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Mycobacterium tuberculosis EspB protein suppresses interferon-γ-induced autophagy in murine macrophages
1Laboratory of Infection and Immunity, School of Basic Medical Sciences, West China Center of Medical Sciences, Sichuan University, Chengdu 610041, China.
Background:
Mycobacterium tuberculosis (Mtb) persists within immature phagosomes by preventing their maturation into phagolysosomes. Although the early secretory antigenic target 6 (ESAT-6) system 1 (ESX-1) secretion-associated protein B (EspB) of Mtb is strongly linked to immunogenicity and virulence of this organism, its mechanism of action remains largely unclear. This study aimed to investigate EspB effects on autophagy in murine ANA-1 macrophage cells.
Methods:
EspB gene was amplified by polymerase chain reaction from Mtb H37Rv genomic DNA to express recombinant EspB protein. Levels of autophagic markers, including Microtubule-associated protein 1 light chain 3 beta (LC3B-I and -II), phosphorylated signal transducer and activator of transcription (STAT)1 and total STAT1 in ANA-1 cells treated with EspB proteins were assessed by Western blotting. In addition, autophagic vacuoles were detected by fluorescence microscopy. Finally, IFN-γR1 expression was evaluated by semiquantitative reverse transcriptase polymerase chain reaction and flow cytometry.
Results:
EspB gene was expressed in Escherichia coli cells to yield a soluble N-terminal glutatione S-transferase tag fusion protein used in subsequent experiments. Preincubation with EspB significantly suppressed autophagosome formation and LC3B expression induced by interferon (IFN)-γ stimulation, in a dose-dependent manner. These results were confirmed by the reduced incorporation of monodansylcadaverine, a marker for the acidic compartment of autolysosomes, after treatment with EspB. Interestingly, we found that IFN-γ receptor 1 mRNA and protein levels were decreased in EspB-stimulated ANA-1 cells in comparison with untreated cells. Finally, EspB protein also inhibited IFN-γ-activated STAT1 phosphorylation, thereby downmodulating macrophage responsiveness to IFN-γ.
Conclusion:
EspB inhibits autophagosome formation in murine macrophages, at least in part by downregulating IFN-γ receptor 1 expression. Overall, EspB should be considered a relevant factor in the pathogenesis of mycobacterial infections in humans.
Insights
Mycobacterium tuberculosis EspB protein inhibits macrophage autophagy by reducing interferon-gamma receptor 1 expression, impacting host defense during infection. This protein is a key factor in mycobacterial pathogenesis.
Area of Science:
- Immunology
- Microbiology
- Cell Biology
Background:
- Mycobacterium tuberculosis (Mtb) evades host immunity by preventing phagosome maturation.
- The Mtb early secretory antigenic target 6 (ESAT-6) system 1 (ESX-1) secretion-associated protein B (EspB) is linked to virulence, but its mechanism is unclear.
- This study investigates EspB's impact on autophagy in murine ANA-1 macrophage cells.
Purpose of the Study:
- To elucidate the mechanism of action of Mtb EspB protein.
- To determine EspB's effect on autophagy in macrophages.
- To investigate EspB's influence on interferon-gamma (IFN-γ) signaling pathways.
Main Methods:
- Recombinant EspB protein was expressed and purified.
- Western blotting was used to assess autophagic markers (LC3B) and STAT1 phosphorylation.
- Autophagic vacuoles and IFN-γ receptor 1 (IFN-γR1) expression were analyzed via fluorescence microscopy, RT-PCR, and flow cytometry.
Main Results:
- EspB suppressed autophagosome formation and LC3B expression induced by IFN-γ in a dose-dependent manner.
- EspB treatment reduced the acidic compartment of autolysosomes.
- EspB decreased IFN-γ receptor 1 expression and inhibited IFN-γ-activated STAT1 phosphorylation, reducing macrophage responsiveness to IFN-γ.
Conclusions:
- EspB inhibits autophagosome formation in macrophages, partly by downregulating IFN-γ receptor 1.
- EspB plays a significant role in the pathogenesis of Mtb infections.
- Understanding EspB's function offers insights into host-pathogen interactions and potential therapeutic targets.
