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Staphylococcal virulence factor HlgB targets the endoplasmic-reticulum-resident E3 ubiquitin ligase AMFR to promote pneumonia

Nature Microbiology volume 8, pages 107–120 (2023)Cite this article

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Abstract

Staphylococcus aureus invades cells and persists intracellularly, causing persistent inflammation that is notoriously difficult to treat. Here we investigated host–pathogen interactions underlying intracellular S. aureus infection in macrophages and discovered that the endoplasmic reticulum (ER) is an important cellular compartment for intracellular S. aureus infection. Using CRISPR–Cas9 guide RNA library screening, we determined that the autocrine motility factor receptor (AMFR), an ER-resident E3 ubiquitin ligase, played an essential role in mediating intracellular S. aureus-induced inflammation. AMFR directly interacted with TAK1-binding protein 3 (TAB3) in the ER, inducing K27-linked polyubiquitination of TAB3 on lysine 649 and promoting TAK1 activation. Moreover, the virulence factor γ-haemolysin B (HIgB) of S. aureus bound to the AMFR and regulated TAB3. Our findings highlight an unknown role of AMFR in intracellular S. aureus infection-induced pneumonia and suggest that pharmacological interruption of AMFR-mediated TAB3 signalling cascades and HIgB targeting may prevent invasive staphylococci-mediated pneumonia.

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Fig. 1: AMFR is critical for intracellular MRSA-induced inflammation in macrophages.
Fig. 2: AMFR deficiency in macrophages ameliorates intracellular MRSA-induced pneumonia.
Fig. 3: AMFR regulates NF-κB signalling pathways and interacts with TAB3 on ER.
Fig. 4: AMFR mediates K27-linked polyubiquitination of TAB3 on Lys649 and promotes TAK1 activation.
Fig. 5: S. aureus HlgB binds and promotes AMFR-mediated NF-κB activation.
Fig. 6: HlgB plays a critical role in AMFR-mediated inflammation.

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Data availability

The authors declare that the data supporting the findings of this study are available within the article. MRSA strain sequences used in this work are available at https://www.ncbi.nlm.nih.gov/. Source data are provided with this paper.

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Acknowledgements

This work was supported by grants from the National Natural Science Foundation of China (grant numbers 81973329 and 82173821 to F.Q. and grant numbers 82073858 and 82273934 to L.S.) and Natural Science Foundation of Shanghai (21ZR1432700, to L.S.).

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Authors and Affiliations

  1. Engineering Research Center of Cell & Therapeutic Antibody, Ministry of Education, Pharm-X Center, Research Center for Small Molecule Immunological Drugs, School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China

    Lei Sun, Haibo Zhang, Huihui Zhang, Xinyi Lou, Zhiming Wang, Xinyi Yang, Daijie Chen, Ao Zhang & Feng Qian

  2. Department of Basic Medicine, Wuxi School of Medicine, Jiangnan University, Wuxi, China

    Yaxian Wu

  3. Institute of Antibiotics, Huashan Hospital, Fudan University, Shanghai, China

    Beining Guo

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Contributions

L.S. and F.Q. conceived the study. Ha.Z., Hu.Z., X.L., Z.W., Y.W., X.Y. and B.G. designed, performed and interpreted experimental data. L.S. D.C., A.Z. and F.Q. wrote the paper. All authors read and approved the final manuscript.

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Correspondence to Lei Sun or Feng Qian.

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Extended data

Extended Data Fig. 1 AMFR deficiency in macrophages ameliorates intracellular MRSA-induced cytokine production.

