Mycobacterium tuberculosis Type VII Secreted Effector EsxH Targets Host ESCRT to Impair Trafficking

Abstract

Mycobacterium tuberculosis (Mtb) disrupts anti-microbial pathways of macrophages, cells that normally kill bacteria. Over 40 years ago, D'Arcy Hart showed that Mtb avoids delivery to lysosomes, but the molecular mechanisms that allow Mtb to elude lysosomal degradation are poorly understood. Specialized secretion systems are often used by bacterial pathogens to translocate effectors that target the host, and Mtb encodes type VII secretion systems (TSSSs) that enable mycobacteria to secrete proteins across their complex cell envelope; however, their cellular targets are unknown. Here, we describe a systematic strategy to identify bacterial virulence factors by looking for interactions between the Mtb secretome and host proteins using a high throughput, high stringency, yeast two-hybrid (Y2H) platform. Using this approach we identified an interaction between EsxH, which is secreted by the Esx-3 TSSS, and human hepatocyte growth factor-regulated tyrosine kinase substrate (Hgs/Hrs), a component of the endosomal sorting complex required for transport (ESCRT). ESCRT has a well-described role in directing proteins destined for lysosomal degradation into intraluminal vesicles (ILVs) of multivesicular bodies (MVBs), ensuring degradation of the sorted cargo upon MVB-lysosome fusion. Here, we show that ESCRT is required to deliver Mtb to the lysosome and to restrict intracellular bacterial growth. Further, EsxH, in complex with EsxG, disrupts ESCRT function and impairs phagosome maturation. Thus, we demonstrate a role for a TSSS and the host ESCRT machinery in one of the central features of tuberculosis pathogenesis.

Role of Metal-Dependent Regulation of ESX-3 Secretion in Intracellular Survival of Mycobacterium tuberculosis

“…Interestingly, esxG and esxH play a role in virulence beyond their requirement for iron acquisition (20). Consistent with these findings, we previously showed that the EsxG-EsxH complex from M. tuberculosis, but not that from the M. smegmatis EsxG-EsxH complex, targets the host hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), a component of the endosomal sorting complexes required for transport (ESCRT) (28). M. tuberculosis EsxG-EsxH inhibits ESCRT, which is required for phagosome maturation.…”

“…EsxG Mt and EsxH Mt were expressed in tandem as a single polypeptide separated by a linker (GLVPRGSTG) as previously described (28 8.0) and incubated at room temperature for 40 min, followed by centrifugation at 10,000 ϫ g for 30 min at 4°C. The supernatant was incubated with nickel-nitrilotriacetic acid (Ni-NTA) Superflow resin (Qiagen) at 4°C for 1.5 h. The Ni-NTA resin was washed with lysis buffer containing up to 60 mM imidazole, followed by elution in lysis buffer containing 250 mM imidazole.…”

The ubiquitin system: a critical regulator of innate immunity and pathogen–host interactions

“…42 Importantly, perturbation of this process adversely affects phagosomal maturation and pathogen eradication. 43 The ESCRT (endosomal sorting complex required for transport) machinery consists of four complexes, ESCRT-0, -I, -II and -III, which operate sequentially to mediate MVB formation via three distinct but connected steps: capturing the ubiquitin-tagged cargo, deforming the phagosomal/endosomal membrane and membrane abscission from the inside. 44 The ubiquitin system has a central role in ESCRT-mediated MVB biogenesis.…”

Manipulation of the Mononuclear Phagocyte System by Mycobacterium tuberculosis

“…Recently, it was shown that the secreted tyrosine phosphatase PtpA directly binds to the H subunit of the v-ATPase, resulting in the dephosphorylation of vacuolar protein sorting protein 33B (VPS33B) and subsequent impairment of v-ATPase recruitment to the vacuole (Wong et al 2011). It was also shown that M. tuberculosis uses its type VII secretion system ESX-3 to export two effector proteins-namely, EsxH and EsxG-that interact with Hrs and impair phagosome maturation through disrupting the endosomal sorting complex required for transport (ESCRT) (Mehra et al 2013). Other mycobacterial secreted proteins are proposed to interfere with the host cell vacuolar sorting machinery, as suggested, for example, by the aberrant trafficking of a M. tuberculosis mutant inactivated in the alternative secretion system component SecA2 (Sullivan et al 2012).…”