The new protein transport system mediates the excretion of toxins by the causative agent of tuberculosis

Six years ago, Dr. Michael Niederweis, described the first toxin ever found for the deadly pathogen Mycobacterium tuberculosis.

This toxin, a toxin that necrotizes tuberculosis, or TNT, has become the founder of a new class of previously unrecognized toxins present in more than 600 bacterial and fungal species, as determined by the similarity of the protein sequence.

The toxin is released as the bacterium M. tuberculosis survives and grows within its human macrophage host, killing the macrophage and allowing the bacteria to escape and spread.

For 132 years, the lack of the identified toxin in M. tuberculosis was at odds with almost all other pathogenic bacteria whose toxins contribute to disease or death. M. tuberculosis infects 9 million people a year and kills more than a million.

Now, in another groundbreaking paper, researchers at the University of Alabama at Birmingham and colleagues describe how two small ESX proteins derived from M. tuberculosis mediate TNT secretion by creating pores in the membranes that envelop the bacteria.

This finding can be widely used because the recognizable three amino acid motif found on EsxE and EsxF – tryptophan / any amino acid / glycine, known by the acronym WXG – is found on many other small mycobacterial proteins and on the large super family WXG100 bacterial proteins resembling EsxE and EsxF.

Here, for the first time, we show that small Esx proteins from the WXG100 family have an important molecular function within the Mtb cell through the secretion of toxins. Our results suggest a dynamic pore-forming mechanism with small Esx proteins that could be applicable to other members of the large WXG100 protein family. Thus, our study not only represents a major advance in our understanding of the secretion of TNT and possibly other proteins in M. tuberculosis, but also describes the biological function of Esx-paralogs in M. tuberculosis and their homologues in the large WXG100 protein family in gram-positive bacteria.. “

Micheal Niederweis, Professor, Department of Microbiology, University of Alabama at Birmingham

TNT is one of two domains in the outer membrane protein of M. tuberculosis CpnT; the activity of the TNT domain of CpnT in the cytosol of macrophages induces macrophage death by NAD + hydrolysis. M. tuberculosis has an inner and an outer membrane, and the protein needs to pass through each layer to be secreted outside the bacterium. It is not known how CpnT reaches the outer membrane.

EsxE and EsxF are part of the same gene segment as CpnT, and UAB researchers hypothesized that two small proteins could be involved in toxin secretion.

Creating different strains lacking EsxE or EsxF, they showed that both proteins are necessary for the translocation of CpnT to the cell surface of M. tuberculosis and for the secretion of TNT into the cytosol of macrophages infected with M. tuberculosis. Furthermore, EsxE and EsxF are surface-available proteins on M. tuberculosis as a membrane-bound complex.

To learn more about the mechanism of this translocation, the UAB team made mutants of each Esx protein, where the amino acid tryptophan of the individual WXG motif on each protein was replaced with the amino acid alanine. The mutants showed that intact WXG motif on EsxE and on EsxF is required for efficient translocation of CpnT to the outer membrane of M. tuberculosis and subsequent secretion of TNT into the cytosol of infected macrophages.

Purification of the water-soluble proteins EsxE and EsxF showed that they formed EsxE-EsxF dimers, and five of these dimers were assembled into star-shaped structures, as seen by electron microscopy. Each was about 10 nanometers wide, with a 3-nanometer central pore.

Experiments with flat bilayer lipids were key to understanding the molecular function of EsxE-EsxF, as they showed that EsxE-EsxF pores form channels through lipid membranes.

Finally, the researchers showed that WXG motifs were required for pore formation and membrane disruption by the EsxE-EsxF complex, and the motifs mediated a set of functional EsxE-EsxF oligomers. This now defines the biochemical role of the previously enigmatic WXG motif.

“EsxE and EsxF form the first known components of the outer membrane that mediate protein secretion in M. tuberculosis,” Niederweis said. “However, EsxE and EsxF are unlikely to be sufficient for TNT secretion because an energy source is required in all known bacterial protein secretion systems. It is therefore possible for EsxE-EsxF to bind to other proteins or protein complexes to achieve export of CpnT and TNT secretions. “

UAB researchers propose two models for transporting CpnT EsxE and EsxF. In the first, EsxE-EsxF heterodimers form pores in the inner membrane and then form a second pore in the outer membrane to create transmembrane channels.

“Alternatively,” Niederweis said, “the inner membrane channel is extended to encompass the periplasm through filament formation and connects to EsxE-EsxF pores in the outer membrane, exposing EsxF to the cell surface. In this model, the alleged EsxE-EsxF channel tunnel allows export CpnT polypeptide to the outer membrane of M. tuberculosis and subsequent secretion of TNT and EsxE-EsxF. “

Co-authors with Niederweis in the study, “Esx pore-forming proteins mediate the excretion of Mycobacterium tuberculosis toxins,” published in Nature Communications, are Uday Tak and Terje Dokland, Department of Microbiology, UAB.

“This work was an outstanding achievement of outstanding graduate student Uday Tak, who performed almost all of these experiments on his own,” Niederweis said. Uday Tak received his doctorate in November 2020 and is now a postdoctoral fellow at the University of Colorado-Boulder.

This paper was selected by the Editor-in-Chief as a prominent event and was published on a special website entitled “Microbiology and Infectious Diseases”.


University of Alabama at Birmingham

Journal reference:

Tak, U., and others. (2021) Esx pore-forming proteins mediate toxin secretion Mycobacterium tuberculosis. Nature Communications.