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. 2016 Aug 30:6:91.
doi: 10.3389/fcimb.2016.00091. eCollection 2016.

MTMR4 Is Required for the Stability of the Salmonella-Containing Vacuole

Wei X Teo  1 , Markus C Kerr  1 , Rohan D Teasdale  1
Affiliations

MTMR4 Is Required for the Stability of the Salmonella-Containing Vacuole

Wei X Teo et al. Front Cell Infect Microbiol. .

Abstract

The intracellular pathogen Salmonella enterica servovar Typhimurium (S.typhimurium) modulates the host cell's phosphoinositide (PI) metabolism to establish its intracellular replicative niche, the Salmonella-containing vacuole (SCV). Upon invasion, phosphoinositide 3-phosphate (PI(3)P) and other early endosomal markers are rapidly recruited to and remain associated with the SCV throughout its early maturation. While the phosphoinositide 3-phosphatase myotubularin 4 (MTMR4) has an established role in regulating autophagy and cellular PI(3)P-content, two processes associated with the intracellular survival of S. typhimurium, a direct role for MTMR4 in Salmonella biology has not been examined. Here we demonstrate that GFP-tagged MTMR4 is recruited to the SCV and infection of cells depleted of endogenous MTMR4 results in a decrease in viable intracellular Salmonella. This reflects a significant increase in the proportion of SCVs with compromised integrity, which targets the compartment for autophagy and consequent bacterial cell death. These findings highlight the importance of PI(3)P regulation to the integrity of the SCV and reveal a novel role for the myotubularins in bacterial pathogenesis.

Keywords: Phosphotidylinositols; Salmonella; Salmonella-containing vacuole; autophagy; myotubularin.

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Figures

Figure 1
Figure 1
Recruitment of PI(3,5)P2 to the SCV and Sifs. (A) Time-lapse video microscopy of WT-SL1344 infected HeLa cells stably expressing GFP-LAMP1 and transiently expressing mCherry 2*ML1N. Montage representative of a 90 min movie captured using Zeiss LSM710 FCS scanning confocal microscope. (B) HeLa cells stably expressing GFP-LAMP1 cells and transiently expressing mCherry-2*ML1N were infected with WT-SL1344 and imaged 6 h p.i live on Zeiss LSM710 FCS scanning confocal microscope. Scale bar: 20 μm. White arrows indicate SCV.
Figure 2
Figure 2
MTMR4 regulates PI(3)P endosomal levels but not PI(3,5)P2. (A) A431 cells were transduced with shRNA targeting MTMR4 and the knockdown efficiently confirmed using RT-PCR. (B) MTMR4-depleted cells were fixed and immunolabelled with EEA1, counterstained with DAPI. (C) The number of EEA1 puncta was analyzed. (D) A431 cells were seeded and transfected with HA-MTMR4 or HA-empty vector, fixed, and immunofluorescence performed using antibodies against EEA1. (E) Quantification of EEA1 puncta following overexpression of HA-MTMR4. (F) MTMR4-depleted cells were pulsed with dextran conjugated to tetramethylrhodamine for 30 min before thorough washing with excess media and fixed, counterstained with DAPI. (G) Quantification of dextran puncta. (H) MTMR4 depleted cells were transfected with Myc-2*ML1N construct, immunolabelled and quantified in (I). (J) A431 cells were co-transfected with MTMR4 or empty vector and Myc-2*ML1N, fixed, immunolabeled and puncta quantified in (K). Data representative of three independent experiments (N = 3; at least 25 cells counted per condition; Error bar denote ± S.E.M., *P < 0.05, Scale bar: 20 μm).
Figure 3
Figure 3
MTMR4 is associated with the SCV. (A) Time-lapse video microscopy of A431 cells transfected with GFP-MTMR4 or GFP-MTMR3 and infected with RFP-SL1344. Montage is representative of a 25 min movie captured using Nikon Deconvolution microscope. (B) A431 cells were transfected with GFP-MTMR4 and infected with either RFP-SL1344 or ΔSopB-SL1344. MTMR4-positive Salmonella were then probed for the presence of EEA1 or SNX1 and quantified over a time course assay (C,D) respectively. (N = 3; Error bar denote mean ± S.D., Scale bar: 20 μm, **p < 0.005).
Figure 4
Figure 4
MTMR4 depletion renders cells less permissible to SCV growth. (A) MTMR4-depleted A431 and control cells were infected with RFP-SL1344. Intracellular replication was monitored by measuring the relative area occupied by RFP-fluorescence in (B) infected cells and colony forming unit (C) assay. (N = 3, Error bar denote mean ± S.D., Scale bar 20 μm, *p < 0.05, **p < 0.005).
Figure 5
Figure 5
Depletion of MTMR4 enhances autophagy. (A) MTMR4-depleted HeLa and control cells were transfected with GFP-LC3, and treated with DMSO, 100 nM rapamycin, 100 nM wortmannin, or 20 mM 3-MA. Coverslips were then fixed and immunolabelled using antibodies against GFP. (B) Representative blot of MTMR4 knockdown and control whole cell lysates were harvested and 20 μg of proteins subjected to western immunoblotting against LC3 and tubulin. Scale bar 20 μm.
Figure 6
Figure 6
The proportion of damaged SCVs increases in MTMR4 depleted cells. MTMR4-depleted A431 cells and non-silencing control cells were infected with RFP-SL1344 or WT-SL1344. Cells were fixed at stipulated time points and probed for association autophagic markers. Quantification of the percentage of (A) galectin-8+, (B) recruitment of GFP-LC3+ or (C) p62+ bacteria. (N = 3, Error bar denote mean ± S.D., *p < 0.05, **p < 0.005).
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