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Mechanistic insight into bacterial entrapment by septin cage reconstitution.

ABSTRACT: Septins are cytoskeletal proteins that assemble into hetero-oligomeric complexes and sense micron-scale membrane curvature. During infection with Shigella flexneri, an invasive enteropathogen, septins restrict actin tail formation by entrapping bacteria in cage-like structures. Here, we reconstitute septin cages in vitro using purified recombinant septin complexes (SEPT2-SEPT6-SEPT7), and study how these recognize bacterial cells and assemble on their surface. We show that septin complexes recognize the pole of growing Shigella cells. An amphipathic helix domain in human SEPT6 enables septins to sense positively curved membranes and entrap bacterial cells. Shigella strains lacking lipopolysaccharide components are more efficiently entrapped in septin cages. Finally, cryo-electron tomography of in vitro cages reveals how septins assemble as filaments on the bacterial cell surface.

SUBMITTER: Lobato-Marquez D

PROVIDER: S-EPMC8302635 | biostudies-literature | 2021 Jul

REPOSITORIES: biostudies-literature

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Mechanistic insight into bacterial entrapment by septin cage reconstitution.

Lobato-Márquez Damián D Xu Jingwei J Güler Gizem Özbaykal GÖ Ojiakor Adaobi A Pilhofer Martin M Mostowy Serge S

Nature communications 20210723 1

Septins are cytoskeletal proteins that assemble into hetero-oligomeric complexes and sense micron-scale membrane curvature. During infection with Shigella flexneri, an invasive enteropathogen, septins restrict actin tail formation by entrapping bacteria in cage-like structures. Here, we reconstitute septin cages in vitro using purified recombinant septin complexes (SEPT2-SEPT6-SEPT7), and study how these recognize bacterial cells and assemble on their surface. We show that septin complexes recog ...[more]

PMID: 34301939

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Project description:The bacterial prepeptidase C-terminal (PPC) domain can be found in the C termini of a wide variety of proteases that are secreted by marine bacteria. However, the functions of these PPC domains remain unknown due to a lack of systematic research. Here, the binding and swelling abilities of eight PPC domains from six different proteases were compared systematically via scanning electron microscopy (SEM), enzyme assays, and fluorescence spectroscopy. These PPC domains all possess the ability to bind and swell insoluble collagen. PPC domains can expose collagen monomers but cannot disrupt the pyridinoline cross-links or unwind the collagen triple helix. This ability can play a synergistic role alongside collagenase in collagen hydrolysis. Site-directed mutagenesis of the PPC domain from Vibrio anguillarum showed that the conserved polar and aromatic residues Y6, D26, D28, Y30, W42, E53, C55, and Y65 and the hydrophobic residues V10, V18, and I57 played key roles in substrate binding. Molecular dynamic simulations were conducted to investigate the interactions between PPC domains and collagen. Most PPC domains have a similar mechanism for binding collagen, and the hydrophobic binding pocket of PPC domains may play an important role in collagen binding. This study sheds light on the substrate binding mechanisms of PPC domains and reveals a new function for the PPC domains of bacterial proteases in substrate degradation.IMPORTANCE Prepeptidase C-terminal (PPC) domains commonly exist in the C termini of marine bacterial proteases. Reports examining PPC have been limited, and its functions remain unclear. In this study, eight PPCs from six different bacteria were examined. Most of the PPCs possessed the ability to bind collagen, feathers, and chitin, and all PPCs could significantly swell insoluble collagen. PPCs can expose collagen monomers but cannot disrupt pyridinoline cross-links or unwind the collagen triple helix. This swelling ability may also play synergistic roles in collagen hydrolysis. Comparative structural analyses and the examination of PPC mutants revealed that the hydrophobic binding pockets of PPCs may play important roles in collagen binding. This study provides new insights into the functions and ecological significance of PPCs, and the molecular mechanism of the collagen binding of PPCs was clarified, which is beneficial for the protein engineering of highly active PPCs and collagenase in the pharmaceutical industry and of artificial biological materials.

Dataset Information

Mechanistic insight into bacterial entrapment by septin cage reconstitution.

Publications

Mechanistic insight into bacterial entrapment by septin cage reconstitution.

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