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

Mehra A, Zahra A, Thompson V, Sirisaengtaksin N, Wells A, Porto M, Köster S, Penberthy K, Kubota Y, Dricot A, Rogan D, Vidal M, Hill DE, Bean AJ, Philips JA - PLoS Pathog. (2013)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Division of Infectious Diseases, Department of Medicine, Department of Pathology and Department of Microbiology, New York University School of Medicine, New York, New York, United States of America.

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.

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3814348&req=5

ppat-1003734-g003: 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.

Mentions: To determine whether EsxHMt interacts with Hrs and alters ESCRT function in mammalian cells, we expressed EsxHMt–FLAG in HEK293 cells. EsxHMt was not detectable unless we co-expressed EsxGMt. (Figure 3A, compare lanes 1 and 3; see Figure S4 for quantification); its abundance was also increased slightly by overexpression of Hrs (compare lane 1′ with 2′ and lane 5 with 6). When expressed alone, EsxHMt could be stabilized by MG132, likely because it is not properly folded without EsxGMt and hence is subject to proteasome-mediated degradation (Figure 3A compare lanes 1 and 5). To determine if there was an interaction between EsxGMt-EsxHMt and Hrs, we performed co-immunoprecipitation experiments in cells co-transfected with EsxGMt, EsxHMt, and Hrs-myc. Hrs was immunoprecipitated with an antibody directed against the myc-tag, and we found that EsxHMt was co-immunoprecipitated (Figure 3B). No EsxHMt was co-immunoprecipitated when an isotype control antibody was used, and as expected, EsxHMs and EsxHMt-H76A E77A were impaired in the interaction with Hrs (Figure 3B and 3C). Interestingly, the co-immunoprecipitation of EsxHMt and Hrs could only be detected when cells were pre-treated with MG132. Thus, one possibility is that the EsxGMt-EsxHMt heterodimer is polyubiquitinated and degraded by the proteasome. In the presence of MG132, the polyubiquitinated species might accumulate, allowing us to detect an interaction between Hrs and an ubiquitinated species of EsxHMt, since Hrs contains an ubiquitin interacting motif (UIM) domain. Arguing against this possibility, when EsxGMt was co-expressed with EsxHMt, there was little, if any, effect of MG132 on EsxHMt protein levels (Figure 3A, compare lanes 3 and 7, Figure S4, and Figure S5). In addition, when we examined mono- and polyubiquitinated proteins using the FK2 antibody, inhibition of the proteasome with MG132 caused the accumulation of high molecular weight proteins as anticipated. However, there was no difference seen in the quantity or mobility of EsxHMt (Figure S5). Further, when we mapped the region of Hrs required for the interaction with EsxGMt-EsxHMt in the Y2H assay, the UIM domain was not required. Amino acids 398–630, which contain a coiled-coil region, were sufficient to mediate the interaction (Figure 2E). We verified that the C-terminal half of Hrs was sufficient to mediate an interaction by co-immunoprecipitation (Fig. 3D). In summary, these data show that EsxGMt stabilizes EsxHMt in the mammalian cytosol and that the heterodimer can bind the C-terminus of Hrs.


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

Mehra A, Zahra A, Thompson V, Sirisaengtaksin N, Wells A, Porto M, Köster S, Penberthy K, Kubota Y, Dricot A, Rogan D, Vidal M, Hill DE, Bean AJ, Philips JA - PLoS Pathog. (2013)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3814348&req=5

ppat-1003734-g003: 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.
Mentions: To determine whether EsxHMt interacts with Hrs and alters ESCRT function in mammalian cells, we expressed EsxHMt–FLAG in HEK293 cells. EsxHMt was not detectable unless we co-expressed EsxGMt. (Figure 3A, compare lanes 1 and 3; see Figure S4 for quantification); its abundance was also increased slightly by overexpression of Hrs (compare lane 1′ with 2′ and lane 5 with 6). When expressed alone, EsxHMt could be stabilized by MG132, likely because it is not properly folded without EsxGMt and hence is subject to proteasome-mediated degradation (Figure 3A compare lanes 1 and 5). To determine if there was an interaction between EsxGMt-EsxHMt and Hrs, we performed co-immunoprecipitation experiments in cells co-transfected with EsxGMt, EsxHMt, and Hrs-myc. Hrs was immunoprecipitated with an antibody directed against the myc-tag, and we found that EsxHMt was co-immunoprecipitated (Figure 3B). No EsxHMt was co-immunoprecipitated when an isotype control antibody was used, and as expected, EsxHMs and EsxHMt-H76A E77A were impaired in the interaction with Hrs (Figure 3B and 3C). Interestingly, the co-immunoprecipitation of EsxHMt and Hrs could only be detected when cells were pre-treated with MG132. Thus, one possibility is that the EsxGMt-EsxHMt heterodimer is polyubiquitinated and degraded by the proteasome. In the presence of MG132, the polyubiquitinated species might accumulate, allowing us to detect an interaction between Hrs and an ubiquitinated species of EsxHMt, since Hrs contains an ubiquitin interacting motif (UIM) domain. Arguing against this possibility, when EsxGMt was co-expressed with EsxHMt, there was little, if any, effect of MG132 on EsxHMt protein levels (Figure 3A, compare lanes 3 and 7, Figure S4, and Figure S5). In addition, when we examined mono- and polyubiquitinated proteins using the FK2 antibody, inhibition of the proteasome with MG132 caused the accumulation of high molecular weight proteins as anticipated. However, there was no difference seen in the quantity or mobility of EsxHMt (Figure S5). Further, when we mapped the region of Hrs required for the interaction with EsxGMt-EsxHMt in the Y2H assay, the UIM domain was not required. Amino acids 398–630, which contain a coiled-coil region, were sufficient to mediate the interaction (Figure 2E). We verified that the C-terminal half of Hrs was sufficient to mediate an interaction by co-immunoprecipitation (Fig. 3D). In summary, these data show that EsxGMt stabilizes EsxHMt in the mammalian cytosol and that the heterodimer can bind the C-terminus of Hrs.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Division of Infectious Diseases, Department of Medicine, Department of Pathology and Department of Microbiology, New York University School of Medicine, New York, New York, United States of America.

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.

Show MeSH
Related in: MedlinePlus