The Pathogenic Mechanism of the Mycobacterium ulcerans Virulence Factor, Mycolactone, Depends on Blockade of Protein Translocation into the ER

Abstract

Infection with Mycobacterium ulcerans is characterised by tissue necrosis and immunosuppression due to mycolactone, the necessary and sufficient virulence factor for Buruli ulcer disease pathology. Many of its effects are known to involve down-regulation of specific proteins implicated in important cellular processes, such as immune responses and cell adhesion. We have previously shown mycolactone completely blocks the production of LPS-dependent proinflammatory mediators post-transcriptionally. Using polysome profiling we now demonstrate conclusively that mycolactone does not prevent translation of TNF, IL-6 and Cox-2 mRNAs in macrophages. Instead, it inhibits the production of these, along with nearly all other (induced and constitutive) proteins that transit through the ER. This is due to a blockade of protein translocation and subsequent degradation of aberrantly located protein. Several lines of evidence support this transformative explanation of mycolactone function. First, cellular TNF and Cox-2 can be once more detected if the action of the 26S proteasome is inhibited concurrently. Second, restored protein is found in the cytosol, indicating an inability to translocate. Third, in vitro translation assays show mycolactone prevents the translocation of TNF and other proteins into the ER. This is specific as the insertion of tail-anchored proteins into the ER is unaffected showing that the ER remains structurally intact. Fourth, metabolic labelling reveals a near-complete loss of glycosylated and secreted proteins from treated cells, whereas cytosolic proteins are unaffected. Notably, the profound lack of glycosylated and secreted protein production is apparent in a range of different disease-relevant cell types. These studies provide a new mechanism underlying mycolactone's observed pathological activities both in vitro and in vivo. Mycolactone-dependent inhibition of protein translocation into the ER not only explains the deficit of innate cytokines, but also the loss of membrane receptors, adhesion molecules and T-cell cytokines that drive the aetiology of Buruli ulcer.

Suppressive effect of mycolactone-containing fraction from Mycobacterium ulcerans on antibody production against co-administered antigens

“…On the other hand, other researchers reported that mycolactone inhibited the function of the Sec61 translocon [7,9,26]. Sec61 mediates antigen transport across endosomal membranes and affects cross-presentation in dendritic cells [27].…”

“…In addition, several in vitro studies have suggested that mycolactone could inhibit cytokine and chemokine production from immune cells such as T cells, macrophages, and dendritic cells [9][10][11][12][13]. These observations clearly suggested that mycolactone presents immunosuppressive effects and may have an important role in the successful infection of M. ulcerans in humans.…”

“…Injection of mycolactone into guinea pig produced an ulcer similar to Buruli ulcer [4]. Mycolactone inhibits the production of various cytokines, chemokines, and other secreted immune modulators [7] from immune cells such as monocytes [8], macrophages [9], dendritic cells [10], and T cells [11][12][13]. When a mycolactone-deficient strain of M. ulcerans was injected into model animals, a granuloma was successfully developed and the bacteria were cleared from the infected regions [14,15], suggesting that mycolactone has a suppressive effect on cell-mediated immune responses.…”

Sec61 blockade by mycolactone inhibits antigen cross-presentation independently of endosome-to-cytosol export

“…Mycolactone is a polyketidederived macrolide produced by the human pathogen Mycobacterium ulcerans, which was recently identified as a potent Sec61 inhibitor (13)(14)(15)(16). Mycolactone diffuses passively across the plasma membrane to target the pore-forming subunit of the translocon (Sec61α) (13), leading to the proteasomal degradation of newly synthesized Sec61 clients blocked in translocation (17). In contrast to previously described Sec61 inhibitors, such as cotransin (18), mycolactone prevents the biogenesis of secretory and transmembrane proteins with minor selectivity toward Sec61 substrates, as well as uniformly high efficacy (13,16).…”

Shaping mycolactone for therapeutic use against inflammatory disorders

“…Contrary to wild-type strains, mycolactone-deficient mutants of M. ulcerans do not impair the capacity of T cells to produce interleukin-2 (IL-2) in animal models, showing that mycolactone is the cause of these cellular response defects (11). In vitro, nanomolar concentrations of mycolactone efficiently blocked the functional maturation of dendritic cells (12) and cytokine production by activated lymphocytes, monocytes, macrophages, and epithelial cells (13)(14)(15)(16)(17). Mycolactone also down-regulated the expression of L-selectin on naïve T cells, profoundly affecting the lymphocyte homing and expansion upon antigenic stimulation in vivo (18).…”