1 Scientific RepoRts | 6:32618 | DOI: 10.1038/srep32618 www.nature.com/scientificreports EsxA membrane-permeabilizing activity plays a key role in mycobacterial cytosolic translocation and virulence: efects of single-residue mutations at glutamine 5 Qi Zhang 1,2 , Decheng Wang 3,4 , Guozhong Jiang 1,† , Wei Liu 1,† , Qing Deng 3 , Xiujun Li 5 , Wei Qian 6 , Hugues Ouellet 1 & Jianjun Sun 1,6 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 signifcantly diminished the membrane-permeabilizing activity, while Q5V enhanced the activity. By taking advantage of the single-residue mutations, we tested the efects 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 signifcantly attenuated virulence, evidenced by intracellular survival and cytotoxicity in mouse macrophages as well as infection of zebra fsh embryos. The attenuated virulence of the Q5K mutant was correlated to the impaired cytosolic translocation. On the contrary, the Q5V mutant had a signifcantly 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. Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is considered one of the most successful pathogens. It is estimated that Mtb infects one-third of the world’s population and kills more than one million people each year 1,2 . At the early stage of infection, Mtb is internalized into the phagosome of host alveolar mac- rophage. Instead of being destroyed in the phagolysosomal compartment, Mtb inhibits phagosome-lysosome fusion, persists and replicates within the phagosome 3–5 . Recent studies have also shown that upon lysosome-phagosome fusion, Mtb progressively translocates from phago-lysosomes into the cytosol 6 , where Mtb replicates and spread to new cells 7,8 . Phagosomal maturation arrest and cytosolic translocation have been attrib- uted, at least in part, to a Type VII secretion system, termed ESX-1 6–11 . Te esx-1 locus was frst revealed by the comparative genomic studies, in which Region of Diference 1 (part of the esx-1 locus) was found to be present in Mtb, but not in the attenuated vaccine strain Mycobacterium bovis Bacille Calmette-Guérin (BCG) 12,13 . Deletion 1 Department of Biological Sciences, Border Biomedical Research Center, University of Texas at El Paso, El Paso, TX 79968, USA. 2 School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou, 45001, China. 3 Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA. 4 Medical College of China Three Gorges University, Yichang, 443002, China. 5 Department of Chemistry, University of Texas at El Paso, El Paso, TX, 79968, USA. 6 Sino-Duth Biomedical Information Engineering School of Northeastern University, Shenyang, China. † Present address: The First Afliated Hospital of Zhengzhou University, Zhengzhou, China. Correspondence and requests for materials should be addressed to J.S. (email: jsun@utep.edu) Received: 09 May 2016 Accepted: 10 August 2016 Published: 07 September 2016 OPEN