Abstract
EsxA (ESAT-6), an important virulence factor of Mycobacterium tuberculosis, plays an essential role in phagosome rupture and bacterial cytosolic translocation within host macrophages. Our previous study showed that EsxA exhibits a unique membrane-interacting activity that is not found in its ortholog from nonpathogenic Mycobacterium smegmatis. However, the molecular mechanism of EsxA membrane insertion remains unknown. In this study, we generated truncated EsxA proteins with deletions of the N- and/or C-terminal flexible arm. Using a fluorescence-based liposome leakage assay, we found that both the N- and C-terminal arms were required for membrane disruption. Moreover, we found that, upon acidification, EsxA converted into a more organized structure with increased α-helical content, which was evidenced by CD analysis and intrinsic tryptophan fluorescence. Finally, using an environmentally sensitive fluorescent dye, we obtained direct evidence that the central helix-turn-helix motif of EsxA inserted into the membranes and formed a membrane-spanning pore. A model of EsxA membrane insertion is proposed and discussed.
Highlights
Mycobacterium tuberculosis EsxA exhibits a pH-dependent membrane-interacting activity. Both the N- and C-terminal flexible arms are required for membrane disruption, and the central helix-turn-helix motif of EsxA inserts into the membrane
Our previous study showed that EsxA exhibits a unique membrane-interacting activity that is not found in its ortholog from nonpathogenic Mycobacterium smegmatis
We characterized the pH-dependent membrane interactions and conformational changes of M. tuberculosis EsxA, and we showed that M. tuberculosis EsxA exhibits a unique membrane-interacting activity that is not found in its ortholog from nonpathogenic Mycobacterium smegmatis [26]
Summary
Mycobacterium tuberculosis EsxA exhibits a pH-dependent membrane-interacting activity. Results: Both the N- and C-terminal flexible arms are required for membrane disruption, and the central helix-turn-helix motif of EsxA inserts into the membrane. Conclusion: EsxA forms a membrane-spanning pore at low pH. Our previous study showed that EsxA exhibits a unique membrane-interacting activity that is not found in its ortholog from nonpathogenic Mycobacterium smegmatis. We generated truncated EsxA proteins with deletions of the N- and/or C-terminal flexible arm. Using a fluorescence-based liposome leakage assay, we found that both the N- and C-terminal arms were required for membrane disruption. Using an environmentally sensitive fluorescent dye, we obtained direct evidence that the central helix-turn-helix motif of EsxA inserted into the membranes and formed a membrane-spanning pore. A model of EsxA membrane insertion is proposed and discussed
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