Golgi-Resident GTPase Rab30 Promotes the Biogenesis of Pathogen-Containing Autophagosomes - PubMed (original) (raw)

Golgi-Resident GTPase Rab30 Promotes the Biogenesis of Pathogen-Containing Autophagosomes

Seiichiro Oda et al. PLoS One. 2016.

Abstract

Autophagy acts as a host-defense system against pathogenic microorganisms such as Group A Streptococcus (GAS). Autophagy is a membrane-mediated degradation system that is regulated by intracellular membrane trafficking regulators, including small GTPase Rab proteins. Here, we identified Rab30 as a novel regulator of GAS-containing autophagosome-like vacuoles (GcAVs). We found that Rab30, a Golgi-resident Rab, was recruited to GcAVs in response to autophagy induction by GAS infection in epithelial cells. Rab30 recruitment was dependent upon its GTPase activity. In addition, the knockdown of Rab30 expression significantly reduced GcAV formation efficiency and impaired intracellular GAS degradation. Rab30 normally functions to maintain the structural integrity of the Golgi complex, but GcAV formation occurred even when the Golgi apparatus was disrupted. Although Rab30 also colocalized with a starvation-induced autophagosome, Rab30 was not required for autophagosome formation during starvation. These results suggest that Rab30 mediates autophagy against GAS independently of its normal cellular role in the structural maintenance of the Golgi apparatus, and autophagosome biogenesis during bacterial infection involves specific Rab GTPases.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1

Fig 1. Golgi-resident GTPase Rab30 is localized to GAS-targeting autophagic structures.

(A) HeLa cell transiently expressing EmGFP-LC3 were infected with GAS (MOI = 100) for 4 h. Cells were then fixed, permeabilized, and immunostained with an anti-Rab30 antibody. Cellular and bacterial DNA was stained with DAPI. (B) HeLa cells transiently expressing FLAG-Atg5 and EmGFP-Rab30 were infected with GAS (MOI = 100) for 2 h. Cells were then immunostained with an anti-FLAG antibody. (C) HeLa cells transiently expressing mCherry-LC3 and EmGFP-Rab30 were infected with GAS for 4 h. Cells were then immunostained with an anti-LAMP1 antibody. Bars, 10 μm. (D) The number of cells containing GcAVs were counted and presented as the percentage of the total number of GAS-infected cells. Cells were transfected and infected with GAS for 4 h, as described in (C). The data shown represent result from >200 infected cells in terms of the mean value ± SD from 3 independent experiments. (E) HeLa cells transiently expressing mCherry-LC3 and EmGFP-Rab30 WT, EmGFP-Rab30 Q68L (QL), or EmGFP-Rab30 T23N (TN) were infected with GAS for 4 h. Bars, 10 μm. (F) Colocalization frequencies of GcAV and Rab30 were counted and presented as the percentage of the total number of GcAVs. The data shown represent the result from >80 GcAVs in terms of the mean value ± SD from 3 independent experiments. ** P < 0.01.

Fig 2

Fig 2. Rab30 is redistributed from the Golgi apparatus to GcAVs upon autophagy induction by GAS.

(A) HeLa cells transiently expressing mCherry-LC3 and EmGFP-Rab30 were infected with GAS (MOI = 100) for 4 h. Cells were immunostained with an anti-GM130 antibody. (B) HeLa cells transiently expressing EmGFP-Rab30, mCherry-Rab7, and FLAG-LC3 were infected with JRS4 WT or JRS4ΔSLO for 4 h. (C) The percentages of cells with Rab30-associated GAS were quantified. Data represent the result of >100 cells in terms of the mean value ± SD from 3 independent experiments. (D) HeLa WT or Atg5 knockout (KO) cells transiently expressing EmGFP-Rab30 and mCherry-LC3 were infected with GAS for 4 h. Bars, 10 μm. (E) The percentages of cells with Rab30-associated GAS were quantified. ** P < 0.01.

Fig 3

Fig 3. Rab30 is involved in autophagy during GAS infection.

