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

Reviewer #1 (Public Review):

The authors have investigated the effect of the toxin mycolactone produced by mycobacterium ulcerans on the endothelium. Mycobacterium ulcerans is involved in Buruli ulcer classified as a neglected disease by WHO. This disease has dramatic consequences on the microcirculation causing important cutaneous lesions. The authors have previously demonstrated that endothelial cells are especially sensitive to mycolactone. The present study brings more insight into the mechanism involved in mycolactone-induced endothelial cells defect and thus in microcirculatory dysfunction. The authors showed that mycolactone directly affected the synthesis of proteoglycans at the level of the golgi with a major consequence on the quality of the glycocalyx and thus on the endothelial function and structure. Importantly, the authors show that blockade of the enzyme involve in this synthesis (galactosyltransferase II) phenocopied the effects of mycolactone. The effect of mycolactone on the endothelium was confirmed in vivo. Finally, the authors showed that exogenous laminin-511 reversed the effects of mycolactone, thus opening an important therapeutic perspective for the treatment of wound healing in patients suffering Buruli ulcer and presenting lesions.

Reviewer #2 (Public Review):

The authors dissected the effects of mycolacton on endothelial cell biology and vessel integrity. The study follows up on previous work by the same group, which highlighted alterations in vascular permeability and coagulation in patients with Buruli ulcer. It provides a mechanistic explanation for these clinical observations, and suggests that blockade of Sec61 in endothelial cells contributes to tissue necrosis and slow wound healing.

Overall, the generated data support their conclusions and I only have two major criticisms:

- Replicating the effects of mycolactone on endothelial parameters with Ipomoeassin F (or its derivative ZIF-80) does not demonstrate that these effects are due to Sec61 blockade. This would require genetic proof, using for example endothelial cells expressing Sec61A mutants that confer resistance to mycolactone blockade. The authors claimed in the Discussion that they could not express such mutants in primary endothelial cells, but did they try expressing mutants in HUVEC cell lines? Without such genetic evidence all statements claiming a causative link between the observed effects on endothelial parameters and Sec61 blockade should be removed or rephrased. The same applies to speculations on the role of Sec61 in epithelial migration defects in discussion. Data corresponding to Ipomoeassin F and ZIF-80 do not add important information, and may be removed or shown as supplemental information.
- While statistical analysis is done and P values are provided, no information is given on the statistical tests used, neither in methods nor results. This must be corrected, to evaluate the repeatability and reproducibility of their data.

Reviewer #3 (Public Review):

Buruli ulcer is a severe skin infection in humans that is caused by a bacterium, Mycobacterium ulcerans. The main clinical sign is a massive tissue necrosis subsequent to an edema stage. The main virulence factor called mycolactone is a polyketide with a lactone core and a long alkyl chain that is released within vesicles by the bacterium. Mycolactone was already shown to account for several disease phenotypes characteristic of Buruli ulcer, for instance tissue necrosis, host immune response modulation and local analgesia. A large number of cellular pathways in various cell types was reported to be impacted by mycolactone. Among those, the Sec61 translocon involved in the transport of certain proteins to the endoplasmic reticulum was first identified by the authors of the study and is currently the most consensual target. Mycolactone disruption of Sec61 function was then shown to directly impact on cell apoptosis in macrophages, limited immune responses by T-cells and increased autophagy in dermal endothelial cells and fibroblasts. In their manuscript, Tzung-Harn Hsieh and their collaborators investigated the Sec61- dependent role of mycolactone on morphology, adhesion and migration of primary human dermal microvascular endothelial cells (HDMEC). They used a combination of sugar and proteomic studies on a live image-based phenotypic assay on HDMEC to characterize the effect of mycolactone. First, they showed that upon incubation of monolayer of HDMEC with mycolactone at low dose (10 ng/mL) for 24h, the cells become elongated before rounding and eventually detached from the culture dish at 48h. Next, mycolactone was probed on a scratch assay and migration of the cells ceased upon a 24h incubation. The same effect as mycolactone on these two assays was observed for two other Sec61 inhibitors Ipomoeassin F and ZIF-80. Then, the authors resorted to the widely established mouse footpad model of M. ulcerans infection to evidence fibrinogen accumulation outside the blood vessel within the endothelium at 28 days post-infection, correlating with severe endothelial cell morphology changes.

