title: Mycobacterium tuberculosis EspB protein suppresses interferon-γ-induced autophagy in murine macrophages.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors: Laboratory of Infection and Immunity, School of Basic Medical Sciences, West China Center of Medical Sciences, Sichuan University, Chengdu 610041, China.
authors: DanDan
authors: D
authors: Huang
authors: Laboratory of Infection and Immunity, School of Basic Medical Sciences, West China Center of Medical Sciences, Sichuan University, Chengdu 610041, China. Electronic address: baolang@scu.edu.cn.
authors: Lang
authors: L
authors: Bao
mesh_terms: D000818:Animals; D001343:Autophagy / Q000276:immunology*; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007371:Interferon-gamma / Q000276:immunology*; D008264:Macrophages / Q000276:immunology* / Q000378:metabolism; D051379:Mice; D009169:Mycobacterium tuberculosis / Q000276:immunology* / Q000378:metabolism; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics; D017471:Receptors, Interferon / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism*; D050794:STAT1 Transcription Factor / Q000378:metabolism; D000096743:Interferon gamma Receptor
chemical_list: D001425:Bacterial Outer Membrane Proteins; D007371:Interferon-gamma; D012333:RNA, Messenger; D017471:Receptors, Interferon; D011994:Recombinant Proteins; D050794:STAT1 Transcription Factor; D000096743:Interferon gamma Receptor; C000615638:EspB protein, Mycobacterium tuberculosis; C494080:Stat1 protein, mouse
keywords: ANA-1 macrophages; EspB; Mycobacterium tuberculosis; autophagy; interferon-γ receptor 1