Mycobacterial dynamin-like protein IniA mediates membrane fission

Wang, Manfu; Yang, Xiuna; Zhang, Bing; Ren, Jie; Liu, Aijun; Ran, Yajun; Yan, Bing; Chen, Fang; Guddat, Luke W.; Hu, Junjie; Li, Jun; Rao, Zihe

doi:10.1038/s41467-019-11860-z

Cited by 38 publications

(47 citation statements)

References 34 publications

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“…Our complementation analyses indicated IniA and IniC have non-redundant functions in EV biogenesis supporting the idea that these DLPs work together. This is in agreement with Wang et al who observed low GTPase activity in monomeric Msm -IniA and suggested that interactions with IniC may be required for high rates of GTP hydrolysis (22). This form of cooperation was described for mitochondrial OPA1 proteins.…”

Section: Discussionsupporting

confidence: 92%

“…Classic dynamin and DLPs form a large family of GTPases that mediate membrane fission and fusion in both eukaryotic and prokaryotic cells. Although the IniA protein in Mtb has not been functionally characterized, the crystal-structure of M. smegmatis ( Msm ) IniA showed conserved structural features with other DLPs (22) including a GTPase domain containing the four consensus elements (G1-G4) and two elongated alpha-helical bundle domains which are structurally contiguous and are also known as neck and trunk domains (Fig 3B). Msm -IniA exhibits GTPase-dependent membrane fission activity in vitro , and it localizes to the plasma membrane causing cell surface wrinkling, when overexpressed in Msm (22).…”

Section: Resultsmentioning

confidence: 99%

“…Although the IniA protein in Mtb has not been functionally characterized, the crystal-structure of M. smegmatis ( Msm ) IniA showed conserved structural features with other DLPs (22) including a GTPase domain containing the four consensus elements (G1-G4) and two elongated alpha-helical bundle domains which are structurally contiguous and are also known as neck and trunk domains (Fig 3B). Msm -IniA exhibits GTPase-dependent membrane fission activity in vitro , and it localizes to the plasma membrane causing cell surface wrinkling, when overexpressed in Msm (22). Computer modeling of Mtb -IniA based on the crystal structure of Msm -IniA suggests conserved folding between the two homologs (Fig 3B-C), although the annotated Mtb protein has an N-terminal extension of 34 amino acids.…”

Section: Resultsmentioning

confidence: 99%

“…Computer modeling of Mtb -IniA based on the crystal structure of Msm -IniA suggests conserved folding between the two homologs (Fig 3B-C), although the annotated Mtb protein has an N-terminal extension of 34 amino acids. At the trunk tip, Msm- IniA has a lipid-binding domain composed of hydrophobic and positively charged residues that are necessary for lipid binding and membrane association (22). These residues are similar in Mtb -IniA.…”

Section: Resultsmentioning

confidence: 99%

“…Early studies showed Mtb -IniA assemble into hexameric-ring like structures upon interaction with lipid monolayers (23) and recent structural and biochemical analysis of Msm -IniA confirmed protein folding and biochemical characteristics of DLPs including GTPase activity, interaction with membrane lipids and GTP-hydrolysis dependent membrane fission activity in vitro (22). Most bacterial DLPs exist as side by side pairs within operons like iniAC or in the case of B. subtillis DnyA, as two DLPs genetically fused into a single unit (21) which suggest interactions of DLP isoforms for membrane remodeling.…”

Section: Discussionmentioning

confidence: 99%

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Dynamin-like proteins are essential for vesicle biogenesis inMycobacterium tuberculosis

Gupta

Palacios

Khataokar

et al. 2020

Preprint

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30 31 Mycobacterium tuberculosis (Mtb) secretes pathogenicity factors and immunologically active 32 molecules via membrane vesicles. However, nothing is known about the mechanisms involved 33 in mycobacterial vesicle biogenesis. This study investigates molecular determinants of 34 membrane vesicle production in Mtb by analyzing Mtb cells under conditions of high vesicle 35 production: iron limitation and VirR restriction. Ultrastructural analysis showed extensive cell 36 envelope restructuring in association with vesicle release that correlated with downregulation 37 of cell surface lipid biosynthesis and peptidoglycan alterations. Comparative transcriptomics 38 showed common upregulation of the iniBAC operon in association with high vesicle production 39 in Mtb cells. Vesicle production analysis demonstrated that the dynamin-like proteins (DLPs) 40 encoded by this operon, IniA and IniC, are necessary for release of EV by Mtb in culture and in 41 infected macrophages. Isoniazid, a first-line antibiotic, used in tuberculosis treatment, was 42 found to stimulate vesicle release in a DLP-dependent manner. Our results provide a new 43 understanding of the function of mycobacterial DLPs and mechanistic insights into vesicle 44 biogenesis. The findings will enable further understanding of the relevance of Mtb-derived 45 extracellular vesicles in the pathogenesis of tuberculosis and may open new avenues for 46 therapeutic research. 47 48 49 3 IMPORTANCE 50 Iron is an essential nutrient that promotes survival and growth of M. tuberculosis, the bacterium 51 that causes human tuberculosis (TB). Limited availability of iron, often encountered in the host 52 environment, stimulates M. tuberculosis to secrete membrane-bound extracellular vesicles 53 containing molecules that may help it evade the immune system. Characterizing the bacterial 54 factors and mechanisms involved in the production of mycobacterial vesicles is important for 55 envisioning ways to interfere with this process. Here, we report the discovery of proteins 56 required by M. tuberculosis for vesicle biogenesis in culture and during host cell infection. We 57 also demonstrate a connection between antibiotic response and extracellular vesicle 58 production. The work provides insights into the mechanisms underlying vesicle biogenesis in 59 M. tuberculosis and permits better understanding of the significance of vesicle production to M. 60 tuberculosis-host interactions and antibiotic stress response. 61 62 63 4 64 65 Extracellular vesicles (EV) are membrane-bound structures actively secreted by most, if not all, 66 cells and play important roles in intercellular communication. Bacteria release EV containing 67 proteins, genetic material, and lipids to communicate with both prokaryotic and eukaryotic cells 68 in their environment (1, 2).69 M. tuberculosis (Mtb), the causative agent of human tuberculosis (TB), releases EV during 70 axenic growth and into the macrophages it infects, both in culture and in the lungs of infected 71 mice (3). Mtb-produced EV (Mtb-EV)...

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Section: Discussionsupporting

confidence: 92%

Section: Resultsmentioning

confidence: 99%