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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.

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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.
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ppat-1003734-g001: 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.

Mentions: We focused on the interaction between EsxH and Hrs because TSSSs, which secrete Esx proteins, are clearly important in virulence but the function of their secreted effectors is largely unknown. In addition, our existing data supported the idea that the ESCRT machinery is important in controlling bacterial replication. Hrs, which plays a central role in the assembly of the initial ESCRT components on endosomes, is recruited to mycobacterial phagosomes [39], and we had previously shown in an RNAi screen in Drosophila that ESCRT restricts the intracellular growth of rapidly growing mycobacteria [40], [41]. Control of bacterial replication appears to be particularly sensitive to ESCRT perturbation, because, in addition, when we screened ∼6500 siRNA pools in RAW 264.7 (RAW) macrophages for their ability to confer enhanced intracellular growth of Msmeg, we found that the two strongest hits were Rab7, known to be involved in late endosome-lysosome fusion, and Tsg101, an ESCRT-I component that is recruited to endosomes by Hrs (data not shown). Hrs was also identified in this screen, although previously we had found no effect with Hrs silencing, which we now attribute to insufficient protein depletion [41]. In the RAW cell RNAi screen that identified Hrs, we used Ambion Silencer siRNA pools, whereas previously we used a Dharmacon siGENOME pool to deplete Hrs [41]. To clarify the discrepancy, we tested a third pool (Dharmacon ON-TARGETplus), which, like the Ambion pool, conferred enhanced growth to Msmeg. We tested the individual Dharmacon ON-TARGETplus siRNAs and found that 2 of 4 targeting Hrs resulted in depletion of Hrs protein, enhanced the growth of Msmeg, and altered trafficking, whereas the other two had no effect (Figure S1 and data not shown). Thus, one possibility is that Mtb secretes EsxH, which binds Hrs and impairs ESCRT function, thereby promoting intracellular bacterial growth. To determine whether ESCRT restricts growth of Mtb, we depleted Hrs and Tsg101 and examined the intracellular growth of Mtb in RAW macrophages. We found no significant effect of silencing on bacterial uptake (data not shown), however when we assessed bacterial colony forming units (CFU) two day post-infection, we observed enhanced intracellular survival of Mtb in cells depleted of Hrs or Tsg101, similar to what was seen with Rab7 silencing (Figure 1A). Intracellular growth of BCG in bone marrow-derived macrophages (BMDMs) was even more strongly effected (Figure S2). Thus, Hrs restricts growth of slow growing and virulent mycobacteria.


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)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3814348&req=5

ppat-1003734-g001: 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.
Mentions: We focused on the interaction between EsxH and Hrs because TSSSs, which secrete Esx proteins, are clearly important in virulence but the function of their secreted effectors is largely unknown. In addition, our existing data supported the idea that the ESCRT machinery is important in controlling bacterial replication. Hrs, which plays a central role in the assembly of the initial ESCRT components on endosomes, is recruited to mycobacterial phagosomes [39], and we had previously shown in an RNAi screen in Drosophila that ESCRT restricts the intracellular growth of rapidly growing mycobacteria [40], [41]. Control of bacterial replication appears to be particularly sensitive to ESCRT perturbation, because, in addition, when we screened ∼6500 siRNA pools in RAW 264.7 (RAW) macrophages for their ability to confer enhanced intracellular growth of Msmeg, we found that the two strongest hits were Rab7, known to be involved in late endosome-lysosome fusion, and Tsg101, an ESCRT-I component that is recruited to endosomes by Hrs (data not shown). Hrs was also identified in this screen, although previously we had found no effect with Hrs silencing, which we now attribute to insufficient protein depletion [41]. In the RAW cell RNAi screen that identified Hrs, we used Ambion Silencer siRNA pools, whereas previously we used a Dharmacon siGENOME pool to deplete Hrs [41]. To clarify the discrepancy, we tested a third pool (Dharmacon ON-TARGETplus), which, like the Ambion pool, conferred enhanced growth to Msmeg. We tested the individual Dharmacon ON-TARGETplus siRNAs and found that 2 of 4 targeting Hrs resulted in depletion of Hrs protein, enhanced the growth of Msmeg, and altered trafficking, whereas the other two had no effect (Figure S1 and data not shown). Thus, one possibility is that Mtb secretes EsxH, which binds Hrs and impairs ESCRT function, thereby promoting intracellular bacterial growth. To determine whether ESCRT restricts growth of Mtb, we depleted Hrs and Tsg101 and examined the intracellular growth of Mtb in RAW macrophages. We found no significant effect of silencing on bacterial uptake (data not shown), however when we assessed bacterial colony forming units (CFU) two day post-infection, we observed enhanced intracellular survival of Mtb in cells depleted of Hrs or Tsg101, similar to what was seen with Rab7 silencing (Figure 1A). Intracellular growth of BCG in bone marrow-derived macrophages (BMDMs) was even more strongly effected (Figure S2). Thus, Hrs restricts growth of slow growing and virulent mycobacteria.

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