Limits...
Evidence for genes associated with the ability of Mycobacterium avium subsp. hominissuis to escape apoptotic macrophages.

Bermudez LE, Danelishvili L, Babrack L, Pham T - Front Cell Infect Microbiol (2015)

Bottom Line: Testing in vivo, however, demonstrated that all the MAH clones were attenuated compared with the wild-type MAC 104 in tissues of mice.Although the mechanism associated with the bacterial inability to leave apoptotic macrophages is unknown, the identification of macrophage cytoplasm targets for the MAH proteins suggest that they interfere either with protein degradation machinery or post-translation mechanisms.The identification of tatC as a MAH protein involved in the ability of MAH to leave macrophages, suggests that secreted effector(s) are involved in the process.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, College of Veterinary Medicine, Oregon State University Corvallis, OR, USA ; Department of Microbiology, College of Science, Oregon State University Corvallis, OR, USA ; Program of Molecular and Cell Biology, Oregon State University Corvallis, OR, USA.

ABSTRACT
Mycobacterium avium subsp. hominissuis (MAH) is an environmental bacteria that infects immunocompromised humans. MAH cases are increasing in incidence, making it crucial to gain knowledge of the pathogenic mechanisms associated with the bacterium. MAH infects macrophages and after several days the infection triggers the phagocyte apoptosis. Many of the intracellular MAH escape the cell undergoing apoptosis leading to infection of neighboring macrophages. We screened a transposon bank of MAH mutants in U937 mononuclear phagocytes for the inability to escape macrophages undergoing apoptosis. Mutations in genes; MAV_2235, MAV_2120, MAV_2410, and MAV_4563 resulted in the inability of the bacteria to exit macrophages upon apoptosis. Complementation of the mutations corrected the phenotype either completely or partially. Testing for the ability of the mutants to survive in macrophages compared to the wild-type bacterium revealed that the mutant clones were not attenuated up to 4 days of infection. Testing in vivo, however, demonstrated that all the MAH clones were attenuated compared with the wild-type MAC 104 in tissues of mice. Although the mechanism associated with the bacterial inability to leave apoptotic macrophages is unknown, the identification of macrophage cytoplasm targets for the MAH proteins suggest that they interfere either with protein degradation machinery or post-translation mechanisms. The identification of tatC as a MAH protein involved in the ability of MAH to leave macrophages, suggests that secreted effector(s) are involved in the process. The study reveals a pathway of escape from macrophages, not shared with Mycobacterium tuberculosis.

No MeSH data available.


Related in: MedlinePlus

(A) The yeast two-hybrid interaction of MAV_2120, MAV_2235, and MAV_4563 proteins with the host target proteins. (a) COP9 signalosome subunit 5 (CSN5), (b) eukaryotic translation termination factor 1 (ETF1), (c) PNN-interacting serine/arginine-rich protein (PNISR), (d) serine/arginine-rich splicing factor 5 (SRSF5), (e) SECIS binding protein 2-like (SECISBP2L), (f) ubiquitin specific peptidase 48 (USP48). (B) Co-immunoprecipitation of MAV_2120 and MAV_4563 from M. smegmatis cell lysates using recombinant 6xHN:CSN5 and 6xHN:USP48 proteins. Bound proteins were captured on His-columns and visualized by western blotting using 6xHN and Flag antibodies.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4548235&req=5

Figure 3: (A) The yeast two-hybrid interaction of MAV_2120, MAV_2235, and MAV_4563 proteins with the host target proteins. (a) COP9 signalosome subunit 5 (CSN5), (b) eukaryotic translation termination factor 1 (ETF1), (c) PNN-interacting serine/arginine-rich protein (PNISR), (d) serine/arginine-rich splicing factor 5 (SRSF5), (e) SECIS binding protein 2-like (SECISBP2L), (f) ubiquitin specific peptidase 48 (USP48). (B) Co-immunoprecipitation of MAV_2120 and MAV_4563 from M. smegmatis cell lysates using recombinant 6xHN:CSN5 and 6xHN:USP48 proteins. Bound proteins were captured on His-columns and visualized by western blotting using 6xHN and Flag antibodies.

Mentions: To demonstrate direct binding of identified host proteins to M. avium proteins, we performed co-immunoprecipitation of Flag-tagged MAV_2120 (Flag:MAV_2120) and MAV_4563 (Flag:MAV_4563) with 6xHN tagged CSN5 (6xHN:CSN5) and USP48 (6xHN:USP48), respectively (Figure 3A). Recombinant 6xHN:CSN5 and 6xHN:USP48 were expressed in the pET system. The Flag:tagged M. avium genes were overexpressed in pMV261 and transformed into M. smegmatis. Bacteria were lysed at a mid-log growth phase and the cleared protein fraction was incubated with the recombinant host proteins for overnight at 4°C. Recombinant host and bacterial protein mixture were passed through His-columns and bound proteins were visualized with 6xHN and Flag antibodies by western blotting. M. smegmatis protein lysate expressing just pMV261 vector was used as a negative control in the co-immunoprecipitation experiment with 6xHN:CSN5 and 6xHN:USP48. As shown in Figure 3B, while no interaction was detected in the control group, purification of recombinant CSN5 and USP48 proteins led to co-purification of MAV_2120 and MAV_4563, respectively, confirming the physical binding between studied host and bacterial proteins.