(a) BMDMs were infected with MRSA43300 (dot) and clinical MRSA strain S1-2-4 (circle), S1-2-12 (triangle), and S1-2-22 (square) with an MOI of 8 for 1 h, extracellular MRSA was removed and killed with 300 μg/mL gentamicin and incubated in medium containing gentamicin (50 µg/mL). The number of gentamicin-protected bacteria was determined at 1, 3, and 5 day post-infection by plating for colony forming units. (b) BMDMs were treated with MRSA43300 (MOI 8:1) for 1 h. The gentamicin-protected intracellular MRSA were examined by TEM at 2 h post-infection. Triangles indicate ER membranes, Arrowheads indicate living cells. (c) THP-1 cells were transfected with lentivirus-mediated AMFR shRNA (AMFR shRNA) for 48 h. The knockdown efficiency was detected by western blot. Scrambled shRNA (Scr shRNA) was used as control. (d) qRT-PCR analysis of TNF, IL1B, and IL6 mRNA expression in THP-1 cells transfected with lentivirus-mediated AMFR shRNA or Scrambled shRNA for 48 h and infected with clinical MRSA strains (S1-2-4, S1-2-12, S1-2-14, and S1-2-22) (MOI = 8). The results are shown as the relative levels of gene transcripts, with that of uninfected cells set as 1. (e) Human CD14+ monocytes were transfected with lentivirus-mediated AMFR shRNA. The knockdown efficiency was detected by western blot. Scrambled shRNA (Scr shRNA) was used as control. Data are representative of three independent experiments (b, c, e) and means ± SEM (a-d), n = 3 biological replicates, unpaired two-tailed Student’s t test.

Source data

Extended Data Fig. 2 AMFR does not regulate the engulf and killing activity of macrophages against MRSA.

(a) BMDMs were isolated from Amfrfl/fl (Amfr+/+) and LysM-Cre Amfrfl/fl (Amfr−/−) mice. Then, cells were incubated with mCherry-MRSA43300 (MOI = 8) for 45 min for phagocytosis assay. (b) Amfr+/+ and Amfr−/− macrophages were infected with mCherry-MRSA43300 (MOI = 8) for 1 h. The cells were then incubated with gentamicin for 1 hours, and intracellular bacterial CFUs were determined at the end of the gentamicin incubation period. Data shown are representative of three independent experiments (a), or presented as means ± SEM (b), n = 3 biological replicates, unpaired two-tailed Student’s t test.

Source data

Extended Data Fig. 3 AMFR deficiency in macrophages ameliorates intracellular MRSA-induced pneumonia.

(a) Gating strategy for flow cytometry analysis of the neutrophils (Neu., Ly-6G+) and alveolar macrophages (AMs, CD11c+ F4/80+) in the BALF of MRSA-infected mice. (b) BALF cells were sorted by Flow cytometry following Cytospin preparations and Giemsa staining. Representative neutrophils (Neu.) and alveolar macrophages (AMs) are shown. (c) BALF neutrophils and AMs’ ratios were estimated. Data shown are representative of three independent experiments, or presented as means ± SD from three independent experiments (n = 5 mice per group per experiment), two-way ANOVA (Bonferroni test).

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Extended Data Fig. 4 AMFR deficiency dose not alter the antimicrobial functions of neutrophils.

(a) Bone marrow neutrophils were isolated from Amfrfl/fl (Amfr+/+) and LysM-Cre Amfrfl/fl (Amfr−/−) mice. Then, cells were infected with mCherry-MRSA43300 (MOI = 8) for 15 min for phagocytosis assay. (b) Amfr+/+ and Amfr−/− neutrophils were infected with mCherry-MRSA43300 (MOI = 8) for 1 h. The cells were then incubated with gentamicin for 1 hours, and intracellular bacterial CFUs were determined at the end of the gentamicin incubation period. (c) The Amfr+/+ and Amfr−/− neutrophils were stimulated with MRSA43300 (MOI = 8), and the superoxide production were measured. Representative tracing showing the production of superoxide. (d) Cumulative superoxide production was quantified based the area under tracing line in c. Data shown are representative of three independent experiments (a, c), or presented as means ± SEM (b, d), n = 3 biological replicates, unpaired two-tailed Student’s t test.

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Extended Data Fig. 5 AMFR deficiency have no effect on UPR and ER stress.

