Mycolactone causes catastrophic Sec61-dependent loss of the endothelial glycocalyx and basement membrane: a new indirect mechanism driving tissue necrosis in Mycobacterium ulcerans infection

The drivers of tissue necrosis in Mycobacterium ulcerans infection (Buruli ulcer disease) have historically been ascribed solely to the directly cytotoxic action of the diffusible exotoxin, mycolactone. However, its role in the clinically-evident vascular component of disease aetiology remains poorly explained. We have now dissected mycolactone’s effects on primary vascular endothelial cells in vitro and in vivo. We show that mycolactone-induced changes in endothelial morphology, adhesion, migration, and permeability are dependent on its action at the Sec61 translocon. Unbiased quantitative proteomics identified a profound effect on proteoglycans, driven by rapid loss of type II transmembrane proteins of the Golgi, including enzymes required for glycosaminoglycan (GAG) synthesis, combined with a reduction in the core proteins themselves. Loss of the glycocalyx is likely to be of particular mechanistic importance, since knockdown of galactosyltransferase II (beta-1,3-galactotransferase 6; B3Galt6), the GAG linker-building enzyme, phenocopied the permeability and phenotypic changes induced by mycolactone. Additionally, mycolactone depleted many secreted basement membrane components and microvascular basement membranes were disrupted in vivo. Remarkably, exogenous addition of laminin-511 reduced endothelial cell rounding, restored cell attachment and reversed the defective migration caused by mycolactone. Hence supplementing mycolactone-depleted extracellular matrix may be a future therapeutic avenue, to improve wound healing rates.

HDMEC membrane fraction proteins significantly upregulated by mycolactone. Video 1. HDMECs were exposed to 10 ng/mL mycolactone. Live cell imaging was performed with using the zenCELL Owl incubator microscope every 30 minutes for 48 hours.
Time-lapse videos (representative of 3 independent experiments) were generated with zencell-owl software. Time stamp and scale bar as indicated.
Video 2. HDMECs were exposed to 0.02% DMSO. Live cell imaging was performed with using the zenCELL Owl incubator microscope every 30 minutes for 48 hours. Time-lapse videos (representative of 3 independent experiments) were generated with zencell-owl software. Time stamp and scale bar as indicated.

Video 3.
A scratch was introduced to HUVEC monolayer exposed to 0.02% DMSO and live cell imaging was performed with the zenCELL Owl incubator microscope every 15 min for 23 hours. Time-lapse videos (representative of 3 independent experiments) were generated with zencell-owl software. Time stamp and scale bar as indicated.

Video 4.
A scratch was introduced to HUVEC monolayer exposed to 10 ng/mL mycolactone and live cell imaging was performed with the zenCELL Owl incubator microscope every 15 min for 23 hours. Time-lapse videos (representative of 3 independent experiments) were generated with zencell-owl software. Time stamp and scale bar as indicated.

Video 5.
HUVECs were seeded on to laminin-511 coated well. The following day, a scratch was introduced to the monolayer and the cells were exposed to 10 ng/mL mycolactone. Live cell imaging was then performed with the zenCELL Owl incubator microscope every 15 min . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted February 21, 2023. ; https://doi.org/10.1101/2023.02.21.529382 doi: bioRxiv preprint for 23 hours. Time-lapse videos (representative of 3 independent experiments) were generated with zencell-owl software. Time stamp and scale bar as indicated.
Video 6. HUVECs were seeded on to laminin-411 coated well. The following day, a scratch was introduced to the monolayer and the cells were exposed to 10 ng/mL mycolactone. Live cell imaging was then performed with the zenCELL Owl incubator microscope every 15 min for 23 hours. Time-lapse videos (representative of 3 independent experiments) were generated with zencell-owl software. Time stamp and scale bar as indicated.
Video 7. HUVECs were seeded on to laminin-111 coated well. The following day, a scratch was introduced to the monolayer and the cells were exposed to 10 ng/mL mycolactone. Live cell imaging was then performed with the zenCELL Owl incubator microscope every 15 min for 23 hours. Time-lapse videos (representative of 3 independent experiments) were generated with zencell-owl software. Time stamp and scale bar as indicated.
. CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in  (ii) TRAP increases translocation of proteins whose signal peptides bear a high GP content. TRAP is a heterotetrameric complex which interacts with the ribosome on the cytosolic side of the ER membrane and with Sec61α on the luminal side, binding to a hinge region between the N-and C-terminal halves of the protein facilitating channel opening [1,2] (iii) The Sec62/63 complex is involved in post-translational translocation but can also assist opening of the channel for proteins with signal peptides that gate slowly due to the presence relatively long but less hydrophobic "H-regions" and lower carboxy terminal polarity [3]. This complex also interacts with the ribosome and the translocon and may also interact directly with the nascent peptide chain [4]. In addition, Sec63 recruits BiP to the translocon to further assist channel opening on the luminal side [3]. (E) Overlap between mycolactone-downregulated endothelial membrane proteome and translocon-dependent proteome. Venn diagram created using JVenn [5] showing overlap in significantly downregulated proteome between the dataset presented here and those obtained in Hela cells treated with siRNA for Sec61α and translocon associated protein TRAP or HEK293 cells with Sec62 or Sec623 knocked out [1,3]. (F) Top significantly over-represented (p<0.05) Gene Ontology groups in upregulated data set, compared to whole genome. Data generated with WebGestalt. . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted February 21, 2023. ; Figure S4. Sec61 blockade suppresses B3Galt6 expression in endothelial cells. (A) HDMECs exposed to 10 ng/mL of mycolactone (MYC), 0.02% DMSO, 20 nM ZIF-80 or remained untreated for 24 hours were fixed, permeabilised and immunostained with anti-B3Galt6 and anti-giantin antibodies and nuclei stained with DAPI. Images are representative of 2 independent experiments. Scale bar = 50 μm.
. CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted February 21, 2023. ; https://doi.org/10.1101/2023.02.21.529382 doi: bioRxiv preprint Figure S6. Mycolactone impacts endothelial cell adhesion molecules. HDMECs exposed to 10 ng/mL of mycolactone (MYC), 0.02% DMSO or remained untreated for indicated times. Cells were lysed and subjected to immunoblotting with anti-fibronectin (A) and anti-integrin α5 (B) antibodies. Each immunoblot intensity was normalised according to GAPDH and untreated controls. Data from 3 independent experiments are presented (mean ± SEM). ns, not significant; **, P < 0.01; ****, P < 0.0001.
. CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted February 21, 2023. ; https://doi.org/10.1101/2023.02.21.529382 doi: bioRxiv preprint Figure S7. Endothelial cell adhesion to various laminin isoforms. (A) HDMECs were harvested and layered to laminin-511, 411, 111 or uncoated wells for one hour. Non-adherent cells were washed away and attached cells per field were counted. mean ± SEM (n = 3). (B) HDMECs seeded onto different laminin isoforms were untreated or exposed to 0.02% DMSO or 10 ng/mL mycolactone (MYC) for 48 hrs. Their viability was assayed using CellEvent detection kit as described in (Ogbechi et al, 2018). The number of live cells (negative for both active caspase 3/7 and PI) in three fields was determined and expressed as a proportion of total cells (Mean ± SEM, n = 3 independent experiments). ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.
. CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted February 21, 2023. ; https://doi.org/10.1101/2023.02.21.529382 doi: bioRxiv preprint