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Published on: September 27, 2018
Identification of Mycobacterium avium subsp. hominissuis secreted proteins using an in vitro system mimicking the
Jessica J Chinison1,2, Lia Danelishvili1, Rashmi Gupta1
1Department of Biomedical Sciences, College of Veterinary Medicine, Oregon State University, Corvallis, OR, USA.
Background:
Mycobacterium avium subsp. hominissuis is a common intracellular pathogen that infects patients with HIV/AIDS and cause lung infection in patients with underlying lung pathology. M.avium preferably infects macrophages and uses diverse mechanisms to alter phagosome maturation. Once in the macrophage, the pathogen can alter the host cellular defenses by secreting proteins into the cytosol of host cells, but despite considerable research, only a few secreted effector proteins have been identified. We hypothesized that the environmental cues inside the phagosome can trigger bacterial protein secretion. To identify M. avium secretome within the phagosome, we utilized a previously established in vitro system that mimics the metal ion concentrations and pH of the M. avium phagosome.
Results:
M. avium was exposed to phagosome metal concentrations for different time points and exported proteins were profiled and analyzed against bacterial proteins secreted in the culture medium. Mass spectrometric analysis of the secreted proteome identified several proteins, of which 46 were unique to bacteria incubated in the metal mixture. Ten of potential effectors were selected and secretion of these proteins was monitored within M. avium infected mononuclear phagocytic cells using the beta-lactamase FRET-based reporter system. In addition, pull-down assay was performed for secreted calmodulin-like protein MAV_1356 protein to evaluate for eukaryotic target. All examined M. avium proteins were secreted into the macrophage cytosol, and gene expression analysis suggested that the metal environment likely stimulates secretion of pre-made proteins. Further investigation of bacterial secreted MAV_1356 protein, lead to the observation that the MAV_1356 interacts with the host proteins Annexin A1 and Protein S100-A8.
Conclusions:
We established an in vitro system for the study if proteins secreted intracellularly, and revealed that the metal mixture mimicking the concentration of metals in the phagosome environment, triggers protein secretion.
Insights
Mycobacterium avium subsp. hominissuis secretes proteins into host cells when exposed to phagosome metal concentrations. This study identified novel secreted proteins, including MAV_1356, which interacts with host proteins.
Area of Science:
- Microbiology
- Cell Biology
- Immunology
Background:
- Mycobacterium avium subsp. hominissuis is a significant intracellular pathogen, particularly in HIV/AIDS patients and those with lung conditions.
- This bacterium infects macrophages and employs various strategies to disrupt phagosome maturation and host defenses.
- Identifying secreted effector proteins is crucial for understanding M. avium pathogenesis, yet few have been discovered.
Purpose of the Study:
- To investigate the hypothesis that phagosomal environmental cues trigger protein secretion in M. avium.
- To identify the M. avium secretome within the phagosome using an in vitro system mimicking phagosomal conditions.
- To characterize the function and host interactions of identified secreted proteins.
Main Methods:
- An in vitro system was developed to mimic phagosomal metal ion concentrations and pH.
- Proteomic analysis (mass spectrometry) was used to profile proteins secreted by M. avium under phagosome-mimicking conditions.
- Secretion of candidate effector proteins was monitored in infected macrophages using a beta-lactamase FRET reporter system, and protein-protein interactions were assessed via pull-down assays.
Main Results:
- Exposure to phagosome metal concentrations triggered protein secretion in M. avium.
- Mass spectrometry identified 46 proteins unique to bacteria incubated in the metal mixture.
- Ten candidate effector proteins were confirmed to be secreted into the macrophage cytosol, with MAV_1356 interacting with host Annexin A1 and Protein S100-A8.
Conclusions:
- An in vitro system successfully identified proteins secreted by M. avium within a phagosome-like environment.
- Phagosomal metal concentrations act as a stimulus for the secretion of pre-made bacterial proteins.
- The identified secreted proteins, such as MAV_1356, represent potential virulence factors that interact with host cellular components.
