Mycobacterium tuberculosis VII secretion system effector molecule Rv2347c blocks the maturation of phagosomes and activates the STING/TBK1 signaling pathway to inhibit cell autophagy

Abstract

The VII secretion system is the main channel for Mycobacterium tuberculosis (MTB) to secrete virulence proteins. The ESAT-like proteins EsxA/B and EsxW/V in the RD region of its genome have been used as targets for vaccine antigens. However, the function of EsxO/P has not been explored, although it was predicted to potentially induce Th1 cell responses as a vaccine development target. In this study, the VII secretion system effector molecule Rv2347c was heterologously expressed in Mycobacterium smegmatis and found to inhibit the expression of the early marker RAB5 of phagosomes, thus preventing the maturation process of phagosomes toward lysosomes, and activated the host cytoplasmic sensing pathway. It inhibited autophagy and activated IFNβ transcription through the STING/TBK1 pathway promoting the host's survival. Therefore, Rv2347c plays an important role in the pathogenesis of MTB with the potential to be utilized as a new target for tuberculosis vaccine development.

Importance: We found that the ESAT-like protein Rv2347c (EsxP) can inhibit the maturation of phagosomes, leading to mycobacterium escape from phagosomes into the cytoplasm, which triggers the host's cytoplasmic sensing pathway STING/TBK1, inhibiting autophagy and upregulating IFNβ transcription, which contributes to the survival of mycobacterium in the host cell. We also found that Rv2347c was able to activate host immunity by activating NF-κB via STING and promoting the transcription of downstream pro-inflammatory factors. Meanwhile, the host also produces IL-1β to repair phagosome maturation arrest via the STING-mediated non-NF-κB pathway.

Keywords: STING; autophagy; phagolysosome.

Fig 1

Rv2347c enhances the viability of bacteria in the host. (A) Loading bacteria quantity determination. THP-1 cells were infected with Ms_Rv2347c and Ms_pMV261, and treated with 0.025% SDS for 10 minutes at 6 hours and 24 hours, respectively, to detect the survival of bacteria in the host. (B) The growth of Ms_Rv2347c and Ms_pMV261 in 7H9 medium was measured at OD₆₀₀ every 3 hours. (C) Analyzing the activity of LDH in THP-1 cell culture filtrate infected with Ms_Rv2347c and Ms_pMV261. All experiments were repeated at least three times. Two-tailed unpaired Student’s t-test was used for statistical analysis, significance: *P < 0.05, **P < 0.01, ns for no difference.

Fig 2

Rv2347c ruptures the integrity of the lysosomal membrane and mitochondrial membrane. (A) The fluorescence localization of bacteria and lysosomes observed by confocal microscope. Bacteria were dyed green with Dio, lysosomes were dyed red, and the fluorescence intensity curve was drawn by ImageJ. The scale bars depict 5 µm. (B) Pearson’s correlation coefficient of (A). (C) The infected macrophages were stained with rhodamine 123, the mitochondrial membrane potential was measured by flow cytometry, and the results were analyzed by 10,000 gated cells. All experiments were repeated at least three times. Two-tailed unpaired Student’s t-test was used for statistical analysis, significance: *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 3

Expression levels of phagosomes’ markers in different periods. (A) RT-qPCR was used to detect the mRNA levels of markers of phagosomes in different periods when THP-1 was infected for 24 hours. (B) Western blot was used to detect the expression level of LAMP1 protein when THP-1 was infected for 24 hours. pMV261 refers to THP-1 cells infected with Ms_pMV261, while Rv2347c refers to THP-1 cells infected with Ms_Rv2347c. (C) Determination of the viability of mycobacteria at different pH levels. The colony formation units of overexpressed and empty vector strains were determined at 0, 3, 6, 9, and 12 hours at pH levels 4.5, 5.5, 6.5, and 7.5, respectively. All experiments were repeated at least three times. Two-tailed unpaired Student’s t-test was used for statistical analysis, significance: *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 4

Expression of autophagy proteins LC3 II and P62 in Ms_Rv2347c and Ms_pMV261 infection. (A and B) After 24 hours of infection with Ms_Rv2347c and Ms_pMV261, the expression levels of LC3 protein and P62 protein of THP-1 were detected by Western blot. (C) RT-qPCR was used to detect the mRNA levels of NDP52, P62, LC3, and BECLIN1 when THP-1 was infected for 24 hours. pMV261 refers to THP-1 cells infected with Ms_pMV261, while Rv2347c refers to THP-1 cells infected with Ms_Rv2347c. All experiments were repeated at least three times. Two-tailed unpaired Student’s t-test was used for statistical analysis, significance: *P < 0.05, **P < 0.01.

Fig 5

Rv2347c inhibits autophagy by promoting phosphorylation of TBK1. (A) After Ms_Rv2347c and Ms_pMV261 were infected with THP-1 for 24 hours, the TBK1 protein and its phosphorylation level of THP-1 were detected by Western blot. (B) Western blot was used to detect STAT1 and its phosphorylation level. (C) RT-qPCR was used to detect the transcription level of IFNβ of THP-1. (D) Ms_Rv2347c and Ms_pMV261 infected THP-1 cells treated with C176 or untreated, and the transcription level of IFNβ of THP-1 was detected by RT-qPCR. (E) Western blot was used to detect the expression of LC3 protein. (F) Western blot was used to detect the expression of P62 protein. pMV261 refers to THP-1 cells infected with Ms_pMV261, while Rv2347c refers to THP-1 cells infected with Ms_Rv2347c. All experiments were repeated at least three times. Two-tailed unpaired Student’s t-test was used for statistical analysis, significance: *P < 0.05, **P < 0.01, ns for no difference.

Fig 6

Rv2347c enhances the expression of inflammatory factors in the host by activating the NF-κB pathway through STING. Ms_Rv2347c and Ms_pMV261 were infected with THP-1 for 24 hours. (A) The protein level of IL-1β of THP-1 was detected by Western blot. (B) Western blot was used to detect the protein level of p-P65 of THP-1. (C) The transcription levels of IL-1β and TNFα of THP-1 were detected by RT-qPCR. (D) THP-1 cells treated with C176 or untreated with MS_ Rv2347c and Ms_pMV261 were infected, and the protein level of p-P65 of THP-1 was detected by Western blot. (E) RT-qPCR was used to detect the transcription levels of IL-1β and TNFα of THP-1. (F) Western blot was used to detect the protein level of IL-1β of THP-1. pMV261 refers to THP-1 cells infected with Ms_pMV261, while Rv2347c refers to THP-1 cells infected with Ms_Rv2347c. All experiments were repeated at least three times. Two-tailed unpaired Student’s t-test was used for statistical analysis, significance: *P < 0.05, **P < 0.01, ***P < 0.001, ns for no difference.

Fig 7

Pattern diagram of interaction mechanism between Rv2347c and host. Rv2347c can inhibit autophagy by inhibiting phagosome maturation. After entering the cytoplasm, IFNβ transcription is upregulated by activating STING and phosphorylating TBK1, which activates the host antibacterial immunity and promotes the survival of mycobacteria in the host cell. In addition, Rv2347c can also activate the transcription of downstream inflammatory factors through STING/NF-κB.