(a) qRT-PCR analysis of Chop, Bip, and Atf4 mRNA expression in BMDMs isolated Amfrfl/fl (Amfr+/+) and LysM-Cre Amfrfl/fl (Amfr−/−) mice and infected with MRSA43300 (MOI = 8) for 1 h following gentamicin incubation for 11 h or ER stress activator thapsigargin (Thap., 1 mM) for 12 h. (b) Immunoblot analysis of the phosphorylation level of IRE1 in BMDMs isolated from Amfrfl/fl (Amfr+/+) and LysM-Cre Amfrfl/fl (Amfr−/−) mice and infected with MRSA (MOI = 8) for 1 h following gentamicin incubation for 11 h or ER stress activator thapsigargin (Thap., 1 mM) for 12 h. Data shown are representative of three independent experiments. (c) qRT-PCR analysis of Edem1 expression in BMDMs isolated Amfrfl/fl (Amfr+/+) and LysM-Cre Amfrfl/fl (Amfr−/−) mice and infected with MRSA as above. The results are shown as the relative levels of gene transcripts, with that of unstimulated cells set as 1. Data are means ± SEM (a and c), n = 3 biological replicates, two-way ANOVA (Sidak’s test).

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Extended Data Fig. 6 AMFR deficience in macrophages attenuates NF-kB and MAPK signaling activation under MRSA stimulation.

(a) Densitometric analysis of immunoblot analysis of phosphorylated (p-) p65, p38, JNK, ERK and IκBa in Fig. 3a. (b) Immunoblot analysis of indicated protein in cell lysates of THP-1 cells transfected with lentivirus-mediated AMFR shRNA (AMFR shRNA) or Scrambled shRNA (Scr shRNA) and infected with MRSA43300 for the indicated time. (c) Densitometric analysis of immunoblot analysis in b. The samples derive from the same experiment and that gels/blots were processed in parallel. (a, c). Data shown are representative of three independent experiments (b), or presented as means ± SD (a, c), n = 3 biological replicates, two-way ANOVA (Sidak’s test).

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Extended Data Fig. 7 AMFR interacts with TAB3 and recruits TAB3 to the ER.

(a) Coimmunoprecipitation and immunoblot analysis of HEK293T cells co-transfected with MYC-TAB3, plus FLAG-AMFR, and FLAG-AMFR mutant vectors. (b) Coimmunoprecipitation and immunoblot analysis of HEK293T cells co-transfected with FLAG-TAB3, plus MYC-TAB3, and MYC-TAB3 mutant vectors. (c) Confocal microscopic imaging of HeLa cells expressing MYC-TAB3 (green) and FLAG-AMFR or FLAG-AMFR mutant vectors (red). Blue indicates ER-Tracker stain of ER. Yellow in merge 1 indicates the co-localization of TAB3 and AMFR. White in merge 2 indicates the co-localization of TAB3, AMFR, and ER. Scale bar, 10 mm. (d) Immunoblot analysis of TAB3 expression in cytoplasm and light membranes (LM, containing ER) of Amfr−/− BMDMs transfected with AMFR, or AMFR mutants infected with MRSA43300. (e) Immunoblot analysis of TAB3 expression in cytoplasm and light membranes (LM, containing ER) of Tab3−/− BMDMs transfected with TAB3, or TAB3 mutants infected with MRSA43300. Data shown are representative of three independent experiments.

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Extended Data Fig. 8 AMFR mediates K27-linked polyubiquitination of TAB3 on Lys649, but not regulates the degradation of TAB3.

