Mycobacterium tuberculosis Ser/Thr Protein Kinase B Mediates an Oxygen-Dependent Replication Switch

The majority of Mycobacterium tuberculosis (Mtb) infections are clinically latent, characterized by drug tolerance and little or no bacterial replication. Low oxygen tension is a major host factor inducing bacteriostasis, but the molecular mechanisms driving oxygen-dependent replication are poorly understood. Here, we tested the role of serine/threonine phosphorylation in the Mtb response to altered oxygen status, using an in vitro model of latency (hypoxia) and reactivation (reaeration). Broad kinase inhibition compromised survival of Mtb in reaeration. Activity-based protein profiling and genetic mutation identified PknB as the kinase critical for surviving hypoxia. Mtb replication was highly sensitive to changes in PknB levels in aerated culture, and even more so in hypoxia. A mutant overexpressing PknB specifically in hypoxia showed a 10-fold loss in viability and gross morphological defects in low oxygen conditions. In contrast, chemically reducing PknB activity during hypoxia specifically compromised resumption of growth during reaeration. These data support a model in which PknB activity is reduced to achieve bacteriostasis, and elevated when replication resumes. Together, these data show that phosphosignaling controls replicative transitions associated with latency and reactivation, that PknB is a major regulator of these transitions, and that PknB could provide a highly vulnerable therapeutic target at every step of the Mtb life cycle—active disease, latency, and reactivation.绝大多数结核分枝杆菌（Mycobacterium tuberculosis, Mtb）感染在临床上呈潜伏状态，其特征为药物耐受、细菌复制微弱或完全停滞。低氧张力是诱导细菌抑菌作用的主要宿主因素，但介导氧依赖性细菌复制的分子机制仍不甚明确。本研究借助潜伏感染（低氧）与复燃（复氧）的体外模型，探究了丝氨酸/苏氨酸磷酸化在结核分枝杆菌应对氧状态改变中的调控作用。广谱激酶抑制会削弱结核分枝杆菌在复氧阶段的存活能力。基于活性的蛋白质谱分析（activity-based protein profiling）与基因突变实验证实，PknB是介导结核分枝杆菌耐受低氧环境的关键激酶。结核分枝杆菌的复制在有氧培养条件下对PknB水平变化高度敏感，在低氧环境中这一敏感性进一步增强。在低氧条件下特异性过表达PknB的突变株，其存活率降低10倍，且在低氧环境中出现显著的形态学缺陷。与之相反，在低氧阶段通过化学手段抑制PknB活性，则会特异性损害复氧阶段的生长恢复能力。上述数据支持如下调控模型：PknB活性需被下调以实现细菌抑菌，而当复制恢复时则需上调其活性。综上，本研究表明：磷酸化信号通路调控与结核分枝杆菌潜伏、复燃相关的复制转换；PknB是这类转换的核心调控因子；且PknB可作为结核分枝杆菌生命周期各阶段——包括活动性疾病、潜伏感染与复燃阶段——的高脆弱性治疗靶点。