Evidence for genes associated with the ability of Mycobacterium avium subsp. hominissuis to escape apoptotic macrophages.

Bermudez LE, Danelishvili L, Babrack L, Pham T - Front Cell Infect Microbiol (2015)

(A) The yeast two-hybrid interaction of MAV_2120, MAV_2235, and MAV_4563 proteins with the host target proteins. (a) COP9 signalosome subunit 5 (CSN5), (b) eukaryotic translation termination factor 1 (ETF1), (c) PNN-interacting serine/arginine-rich protein (PNISR), (d) serine/arginine-rich splicing factor 5 (SRSF5), (e) SECIS binding protein 2-like (SECISBP2L), (f) ubiquitin specific peptidase 48 (USP48). (B) Co-immunoprecipitation of MAV_2120 and MAV_4563 from M. smegmatis cell lysates using recombinant 6xHN:CSN5 and 6xHN:USP48 proteins. Bound proteins were captured on His-columns and visualized by western blotting using 6xHN and Flag antibodies.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4548235&req=5

Figure 3: (A) The yeast two-hybrid interaction of MAV_2120, MAV_2235, and MAV_4563 proteins with the host target proteins. (a) COP9 signalosome subunit 5 (CSN5), (b) eukaryotic translation termination factor 1 (ETF1), (c) PNN-interacting serine/arginine-rich protein (PNISR), (d) serine/arginine-rich splicing factor 5 (SRSF5), (e) SECIS binding protein 2-like (SECISBP2L), (f) ubiquitin specific peptidase 48 (USP48). (B) Co-immunoprecipitation of MAV_2120 and MAV_4563 from M. smegmatis cell lysates using recombinant 6xHN:CSN5 and 6xHN:USP48 proteins. Bound proteins were captured on His-columns and visualized by western blotting using 6xHN and Flag antibodies.
Mentions: To demonstrate direct binding of identified host proteins to M. avium proteins, we performed co-immunoprecipitation of Flag-tagged MAV_2120 (Flag:MAV_2120) and MAV_4563 (Flag:MAV_4563) with 6xHN tagged CSN5 (6xHN:CSN5) and USP48 (6xHN:USP48), respectively (Figure 3A). Recombinant 6xHN:CSN5 and 6xHN:USP48 were expressed in the pET system. The Flag:tagged M. avium genes were overexpressed in pMV261 and transformed into M. smegmatis. Bacteria were lysed at a mid-log growth phase and the cleared protein fraction was incubated with the recombinant host proteins for overnight at 4°C. Recombinant host and bacterial protein mixture were passed through His-columns and bound proteins were visualized with 6xHN and Flag antibodies by western blotting. M. smegmatis protein lysate expressing just pMV261 vector was used as a negative control in the co-immunoprecipitation experiment with 6xHN:CSN5 and 6xHN:USP48. As shown in Figure 3B, while no interaction was detected in the control group, purification of recombinant CSN5 and USP48 proteins led to co-purification of MAV_2120 and MAV_4563, respectively, confirming the physical binding between studied host and bacterial proteins.

Bottom Line: Testing in vivo, however, demonstrated that all the MAH clones were attenuated compared with the wild-type MAC 104 in tissues of mice.Although the mechanism associated with the bacterial inability to leave apoptotic macrophages is unknown, the identification of macrophage cytoplasm targets for the MAH proteins suggest that they interfere either with protein degradation machinery or post-translation mechanisms.The identification of tatC as a MAH protein involved in the ability of MAH to leave macrophages, suggests that secreted effector(s) are involved in the process.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, College of Veterinary Medicine, Oregon State University Corvallis, OR, USA ; Department of Microbiology, College of Science, Oregon State University Corvallis, OR, USA ; Program of Molecular and Cell Biology, Oregon State University Corvallis, OR, USA.

ABSTRACT
Mycobacterium avium subsp. hominissuis (MAH) is an environmental bacteria that infects immunocompromised humans. MAH cases are increasing in incidence, making it crucial to gain knowledge of the pathogenic mechanisms associated with the bacterium. MAH infects macrophages and after several days the infection triggers the phagocyte apoptosis. Many of the intracellular MAH escape the cell undergoing apoptosis leading to infection of neighboring macrophages. We screened a transposon bank of MAH mutants in U937 mononuclear phagocytes for the inability to escape macrophages undergoing apoptosis. Mutations in genes; MAV_2235, MAV_2120, MAV_2410, and MAV_4563 resulted in the inability of the bacteria to exit macrophages upon apoptosis. Complementation of the mutations corrected the phenotype either completely or partially. Testing for the ability of the mutants to survive in macrophages compared to the wild-type bacterium revealed that the mutant clones were not attenuated up to 4 days of infection. Testing in vivo, however, demonstrated that all the MAH clones were attenuated compared with the wild-type MAC 104 in tissues of mice. Although the mechanism associated with the bacterial inability to leave apoptotic macrophages is unknown, the identification of macrophage cytoplasm targets for the MAH proteins suggest that they interfere either with protein degradation machinery or post-translation mechanisms. The identification of tatC as a MAH protein involved in the ability of MAH to leave macrophages, suggests that secreted effector(s) are involved in the process. The study reveals a pathway of escape from macrophages, not shared with Mycobacterium tuberculosis.

No MeSH data available.


Related in: MedlinePlus