with a control strain. In vitro, the AC-overexpressing pMindRv2212 strain demonstrated faster growth in a liquid medium, prolonged capacity to form CFUs and a significant delay or even prevention of transition toward dormancy. AC-overexpressing cells exhibited easier recovery from dormancy. In vivo, AC-overexpressing bacteria demonstrated significantly higher growth rates (virulence) in the lungs and spleens of infected mice compared to the control strain, and, unlike the latter, killed mice in the TB-resistant strain before month 8 of infection. Even in the absence of selecting hygromycin B, all pMindRv2212 CFUs retained the Rv2212 insert during in vivo growth, strongly suggesting that AC overexpression is beneficial for bacteria. Taken together, our results indicate that cAMP supports the maintenance of Mtb cells vitality under unfavorable conditions in vitro and their virulence in vivo.