EsxA membrane-permeabilizing activity plays a key role in mycobacterial cytosolic translocation and virulence: effects of single-residue mutations at glutamine 5.

Qi Zhang,Qing Deng,Jianjun Sun,Guozhong Jiang,Xiujun Li,Wei Qian,Hugues Ouellet,Decheng Wang,Wei Liu

doi:10.1038/srep32618

Abstract

EsxA is required for virulence of Mycobacterium tuberculosis (Mtb) and plays an essential role in phagosome rupture and translocation to the cytosol of macrophages. Recent biochemical studies have demonstrated that EsxA is a membrane-permeabilizing protein. However, evidence that link EsxA membrane-permeabilizing activity to Mtb cytosolic translocation and virulence is lacking. Here we found that mutations at glutamine 5 (Q5) could up or down regulate EsxA membrane-permeabilizing activity. The mutation Q5K significantly diminished the membrane-permeabilizing activity, while Q5V enhanced the activity. By taking advantage of the single-residue mutations, we tested the effects of EsxA membrane-permeabilizing activity on mycobacterial virulence and cytosolic translocation using the esxA/esxB knockout strains of Mycobacterium marinum (Mm) and Mtb. Compared to wild type (WT), the Q5K mutant exhibited significantly attenuated virulence, evidenced by intracellular survival and cytotoxicity in mouse macrophages as well as infection of zebra fish embryos. The attenuated virulence of the Q5K mutant was correlated to the impaired cytosolic translocation. On the contrary, the Q5V mutant had a significantly increased cytosolic translocation and showed an overall increased virulence. This study provides convincing evidence that EsxA contributes to mycobacterial virulence with its membrane-permeabilizing activity that is required for cytosolic translocation.

Highlights

Of esx-1 from Mycobacterium tuberculosis (Mtb) resulted in attenuation of virulence and retention of mycobacteria in the phagosome, while transfer of esx-1 into Bacille Calmette-Guérin (BCG) partially restored virulence[14,15,16,17,18,19,20,21]
It was found that Mtb and Mycobacterium smegmatis (Ms). leprae were able to translocate from the phagolysosomal compartment into the cytosol of myeloid cells, and the cytosolic translocation was dependent on secretion of EsxA and EsxB6
In a systematic mutagenesis analysis, we serendipitously found that mutations at Q5 were able to up- or down-regulate its membrane-permeabilizing activity using the ANTS/DPX liposome leakage assay (Fig. 1b), in which the liposomes are pre-loaded with ANTS/DPX and the ANTS fluorescence is de-quenched when EsxA forms pores on the liposomal membrane to release DPX30,33

Summary

Introduction

Of esx-1 from Mtb resulted in attenuation of virulence and retention of mycobacteria in the phagosome, while transfer of esx-1 into BCG partially restored virulence[14,15,16,17,18,19,20,21]. We present evidence that single-residue mutations at Q5 of EsxA either up or down regulate the membrane-permeabilizing activity, which enhances or attenuates mycobacterial virulence in macrophages and in zebra fish embryos through up or down regulation of mycobacterial cytosolic translocation. In the rest of the study, we used Q5K and Q5V to test the effects of EsxA membrane-permeabilizing activity on mycobacterial virulence and cytosolic translocation.

Results

Conclusion