(A) HeLa cells were transfected with a control siRNA or Rab30 siRNA (siRab30 #1), as well as mCherry-LC3, and EmGFP-p62, or EmGFP-NDP52 expression vector and then infected with GAS for 4 h. Cells were then fixed, permeabilized, and immunostained with an anti-ubiquitinated protein (Ub) antibody. Yellow bars, 2 μm. (B) The number of cells containing Ub/NDP52/p62-positive GAS were counted and presented as the percentage of the total number of GAS-infected cells. Data represent the results of >100 cells in terms of the mean value ± SD from 3 independent experiments. (C) HeLa cells transfected with control siRNA or Rab30 siRNA (siRab30 #1), as well as an EmGFP-LC3 expression vector were infected with GAS for 4 h. The cells were then fixed, permeabilized, and stained with DAPI. Yellow arrows indicate GcAV. Bars, 10 μm. (D) The number of cells containing GcAVs was counted and presented as the percentage of the total number of GAS. Data represent the results of >100 cells in terms of the mean value ± SD from 3 independent experiments. * P < 0.05. (E) HeLa cells were transfected with control or Rab30 siRNA (siRab30 #1). At 48 h post-transfection, HeLa cells were infected with GAS for 1, 2, or 6 h. Recovered bacteria were measured in GAS viability assays. The data shown represent the mean value ± SD from 3 independent experiments.

Fig 4

Fig 4. Involvement of Rab30 in GAS-induced autophagy is independent of its role in maintaining the structural integrity of the Golgi apparatus.

(A) HeLa cells were transfected with EmGFP-LC3 and siRNAs against the indicated target mRNAs, or were treated with BFA or GA, as indicated. Subsequently, the cells were infected with GAS (MOI = 100). The cells were then fixed, permeabilized, and stained with DAPI. The percentage of cells with GcAVs was quantified. (B) HeLa cells were transfected with siRNAs against indicated target mRNAs, or treated with BFA or GA, after which they were infected with GAS. Cells were fixed, permeabilized, and immunostained with an anti-GM130 antibody. The percentage of cells with intact Golgi structure was quantified. The data shown represent the results of >200 cells in terms of the mean value ± SD from 3 independent experiments. ** P < 0.01.

Fig 5

Fig 5. Involvement of Rab30 in starvation-induced autophagosome formation.

(A) Confocal microscopic images of mCherry–LC3 puncta with EmGFP–Rab30 in starvation conditions. HeLa cells that expressed mCherry–LC3 and EmGFP–Rab30 were cultured in starvation medium for 2 h and fixed. Yellow arrows indicate Rab30-colocalized LC3 puncta. (B) Confocal microscopic images of EmGFP–LC3 puncta in Rab30 knockdown cells. HeLa cells stably expressing EmGFP–LC3 were transfected with a control siRNA or Rab30 siRNA and cultured in regular medium or starvation medium for 2 h and fixed. Bars, 10 μm. (C) Effect of knockdown of the Rab30 on starvation-induced autophagosome formation. The number of LC3 dots was quantified in confocal microscopic images. The data shown represent the results for >10 images and each percentage represents the mean value ± SD based on three independent experiments. NS, not significant.

Similar articles

Cited by

References

    1. Levine B, Kroemer G (2008) Autophagy in the pathogenesis of disease. Cell 132: 27–42. 10.1016/j.cell.2007.12.018 - DOI - PMC - PubMed
    1. Shibutani ST, Yoshimori T (2014) A current perspective of autophagosome biogenesis. Cell Res 24: 58–68. 10.1038/cr.2013.159 - DOI - PMC - PubMed
    1. Mizushima N, Komatsu M (2011) Autophagy: renovation of cells and tissues. Cell 147: 728–741. 10.1016/j.cell.2011.10.026 - DOI - PubMed
    1. Huang J, Brumell JH (2014) Bacteria-autophagy interplay: a battle for survival. Nat Rev Microbiol 12: 101–114. 10.1038/nrmicro3160 - DOI - PMC - PubMed
    1. Campoy E, Colombo MI (2009) Autophagy in intracellular bacterial infection. Biochim Biophys Acta 1793: 1465–1477. 10.1016/j.bbamcr.2009.03.003 - DOI - PubMed

Publication types

MeSH terms

Substances

Grants and funding

This work was supported by the Funding Programs for Next Generation World-Leading Researchers (LS041) and JSPS KAKENHI Grant Numbers 25293370, 70294113 (to I.N.) and 10598858 (to T.N.).

LinkOut - more resources