To dissect the molecular pathways involved in these phenotypes, the authors performed an HDMEC membrane protein analysis and showed a decrease in the numbers of proteins involved in glycosylation and adhesion. As protein glycosylation mainly occurs in the Golgi apparatus, a deeper analysis revealed that enzymes involved in glycosaminoglycan (GAG) synthesis were lost in mycolactone treated HDMEC. A combination of immunofluorescence and flow cytometry approaches confirmed the impact of mycolactone on the ability of endothelial cells to synthesize GAG chains. The mycolactone effect on cell elongation was phenocopied by knock-down of galactosyltransferase II (B3Galt6) involved in GAG biosynthesis. A second extensive analysis of the endothelial basement membrane component and their ligands identified multiple laminins affected by mycolactone. Using similar functional studies as for GAG, the impact of mycolactone on cell rounding and migration could be reversed by the addition of laminin α5.

The major strengths of the study relies on a combination of cleverly designed phenotypic assays and in-depth cleverly designed membrane proteomic studies and follow-up analysis.
The results really support the conclusions. Congratulations!
The discussion takes into account the current state of the art, which has mostly been established by the authors of the present manuscript.

Author Response

Reviewer #2 (Public Review):

The authors dissected the effects of mycolacton on endothelial cell biology and vessel integrity. The study follows up on previous work by the same group, which highlighted alterations in vascular permeability and coagulation in patients with Buruli ulcer. It provides a mechanistic explanation for these clinical observations, and suggests that blockade of Sec61 in endothelial cells contributes to tissue necrosis and slow wound healing. Overall, the generated data support their conclusions and I only have two major criticisms:

• Replicating the effects of mycolactone on endothelial parameters with Ipomoeassin F (or its derivative ZIF-80) does not demonstrate that these effects are due to Sec61 blockade. This would require genetic proof, using for example endothelial cells expressing Sec61A mutants that confer resistance to mycolactone blockade. The authors claimed in the Discussion that they could not express such mutants in primary endothelial cells, but did they try expressing mutants in HUVEC cell lines? Without such genetic evidence all statements claiming a causative link between the observed effects on endothelial parameters and Sec61 blockade should be removed or rephrased. The same applies to speculations on the role of Sec61 in epithelial migration defects in discussion. Data corresponding to Ipomoeassin F and ZIF-80 do not add important information, and may be removed or shown as supplemental information.

• While statistical analysis is done and P values are provided, no information is given on the statistical tests used, neither in methods nor results. This must be corrected, to evaluate the repeatability and reproducibility of their data.

We respectfully but fundamentally disagree with the comments regarding the Sec61 dependence of the effects that we observed. We showed that loss of glycocalyx and basement membrane components underpinned the phenotypic changes in endothelial cells (morphological changes, loss of adhesion, increased permeability, and reduced ability to repair scratch wounds). We demonstrated that we could phenocopy permeability increases and elongation phenotype by knocking down the type II membrane protein B3Galt6, and reverse the adhesion defect by exogenous provision of the secreted laminin-511 heterotrimer.

Our conclusion that mycolactone mediates these effects via Sec61 inhibition is not based solely on the use of alternative inhibitors but is built on several pillars of evidence:

First, the proteomics data conforms entirely to predictions based on the topology of affected vs. non-effected proteins, and agrees with independently published proteomic datasets from T lymphocytes, dendritic cells and sensory neurons (ref.12), as well as biochemical studies performed using in vitro translocation assays (ref.11,34). Furthermore, the pattern of membrane protein down regulation observed in our experiments fits perfectly with established models of protein translocation mechanisms, particularly with respect to the lack of effect on specific topologies of multipass membrane proteins, tail anchored- and type III membrane proteins (ref.34-36).

Second, since Sec61 very highly conserved amongst mammals and is found in all nucleated cells, it is hard to conceptualise a framework in which mycolactone targets Sec61 in some cells and not others, as this reviewer suggests might be the case for epithelial cells [noting that the work being referred to (ref.29) predates our 2014 work showing that mycolactone is a Sec61 inhibitor (ref.7)]. Indeed, mycolactone has been shown to target Sec61 in multiple independent approaches including forward genetic screens involving random mutagenesis and CRISPR/Cas9 (ref.10, PMID: 35939511). Genetic evidence has previously been provided for the Sec61 dependence of mycolactone effects in epithelial cells (ref.10,17). We have unpublished genetic evidence that the rounding and detachment of epithelial cells due to mycolactone is reduced when resistance mutations are over expressed, and will consider including this in the next version of the manuscript.

