Date Published: October 31, 2013
Publisher: Public Library of Science
Author(s): Alka Mehra, Aleena Zahra, Victor Thompson, Natalie Sirisaengtaksin, Ashley Wells, Maura Porto, Stefan Köster, Kristen Penberthy, Yoshihisha Kubota, Amelie Dricot, Daniel Rogan, Marc Vidal, David E. Hill, Andrew J. Bean, Jennifer A. Philips, Christopher M. Sassetti.
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.
An important virulence property of Mycobacterium tuberculosis (Mtb)- the causative agent of the disease tuberculosis- is its ability to avoid delivery to the lysosome. It has long been appreciated that Mtb alters phagosome maturation, such that internalized bacteria are not transported to the lysosome but instead reside in an early endosome-like compartment , . The Mtb-induced block in phagosome-lysosome fusion has been attributed to a wide array of lipid and protein effectors ,  but the mechanism remains poorly understood. More recently, the ability of Mtb to permeabilize the phagosomal membrane, which allows bacterial products and in some cases intact bacteria to access the cytosol, has been described –. The TSSS Esx-1 and its secreted effectors, EsxA/ESAT-6 and EsxB/CFP-10, are critical for this process. Esx-1 has been investigated intensively because its absence in the vaccine strain Mycobacterium bovis-BCG (BCG) largely accounts for attenuation of that strain –. Mtb encodes five loci resembling Esx-1 (Esx-1-Esx-5), as well as 11 tandem pairs of proteins similar to EsxA and EsxB (EsxA-EsxW), but their cellular targets, if any, are unknown . Esx-3 plays a role in iron acquisition in Mtb, as well as in a non-pathogenic strain, Mycobacterium smegmatis (Msmeg) , . Esx-3 is a focus of vaccine efforts because it secretes EsxG/TB9.8 and EsxH/TB10.4, which are highly antigenic , , and because introduction of the Mtb ESX-3 locus into an Msmeg strain lacking the endogenous ESX-3 region generates highly protective immunity . The ESX-5 locus is required for transport of proteins with conserved proline-glutamic acid (PE) and proline-proline-glutamic acid (PPE) motifs ,  and modulates macrophage responses . Thus, TSSSs and their putative effectors appear to be important in virulence and modulating host cells, however, their mechanism of action and molecular targets are unclear.
We used high throughput Y2H interactome mapping to identify interactions between secreted Mtb proteins and human proteins, identifying 99 new potential interactions. We made use of a large body of literature that has attempted to catalogue the secretome of Mtb. Our study is subject to the uncertainty around the definition of the Mtb secretome. For example, proteins can be in the culture filtrate due to bacterial lysis, rather than secretion, and bioinformatics predictions may be inaccurate. In addition, many secreted proteins play an intrinsic role in the bacterial lifecycle and are unlikely to make a biologically meaningful interaction with host proteins. Thus, to estimate a false positive hit rate of our system, we included a non-secreted control collection. We found approximately two-fold enrichment in the rate of interactions comparing the secretome collection to the control collection, suggesting that true interactions were identified, but that there also may be a relatively high rate of “pseudo-interactions,” which may be valid biophysically but never occur in vivo because the involved proteins are separated spatially or temporally. In addition, the interactome list is by no means complete. We did not screen the entire putative secretome, but rather imposed criteria to try to arrive at a set that was enriched for true secreted proteins likely to play a role in virulence. In addition, the screen was not performed to saturation, and only a fraction of verifiable interactions can be detected by a single method to detect PPIs . Therefore, the list is not comprehensive and likely contains false-positives, but given the paucity of data on host-pathogen interactions in Mtb, it has likely significantly expanded the known Mtb-human protein-protein interactome. It represents a resource for investigators working on Mtb; the confirmation and significance of such interactions will require further validation.
Detailed methods, including description of Y2H interaction mapping, plasmids, siRNAs, and Hill plot analysis, are provided in Text S1.