EspB is a membrane binding effector of the Mycobacterial Type VII secretion system

Nayanika Sengupta, Somnath Dutta


EspB, denoted by Rv3881c, is a PE/PPE family secreted substrate of the ESX-1 apparatus of the T7SS. Full length EspB is a 48 kDa polypeptide (460 amino acids) chain which is cleaved at its C-terminal domain by MycP protease in the periplasm of Mycobacterial cells. In 2013, Chen et al. showed that recombinantly purified mature form of EspB selectively bound phosphatidic acid (PA) and phosphatidylserine (PS) in contrast with the full length EspB. However, till date further knowledge representing the mode of interaction between processed EspB and PA or PS remains unaddressed. This poses an important structural question – does mature EspB have a specific binding pocket for PA and PS or is the binding mediated between EspB and biological membranes composed of PA and PS? Based on the long withstanding debate on the functional role of EspB, we designed a cryo-EM based study to elucidate EspB-lipid affinity. Recombinant mature EspB was purified by two-step affinity and SEC purification. The peak fractions were analysed by NS-TEM and the fraction corresponding to discrete heptameric particles was vitrified to perform cryo-EM structural analysis. Cryo-EM data were collected in the presence of 0.03% fluorinated octyl maltoside to address the challenge of strong preferred orientation in vitreous ice. Through single particle reconstruction, we were able to resolve heptameric EspB at 5.9 Å. We deployed several biophysical tools to screen different target lipids. Microscale thermophoresis (MST) and NS-TEM illustrated strongest binding with PA. Intriguingly, cryo-EM micrographs revealed
PA vesicles decorated with side views of EspB. Our current cryo-EM 3D reconstruction of EspB with PA shows the presence of extra densities near the otherwise unstructured C-terminal disordered region (CTD). We hypothesise that lipid binding may induce order in the intrinsically disordered CTD and thus EspB may elicit pathophysiological response by PA and PS mediated host membrane binding.

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Sengupta ET AL 2022


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