Mycobacterium tuberculosis EspK Has Active but Distinct Roles in the Secretion of EsxA and EspB.

The Mycobacterium tuberculosis type-7 protein secretion system ESX-1 is a major driver of its virulence. While the functions of most ESX-1 components are characterized, many others remain poorly defined. In this study, we examined the role of EspK, an ESX-1-associated protein that is thought to be dispensable for ESX-1 activity in members of the Mycobacterium tuberculosis complex. We show that EspK is needed for the timely and optimal secretion of EsxA and absolutely essential for EspB secretion in M. tuberculosis Erdman. We demonstrate that only the EsxA secretion defect can be alleviated in EspK-deficient M. tuberculosis by culturing it in media containing detergents like Tween 80 or tyloxapol. Subcellular fractionation experiments reveal EspK is exported by M. tuberculosis in an ESX-1-independent manner and localized to its cell wall. We also show a conserved W-X-G motif in EspK is important for its interaction with EspB and enabling its secretion. The same motif, however, is not important for EspK localization in the cell wall. Finally, we show EspB in EspK-deficient M. tuberculosis tends to adopt higher-order oligomeric conformations, more so than EspB in wild-type M. tuberculosis. These results suggest EspK interacts with EspB and prevents it from assembling prematurely into macromolecular complexes that are presumably too large to pass through the membrane-spanning ESX-1 translocon assembly. Collectively, our findings indicate M. tuberculosis EspK has a far more active role in ESX-1-mediated secretion than was previously appreciated and underscores the complex nature of this secretion apparatus. IMPORTANCE Mycobacterium tuberculosis uses its ESX-1 system to secrete EsxA and EspB into a host to cause disease. We show that EspK, a protein whose role in the ESX-1 machinery was thought to be nonessential, is needed by M. tuberculosis for optimal EsxA and EspB secretion. Culturing EspK-deficient M. tuberculosis with detergents alleviates EsxA but not EspB secretion defects. We also show that EspK, which is exported by M. tuberculosis in an ESX-1-independent manner to the cell wall, interacts with and prevents EspB from assembling into large structures inside the M. tuberculosis cell that are nonsecretable. Collectively, our observations demonstrate EspK is an active component of the ESX-1 secretion machinery of the tubercle bacillus.

Domestic pigs experimentally infected with Mycobacterium bovis and Mycobacterium tuberculosis exhibit different disease outcomes.

The domestic pig shares many similarities with humans in anatomy, physiology, and immunology. As such it is an attractive animal model to study human tuberculosis (TB). In this study, we examined disease outcome in pigs challenged via two different routes with either the human TB bacillus Mycobacterium tuberculosis Erdman (M. tb) or bovine TB bacillus M. bovis AF2122/97 in head-to-head comparisons. Pigs challenged intravenously with M. bovis exhibited greater morbidity and rapid onset of mortality, higher bacterial burden and tissue necrosis compared to pigs challenged similarly with M. tb. Concordantly, pigs challenged with aerosolized M. bovis exhibited reduced weight gain and more severe pathology than pigs challenged similarly with M. tb. Specifically, M. bovis challenged pigs presented a spectrum of granulomatous lung lesions similar to that in human TB. In contrast, pigs challenged with M. tb presented mostly early-stage granulomas. Irrespective of challenge dose and pathology however, peripheral IFN-γ responses were similar in both M. bovis and M. tb aerosol challenged pigs. Although M. bovis appears to be more virulent than M. tb, both can be used to model different facets of human TB in pigs, depending on whether one seeks to recapitulate active or latent forms of the disease.

Protein Synthesis and Degradation Inhibitors Potently Block Mycobacterium tuberculosis type-7 Secretion System ESX-1 Activity.

Mycobacterium tuberculosis (M. tb) uses its type-7 secretion system ESX-1 to translocate key virulence effector proteins. Taking a chemical genetics approach, we demonstrate for the first time the importance of mycobacterial proteostasis to ESX-1. We show that individual treatment with inhibitors of protein synthesis (chloramphenicol and kanamycin) and protein degradation (lassomycin and bortezomib), at concentrations that only reduce M. tb growth by 50% and less, specifically block ESX-1 secretion activity in the tubercle bacillus. In contrast, the mycobacterial cell-wall synthesis inhibitor isoniazid, even at a concentration that reduces M. tb growth by 90% has no effect on ESX-1 secretion activity. We also show that chloramphenicol but not isoniazid at subinhibitory concentrations specifically attenuates ESX-1-mediated M. tb virulence in macrophages. Taken together, the results of our study identify a novel vulnerability in the ESX-1 system and offer new avenues of anti-TB drug research to neutralize this critical virulence-mediating protein secretion apparatus.