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Mycobacterium tuberculosis PE25/PPE41 protein complex induces necrosis in macrophages: Role in virulence and disease reactivation?

Tundup S, Mohareer K, Hasnain SE - FEBS Open Bio (2014)

Bottom Line: Necrotic cell death during TB infection is an important prerequisite for bacterial dissemination and virulence.The underlying mechanisms and the bacterial factors involved therein are not well understood.The Mycobacterium tuberculosis (M. tuberculosis) co-operonic PE25/PPE41 protein complex, similar to ESAT-6/CFP-10, belonging to the PE/PPE and ESAT-6 families of genes has co-expanded and co-evolved in the genomes of pathogenic mycobacteria.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, University of Chicago, Chicago, IL, 60637, USA.

ABSTRACT
Necrotic cell death during TB infection is an important prerequisite for bacterial dissemination and virulence. The underlying mechanisms and the bacterial factors involved therein are not well understood. The Mycobacterium tuberculosis (M. tuberculosis) co-operonic PE25/PPE41 protein complex, similar to ESAT-6/CFP-10, belonging to the PE/PPE and ESAT-6 families of genes has co-expanded and co-evolved in the genomes of pathogenic mycobacteria. We report a novel role of this highly immunogenic PE25/PPE41 protein complex in inducing necrosis, but not apoptosis, in macrophages. We propose that these protein complexes of M. tuberculosis, secreted by similar/unique transport system (Type VII), have an important role in M. tuberculosis virulence and disease reactivation.

No MeSH data available.


Related in: MedlinePlus

The PE25/PPE41 complex induces necrosis, but not apoptosis. Necrosis of macrophages stimulated with the recombinant protein was monitored by propidium iodide (PI) exclusion assay. (A) RAW264.7 macrophages were stimulated with different concentrations of the PE25/PPE41 protein complex, as indicated. The peaks in the M2 region represent the uptake of PI dye by the necrotic cells when stimulated with increased concentration of the recombinant PE25/PPE41 protein complex. The number mentioned in M2 region of each of the graph represents the percentage of cells undergoing necrosis in response to the stimulation with PE25/PPE41 protein complex in a dose dependent manner. Data represent results of 3 independent experiments. (B) RAW264.7 macrophages were stimulated with 10 μg/ml of the PE25/PPE41 protein complex for 0, 2, 8 and 12 h, as indicated. Staining with PI/Annexin-V was carried out, cells acquired and data analyzed on flow cytometer (BD Vantage, SE). Necrotic (PI+/Annexin V+) or apoptotic (PI−Annexin V+) populations were gated and compared between cells stimulated for 0, 2, 8 and 12 h. Statistical significance was determined using student’s t test (∗∗∗p < 0.001). (C) TUNEL assay with macrophages stimulated with 10 μg/ml of recombinant PE25/PPE41 protein complex show absence of TUNEL staining in the presence of the PE25/PPE41 complex. Positive control (cells treated with DNase A) shows staining with fluorescein tagged nucleotides at the nicked ends of nucleotides. DAPI staining shows that the cells were alive.
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f0020: The PE25/PPE41 complex induces necrosis, but not apoptosis. Necrosis of macrophages stimulated with the recombinant protein was monitored by propidium iodide (PI) exclusion assay. (A) RAW264.7 macrophages were stimulated with different concentrations of the PE25/PPE41 protein complex, as indicated. The peaks in the M2 region represent the uptake of PI dye by the necrotic cells when stimulated with increased concentration of the recombinant PE25/PPE41 protein complex. The number mentioned in M2 region of each of the graph represents the percentage of cells undergoing necrosis in response to the stimulation with PE25/PPE41 protein complex in a dose dependent manner. Data represent results of 3 independent experiments. (B) RAW264.7 macrophages were stimulated with 10 μg/ml of the PE25/PPE41 protein complex for 0, 2, 8 and 12 h, as indicated. Staining with PI/Annexin-V was carried out, cells acquired and data analyzed on flow cytometer (BD Vantage, SE). Necrotic (PI+/Annexin V+) or apoptotic (PI−Annexin V+) populations were gated and compared between cells stimulated for 0, 2, 8 and 12 h. Statistical significance was determined using student’s t test (∗∗∗p < 0.001). (C) TUNEL assay with macrophages stimulated with 10 μg/ml of recombinant PE25/PPE41 protein complex show absence of TUNEL staining in the presence of the PE25/PPE41 complex. Positive control (cells treated with DNase A) shows staining with fluorescein tagged nucleotides at the nicked ends of nucleotides. DAPI staining shows that the cells were alive.

