Tuberculosis is an infernal machine, and it keeps spreading thanks to the operations of many tiny infernal machines—not the pathogens, mind you. Look more closely. Each tiny Mycobacterium tuberculosis pathogen is studded with even tinier machines, secretion machines. Embedded in the pathogen’s membrane, these secretion machines inject virulence factors into the cells of the immune system, helping M. tuberculosis evade the host’s defenses.

If the smooth operation of secretion machines were disrupted, scientists have reasoned, then the spread of tuberculosis, one of the world’s top ten causes of death, might grind to a halt. But lacking blueprints for secretion machines, scientists hardly knew where to look for the machines’ vulnerabilities.

Now something like blueprints are in hand, thanks to the work of scientists from the University of Würzburg and the Spanish Cancer Research Centre (the Centro Nacional de Investigaciones Oncológicas, or CNIO). Using cryo-electron microscopy and digital image processing, these scientists have uncovered the molecular architecture of a representative secretion machine, a type VII secretion system (T7SS) common to M. tuberculosis and M. smegmatis.

Previously, only very low-resolution structural information was available for T7SS, which appeared to be a hexameric complex with a central channel through which virulence factors could be ejected. Atomic-level details, however, were lacking. And so, it remained unclear how new therapeutic strategies against tuberculosis could be developed based on attacking the secretion system.

The new structural information is much clearer. In fact, it is at a resolution of 3.7 Å, showing elements of the nanomachine that form the transport pore, as well as elements that convert chemical energy into motion and thus drive the transport of effector proteins through the pore. These and other details were described in a paper (“Architecture of the mycobacterial type VII secretion system”) that appeared October 9 in Nature.

“The core of the secretion machine consists of four protein components, EccB3:EccC3:EccD3:EccE3 in a 1:1:2:1 stoichiometry, building two identical protomers,” the article’s authors indicated. “The EccC3 coupling protein comprises a flexible array of four ATPase domains, which are linked to the membrane through a stalk domain. The ‘domain of unknown function’ (DUF) adjacent to the stalk is identified as an ATPase domain essential for secretion.

“EccB3 is predominantly periplasmatic, but a small segment crosses the membrane and contacts the stalk domain, suggesting that conformational changes in the stalk domain triggered by substrate binding at the distal end of EccC3 and subsequent ATP hydrolysis in the DUF could be coupled to substrate secretion to the periplasm.”

Over the past five years, a research group at the University of Würzburg led by Sebastian Geibel, PhD, has worked intensively on the stable reconstitution of a secretion machine and the preparation of samples that could be subjected to cryo-electron microscopy, which requires the protein complexes to be shock frozen under defined conditions. Images of the samples were processed by a CNIO research group led by Oscar Llorca, PhD. The resulting three-dimensional maps of the protein complex allowed the collaborators to create a model of the secretion machine’s molecular structure.

“We were able to see that the components that until now appeared blurred with other techniques are in fact elements that are in constant motion,” explained Llorca. “Thus, we saw that the hexamer of T7SS is composed of a subcomplex of four proteins and that six identical copies of this subcomplex are needed to shape the six-pointed star around a central pore, through which the virulence factors that block the defensive response of the infected organism are ejected.”

Subsequently, the proposed mechanism was successfully tested by the Würzburg University group using different mutated versions of the system. The system used by the German group to test the mechanism is also of great interest to the research community: “It will be very useful to test the effect of new molecules directed against this secretion mechanism, which the bacteria of the genus Mycobacterium need to successfully carry out the infection,” noted Ángel Rivera-Calzada, a member of Llorca’s team and a co-author of the current study.

The new findings may help researchers develop new ways of fighting bacterial infections. Given the rise of antibiotic resistance and the lack of effective vaccines against tuberculosis, researchers may resort to developing molecules that could disrupt the assembly or function of secretion systems.

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