(a) Immunoblot analysis of ubiquitination of TAB3 in HEK293T cells co-transfected with MYC-TAB3 mutant (K649R) and FLAG-AMFR, along with the mutant ubiquitin HA-Ub (K63). (b) Luciferase assay of NF-κB activation in HEK293T cells transfected with indicated plasmids. (c) Immunoblot analysis of the expression of TAB3 in cell lysates of HEK293T cells co-transfected with MYC-TAB3 along with increasing amount of FLAG-AMFR for 48 h. Before harvest, cells were treated with or without proteasome inhibitor MG-132 (25 mM) or lysosomal inhibitor (NH4CL 10 mM or leupeptin 20 mM) for 6 h. Densitometric analysis of immunoblot analysis was shown below. (d) Coimmunoprecipitation and immunoblot analysis of HEK293T cells transfected with indicated plasmids. Data shown are representative of three independent experiments, or presented as means ± SD, n = 3 biological replicates, two-way ANOVA (Sidak’s test).

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Extended Data Fig. 9 MRSA g-hemolysin B (HlgB) binds and promotes AMFR-mediated NF-kB activation.

(a) qRT-PCR analysis of Tnf, Il1b, and Il6 mRNA expression in BMDMs isolated from Amfrfl/fl (Amfr+/+) and LysM-Cre Amfrfl/fl (Amfr−/−) mice and treated with heat-killed MRSA43300 (HKSA) (MOI = 8). (b) qRT-PCR analysis of Tnf, Il1b, and Il6 mRNA expression in BMDMs isolated from Amfrfl/fl (Amfr+/+) and LysM-Cre Amfrfl/fl (Amfr−/−) mice and treated with LPS, or E.coli. (c) qRT-PCR analysis of Tnf, Il1b, and Il6 mRNA expression in BMDMs isolated from Amfrfl/fl (Amfr+/+) and LysM-Cre Amfrfl/fl (Amfr−/−) mice and treated with Poly(I:C). The results are shown as the relative levels of gene transcripts, with that of unstimulated cells set as 1. (d) Luciferase assay of NF-kB activation in HEK293T cells transfected with TAB3 and together with different MRSA leucotoxins expression vector (FLAG-tagged). The expression of each protein was shown below. (e) Densitometric analysis of immunoblot analysis of phosphorylated (p-) p65, p38, JNK, and ERK in Fig. 5c. The samples derive from the same experiment and that gels/blots were processed in parallel. (f) Coimmunoprecipitation and immunoblot analysis of HEK293T cells co-transfected with MYC-AMFR plus FLAG-Hld, or FLAG-HlgC, followed by IP with an anti-MYC Ab. (g) qRT-PCR analysis of Tnf, Il1b, and Il6 mRNA expression in Amfr+/+ and Amfr−/− BMDMs and infected with 43300∆hld. Data are representative of three independent experiments (f), means ± SEM in a-d and g, or means ± SD in e, one-way ANOVA (Tukey’s test), two-way ANOVA (Sidak’s test), or unpaired two-tailed Student’s t test.

Source data

Extended Data Fig. 10 Staphylococcus aureus g-hemolysin B (HlgB) plays a critical role in AMFR-mediated pneumonia.

(a) Hematoxylin and eosin staining of lung tissues from indicated mice after MRSA USA300 or USA300∆hlgB exposure (n = 5 per group). (b) Lung injury was assessed by histological scores in different groups. (c) Neutrophils (Neu., Ly-6G+) and alveolar macrophages (AMs, CD11c+ F4/80+) in BALF were analyzed using flow cytometry. (d) Total cell, neutrophils, and alveolar macrophages counts in BALF were estimated. (e) BALF protein were measured. (f) Bacterial burden in lung tissues from Amfrfl/fl and LysM-Cre Amfrfl/fl mice intratracheally injected with USA300 or USA300∆hlgB for the indicated times. Data are representative of three independent experiments (means ± SD) (n = 5 mice per group per experiment), two-way ANOVA (Sidak’s test).

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Sun, L., Zhang, H., Zhang, H. et al. Staphylococcal virulence factor HlgB targets the endoplasmic-reticulum-resident E3 ubiquitin ligase AMFR to promote pneumonia. Nat Microbiol 8, 107–120 (2023). https://doi.org/10.1038/s41564-022-01278-7

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  • DOI: https://doi.org/10.1038/s41564-022-01278-7

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