Third, given this weight of evidence, one would be hard-pressed to provide an alternative explanation for the specific down-regulation of glycosaminoglycan-synthesising enzymes and adhesion/basement membrane molecules while most cytosolic and non-Sec61 dependent membrane proteins are unchanged or upregulated. However, seeking to be as rigorous as possible we have here shown that a completely independent Sec61 inhibitor produces the same phenotype at the gross and molecular level. Ipomoeassin F (Ipom-F) is a glycolipid, not a polyketide lactone, yet they both compete for binding with cotransin in Sec61α (ref.6). There is significant overlap in the cellular responses to mycolactone and Ipom-F, including the induction of the integrated stress response (ref.17, PMID: 34079010), which we observed again in the current data, providing further evidence that this approach is useful when genetic approaches are technically unattainable.

Therefore, we are confident the effects seen on endothelial cells are Sec61-dependent. We are happy to provide more detail on our lengthy attempts at over-expressing mycolactone resistant SEC61A1 genes in HUVECs; primary endothelial cells derived from the umbilical vein. We are highly experienced in this area, and have previously stably expressed these proteins in epithelial cell lines, reproducing the resistance profile (ref.10,17). Notably though, these cells do not have normal ‘fitness’ in the absence of challenge. Since endothelial cells (and endothelial cell lines; PMID: 12560236) are extremely hard to transfect with plasmids, with efficiency routinely 5-10% (including in our hands), we developed a lentivirus system. We were eventually (after multiple attempts using different protocols) able to transduce primary HUVECs with constructs expressing GFP (at an efficiency of about 10-20%) and select/expand these under puromycin selection. Never-the-less, we never recovered any cells that expressed the flag-tagged SEC61A1 wild type or SEC61A1 carrying the resistance mutant D60G. We also attempted to select D60G-transduced cells with mycolactone epimers, an approach that can help the cells compete against non-transduced cells in culture flasks (ref.10). We concluded that primary endothelial cells are unable to tolerate the expression of additional Sec61α, and this was incompatible with survival.

It’s also important to note that most endothelial cell specialists would agree that endothelial cell lines are not good models of endothelial behaviour. We tested the HMEC-1 cell line, but found it did not express prototypical endothelial marker vWF in the expected way. Therefore we focussed our efforts on primary endothelial cells. Should we be able to overcome the dual challenge of the necessity to work in primary cells, and the difficulty of over-expressing Sec61, we will update this paper at a later date with this data, and will also expand the above arguments.

We apologise for the embarrassing oversight of not including information about the statistical analyses we used, which of course we will correct in full in the revised version. However, we would like to provide this information to readers of the current version of the manuscript. All data were analysed using GraphPad Prism Version 9.4.1:

Figure 1: one-way ANOVA with Dunnett’s (panel A) or Tukey’s (panel B) correction for multiple comparisons

Figure 2 supplement: one-way ANOVA with Tukey’s correction for multiple comparisons (analysed panel)

Figure 3: one-way ANOVA with Tukey’s (panel B) or Dunnett’s (panel E&F) correction for multiple comparisons

Figure 4: one-way ANOVA with Dunnett’s correction for multiple comparisons (all analysed panels)

Figure 5 and supplement: one-way ANOVA with Dunnett’s correction for multiple comparisons (all analysed panels)

Figure 6: one-way ANOVA with Dunnett’s correction for multiple comparisons (analysed panel)

Figure 6 supplement: one-way ANOVA with Dunnett’s correction for multiple comparisons (all analysed panels)

Figure 7: two-way ANOVA with Tukey’s correction for multiple comparisons (all analysed panels; panels B&C also included the Geisser Greenhouse correction for sphericity)

Figure 7 supplement: Panels A&D used a repeated measures one-way ANOVA with Dunnett’s correction for multiple comparisons (panel D also included the Geisser Greenhouse correction for sphericity). Panels B,C&E used a two-way ANOVA with Tukey’s correction for multiple comparisons (panels B&C also included the Geisser Greenhouse correction for sphericity)