A Novel Mycobacterial Starvation Response Mechanism Mediated By Global Regulatory Protein CarD

Mycobacterium tuberculosis is the causative agent of tuberculosis,which kills ～1.5million people worldwide every year.M.tuberculosis usually enters into a dormant state in response to various host stresses(such as nutritional starvation,hypoxic and acidic environment,etc.),which renders M.tuberculosis extremely resistant to host defenses,making it notoriously difficult to be completely eliminated,resulting in a difficult cure of tuberculosis.Under starvation conditions,bacterial cells tend to slow down their translation rate by reducing r RNA synthesis,but the way they accomplish that may vary in different bacteria.In mycobacteria,transcription of r RNA is activated by the RNA polymerase(RNAP)accessory transcription factor Car D,which interacts directly with RNAP to stabilize the RNAP-promoter open complex formed on r RNA genes.Car D plays an important role in transcription initiation and is an essential global transcription factor in mycobacteria.At present,the structures and functions of Car D have been extensively studied,but the mechanisms that control its expression remain obscure.In this thesis,we found that the Car D levels in a variety of mycobacterial cells were dramatically decreased in response to several distinct host-like stresses of nutritional starvation,hypoxic,and acidic conditions and the levels quickly returned to normal after the stresses were terminated.This indicates that Car D plays an important regulatory role in the survival of mycobacterial cells.Then,taking the condition of nutritional starvation as an example,this thesis deeply studied the regulation mechanism of Car D.Our results demonstrated that when mycobacterial cells switched from nutrient-rich to nutrientdeprived conditions the level of Car D was tightly regulated at multiple levels:(1)at the translational level,the antisense RNA Ascar D inhibited the continued translation of car D m RNA;We found a noncoding RNA in the antisense of car D,identified and characterized it,and named it Ascar D.Further studies showed that Ascar D was regulated by Sig F and specifically expressed under stationary phase and starvation conditions,and the induced Ascar D could bind with car D m RNA and inhibit its translation.(2)at the post-translational level,the Clp protease was activated and quickly degrades the residual Car D;Through gene knockout,pull-down and in vitro protease degradation experiments,we found that Clp C1P2P1 complex efficiently recognizes and degrades Car D.Further study showed that Clp C1 protein level increased significantly during stationary phase,and the active Clp complex was assembled in large quantities during stationary phase and starvation conditions,which mediate the rapid degradation of Car D during starvation conditions.(3)(p)pp Gpp content significantly affected Car D protein level;The(p)pp Gpp-mediated stringent response plays an important role in bacterial starvation response.In this thesis,we found a significant reduction in Car D levels after(p)pp Gpp synthesis was induced by the addition of a stringent response inducer.Furthermore,we knocked out and overexpressed the(p)pp Gpp synthase encoding gene rel A and found that Car D level was significantly reduced after rel A was overexpressed,while Car D level was significantly increased after rel A was knocked out.Therefore,under starvation conditions,the three regulators of antisense RNA,Clp protease and(p)pp Gpp worked synergistically to maintain Car D at the minimal level,and diminishes the synthesis of r RNA,to slow down the rate of transcription and translation of mycobacterial cells to help mycobacterial cells cope with the nutritional stress.In summary,this thesis reveals the multilayered regulation mode of Car D and clarifies a novel mechanism for the mycobacterial starvation response mediated by Car D.This not only expands our understanding of the global transcription regulator Car D,but also helps us better understand the adaptation and survival of the pathogenic mycobacteria in the host cells and provide new ideas for the treatment of tuberculosis.