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. 2013 Oct;9(10):e1003734.
doi: 10.1371/journal.ppat.1003734. Epub 2013 Oct 31.

Mycobacterium tuberculosis type VII secreted effector EsxH targets host ESCRT to impair trafficking

Alka Mehra  1 , 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
Affiliations

Mycobacterium tuberculosis type VII secreted effector EsxH targets host ESCRT to impair trafficking

Alka Mehra et al. PLoS Pathog. 2013 Oct.

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.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. ESCRT is required to traffic Mtb to the lysosome.
(A) RAW264.7 cells were treated with control siRNA (Con), individual siRNAs targeting Hrs (#9 or #12), or siRNA pools targeting Tsg101 or Rab7 and infected with Mtb. Bacterial colony forming units (CFU) were enumerated 2 d post-infection and are normalized to the average number of CFU in control wells from three independent experiments. Results reflect the mean +/− SEM. *p = 0.018; ***p = 0.0002; ****p<0.0001, unpaired Student's t-test. (B) Composite images and quantification of Mtb-GFP or BCG-GFP (in green) and RAW cell LAMP1, TfR, or LysoTracker (in red) at 24 hpi. Regions indicated by yellow circles are shown in higher magnification in adjacent panels. In graphs, data points are the mean fluorescence intensity (MFI) around at least 100 phagosomes for each condition; bars show mean +/− SEM. Data are representative of at least three experiments; p<0.0001 for all siRNAs compared to controls.
Figure 2
Figure 2. EsxHMt binds Hrs.
(A) Gal4 DNA-binding domain (DB) fusions of EsxHMt, EsxHMs, or mutant EsxHMt were tested for Y2H interactions with Gal4 activation-domain (AD) fusions of EsxGMt, human (Hs), mouse (Mm), or zebrafish (Dr) Hrs. (B) Y2H interaction between indicated DB and AD constructs. Hrs is human. EsxGMt-EsxHMt-DB did not interact with EsxGMt-AD or EsxHMt-AD, presumably because of the intramolecular interaction in the DB construct. (C) Increasing amounts of Hrs were incubated with a constant amount of immobilized EsxGMt-EsxHMt and bound fraction examined by Coomassie blue. (D) Average binding (n = 3) was fitted with the Hill function, revealing a Hill coefficient of ∼1.7 and a KD of 5.4 µM. (E) EsxGMt-EsxHMt-DB was tested in the Y2H for interactions with human Hrs-AD deletion constructs. The domain structure of Hrs is indicated.
Figure 3
Figure 3. EsxGMt and EsxHMt interact with Hrs and disrupt ESCRT function in mammalian cells.
(A) EsxHMt-FLAG, EsxGMt-His, and Hrs-myc expressed in HEK293 cells. DMSO or MG132 were added 3 h prior to protein harvest and samples were analyzed by western blotting. Lanes 1′ and 2′ are identical to 1 and 2 except that twice the amount of protein was loaded. Quantification from three independent experiments is shown in Figure S4. (B) Immunoprecipitation (IP) of Hrs using antibody recognizing myc tag or isotype control from HEK293 cells expressing Hrs-myc and either EsxGMt-His EsxHMt-FLAG or EsxGMs-His EsxHMs-FLAG. MG132 was used as pre-treatment. Western blot of IP and input were probed with antibodies as indicated. (C) IP of Hrs-myc in HEK293 cells with antibody recognizing myc tag or isotype control from HEK293 cells expressing Hrs-myc, EsxGMt-His, and either EsxHMt-FLAG or EsxHMt-H76A-E77A-FLAG. MG132 was used as pre-treatment. Western blot of IP and input were probed with antibodies as indicated. (D) IP of C-terminal fragment of Hrs (amino acids 398–777) using antibody recognizing V5-tag or isotype control from HEK293 cells expressing Hrs-398–777-V5, EsxGMt-His, EsxHMt-FLAG. MG132 was used as pre-treatment. Western blot of IP and input were probed with antibodies as indicated. (E) HEK293 cells transfected with indicated plasmids were incubated with EGF for 0 or 90 min prior to western analysis. (F–H) A549 cells transfected with plasmids or siRNAs were imaged 90 min after incubation with Alexa-488 EGF. In F, white lines indicate cell borders. (G) and (H), MFI of at least 800 endosomes from at least 30 cells. Black bars show mean +/− SEM. ****p<0.0001 between indicated conditions, unpaired Student's t-test. No MG132 was used in experiments E–H. Data are representative of at least three independent experiments.
Figure 4
Figure 4. EsxGMt EsxHMt arrests phagosome maturation.
(A) H37Rv transformed with empty vector, EsxGMt EsxHMt-FLAG, EsxGMs EsxHMs-FLAG or EsxGMt EsxHMt- H76A-E77A-FLAG were analyzed for the presence of EsxH in the pellet and culture filtrate (CF). DlaT (Rv2215), a cytosolic protein, was used as a loading control and to indicate the degree of bacterial lysis. (B) MFI of phagosomal LAMP1 24 hpi in RAW cells treated with siRNAs and infected with Mtb containing EsxGMt-EsxHMt plasmid (red) or vector control (black); data points are the mean fluorescence intensity (MFI) around at least 70 phagosomes for each condition; p<0.0001 between the two Mtb strains for all conditions, unpaired Student's t-test. (C) Composite images of cells infected with Mtb with autofluorescence of Mtb (blue) and LAMP1 (red). Regions indicated by yellow circles are shown in higher magnification in adjacent panels. (D) MFI of phagosomal TfR 24 hpi in RAW cells treated with siRNAs and infected with Mtb containing EsxGMt-EsxHMt plasmid (red) or vector control (black); data points are the mean fluorescence intensity (MFI) around at least 50 phagosomes for each condition; p<0.0001 between the two Mtb strains for all conditions, except Rab7 (p = 0.0005), unpaired Student's t-test. (E) Composite images of cells infected with Mtb with autofluorescence of Mtb (blue) and TfR (red). Regions indicated by yellow circles are shown in higher magnification. (F) MFI of phagosomal LAMP1 in RAW cells infected with Mtb containing the indicated plasmids 24 hpi. Bars show mean +/− SEM. ****p<0.0001, unpaired Student's t-test.
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