Mentions: In order to differentiate necrotic cell death from apoptosis, propidium iodide (PI)/Annexin V staining was carried out using macrophages stimulated with PE25/PPE41 complex (Fig. 4A and B). Propidium iodide easily passes through the ruptured membrane of dead cells and stains nucleic acids, but live cells or cells in early apoptotic phase are impermeable to PI dye. Unlike PI, Annexin V binds to phosphatidylserine with high affinity, which is externalized on the surface of apoptosed or dead cells, thus recognizing cells undergoing apoptosis even at early stage of cell death. Using flow cytometry staining, it could be seen that while PI uptake by the macrophages was a direct function of increasing concentration of the PE/PPE complex (Fig. 4A), the necrotic population of macrophages (PI+/AnnexinV+) increased at 2, 8 and 12 h of incubation, respectively (Fig. 4B). Absence of apoptotic population (PI−/AnnexinV+) suggests that PE/PPE protein complex induces necrosis of macrophages but not apoptosis (Fig. 4B). These results clearly suggest that the PE25/PPE41 protein complex induces necrosis in macrophages in a time dependent manner. Apoptotic cell death was also ruled out by TUNNEL staining. Macrophages incubated with PE25/PPE41 complex at a concentration as high as 10 μg/ml were negative for TUNEL staining (Fig. 4C). Necrotic cell death was further validated by lactate dehydrogenase (LDH) release assay. LDH assay involves measurement of NADH oxidation to NAD by LDH (released in to the medium from ruptured cells) in presence of pyruvic acid. RAW264.7 macrophages were stimulated with PE25/PPE41 at two different concentrations of PE25/PPE41 protein complex and LDH released was measured in the culture supernatant. It could be seen that the PE25/PPE41 protein complex induced release of LDH into the supernatant of RAW264.7 macrophages in a concentration dependent manner (Fig. 5). Very low oxidation of NADH was seen in the supernatant of cells left unstimulated (Fig. 5) or stimulated with concentration as high as 5 μg/ml of LPS (Fig. 5). Taken together these results demonstrate that the PE25/PPE41 protein complex induces necrosis, but not apoptosis, in RAW 264.7 macrophage cells.


Mycobacterium tuberculosis PE25/PPE41 protein complex induces necrosis in macrophages: Role in virulence and disease reactivation?

Tundup S, Mohareer K, Hasnain SE - FEBS Open Bio (2014)

The PE25/PPE41 complex induces necrosis, but not apoptosis. Necrosis of macrophages stimulated with the recombinant protein was monitored by propidium iodide (PI) exclusion assay. (A) RAW264.7 macrophages were stimulated with different concentrations of the PE25/PPE41 protein complex, as indicated. The peaks in the M2 region represent the uptake of PI dye by the necrotic cells when stimulated with increased concentration of the recombinant PE25/PPE41 protein complex. The number mentioned in M2 region of each of the graph represents the percentage of cells undergoing necrosis in response to the stimulation with PE25/PPE41 protein complex in a dose dependent manner. Data represent results of 3 independent experiments. (B) RAW264.7 macrophages were stimulated with 10 μg/ml of the PE25/PPE41 protein complex for 0, 2, 8 and 12 h, as indicated. Staining with PI/Annexin-V was carried out, cells acquired and data analyzed on flow cytometer (BD Vantage, SE). Necrotic (PI+/Annexin V+) or apoptotic (PI−Annexin V+) populations were gated and compared between cells stimulated for 0, 2, 8 and 12 h. Statistical significance was determined using student’s t test (∗∗∗p < 0.001). (C) TUNEL assay with macrophages stimulated with 10 μg/ml of recombinant PE25/PPE41 protein complex show absence of TUNEL staining in the presence of the PE25/PPE41 complex. Positive control (cells treated with DNase A) shows staining with fluorescein tagged nucleotides at the nicked ends of nucleotides. DAPI staining shows that the cells were alive.
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Related In: Results  -  Collection

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f0020: The PE25/PPE41 complex induces necrosis, but not apoptosis. Necrosis of macrophages stimulated with the recombinant protein was monitored by propidium iodide (PI) exclusion assay. (A) RAW264.7 macrophages were stimulated with different concentrations of the PE25/PPE41 protein complex, as indicated. The peaks in the M2 region represent the uptake of PI dye by the necrotic cells when stimulated with increased concentration of the recombinant PE25/PPE41 protein complex. The number mentioned in M2 region of each of the graph represents the percentage of cells undergoing necrosis in response to the stimulation with PE25/PPE41 protein complex in a dose dependent manner. Data represent results of 3 independent experiments. (B) RAW264.7 macrophages were stimulated with 10 μg/ml of the PE25/PPE41 protein complex for 0, 2, 8 and 12 h, as indicated. Staining with PI/Annexin-V was carried out, cells acquired and data analyzed on flow cytometer (BD Vantage, SE). Necrotic (PI+/Annexin V+) or apoptotic (PI−Annexin V+) populations were gated and compared between cells stimulated for 0, 2, 8 and 12 h. Statistical significance was determined using student’s t test (∗∗∗p < 0.001). (C) TUNEL assay with macrophages stimulated with 10 μg/ml of recombinant PE25/PPE41 protein complex show absence of TUNEL staining in the presence of the PE25/PPE41 complex. Positive control (cells treated with DNase A) shows staining with fluorescein tagged nucleotides at the nicked ends of nucleotides. DAPI staining shows that the cells were alive.
Mentions: In order to differentiate necrotic cell death from apoptosis, propidium iodide (PI)/Annexin V staining was carried out using macrophages stimulated with PE25/PPE41 complex (Fig. 4A and B). Propidium iodide easily passes through the ruptured membrane of dead cells and stains nucleic acids, but live cells or cells in early apoptotic phase are impermeable to PI dye. Unlike PI, Annexin V binds to phosphatidylserine with high affinity, which is externalized on the surface of apoptosed or dead cells, thus recognizing cells undergoing apoptosis even at early stage of cell death. Using flow cytometry staining, it could be seen that while PI uptake by the macrophages was a direct function of increasing concentration of the PE/PPE complex (Fig. 4A), the necrotic population of macrophages (PI+/AnnexinV+) increased at 2, 8 and 12 h of incubation, respectively (Fig. 4B). Absence of apoptotic population (PI−/AnnexinV+) suggests that PE/PPE protein complex induces necrosis of macrophages but not apoptosis (Fig. 4B). These results clearly suggest that the PE25/PPE41 protein complex induces necrosis in macrophages in a time dependent manner. Apoptotic cell death was also ruled out by TUNNEL staining. Macrophages incubated with PE25/PPE41 complex at a concentration as high as 10 μg/ml were negative for TUNEL staining (Fig. 4C). Necrotic cell death was further validated by lactate dehydrogenase (LDH) release assay. LDH assay involves measurement of NADH oxidation to NAD by LDH (released in to the medium from ruptured cells) in presence of pyruvic acid. RAW264.7 macrophages were stimulated with PE25/PPE41 at two different concentrations of PE25/PPE41 protein complex and LDH released was measured in the culture supernatant. It could be seen that the PE25/PPE41 protein complex induced release of LDH into the supernatant of RAW264.7 macrophages in a concentration dependent manner (Fig. 5). Very low oxidation of NADH was seen in the supernatant of cells left unstimulated (Fig. 5) or stimulated with concentration as high as 5 μg/ml of LPS (Fig. 5). Taken together these results demonstrate that the PE25/PPE41 protein complex induces necrosis, but not apoptosis, in RAW 264.7 macrophage cells.

Bottom Line: Necrotic cell death during TB infection is an important prerequisite for bacterial dissemination and virulence.The underlying mechanisms and the bacterial factors involved therein are not well understood.The Mycobacterium tuberculosis (M. tuberculosis) co-operonic PE25/PPE41 protein complex, similar to ESAT-6/CFP-10, belonging to the PE/PPE and ESAT-6 families of genes has co-expanded and co-evolved in the genomes of pathogenic mycobacteria.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, University of Chicago, Chicago, IL, 60637, USA.

ABSTRACT
Necrotic cell death during TB infection is an important prerequisite for bacterial dissemination and virulence. The underlying mechanisms and the bacterial factors involved therein are not well understood. The Mycobacterium tuberculosis (M. tuberculosis) co-operonic PE25/PPE41 protein complex, similar to ESAT-6/CFP-10, belonging to the PE/PPE and ESAT-6 families of genes has co-expanded and co-evolved in the genomes of pathogenic mycobacteria. We report a novel role of this highly immunogenic PE25/PPE41 protein complex in inducing necrosis, but not apoptosis, in macrophages. We propose that these protein complexes of M. tuberculosis, secreted by similar/unique transport system (Type VII), have an important role in M. tuberculosis virulence and disease reactivation.

No MeSH data available.


Related in: MedlinePlus