Article Figures & Data
Figures
- FIG 1
Expression of TFEB in A549 cells by A. baumannii. (A) Western blot analysis of TFEB in A549 cells after incubation with A. baumannii ATCC 17978 for 0.5, 2, and 6 h. The solid black lines in the blots separate the spliced portions of the blots between 2 and 6 h. Values shown in the bar graph are the percentage of TFEB expression in control (CTL) and infected A549 cells. (B) TFEB in A549 cells after incubation with A. baumannii ATCC 17978 for 0.5 and 2 h, immunostaining, and imaging by immunofluorescence microscopy. TFEB detected with rabbit anti-TFEB antibodies and labeled with Alexa Fluor 594-tagged secondary antibodies (red). Blue staining with DAPI shows the location of nuclei of A549 cells. The percentage of TFEB expression in the nuclei of A549 cells was calculated as follows: (number of A549 cells that expressed TFEB in the nuclei of A549 cells/total number of A549 cells) × 100. Results are from three independent experiments, and data are means plus standard errors of the means (SEM) (error bars). Values that are significantly different (P < 0.05) between untreated (control [CTL]) and treated groups are indicated by an asterisk.
- FIG 2
Role of TFEB in A. baumannii internalization by A549 cells. (A and C) Immunoblot analysis of A549 cells transfected with scrambled (SC) and TFEB siRNA and pEGFP-N1-TFEB for 48 and 24 h, respectively. Values in the bar graphs are the percentages of TFEB level in control (CTL) and transfected A549 cells. (B, D, and E) A549 cells were transfected with SC and TFEB siRNA and pEGFP-N1-TFEB and infected with 108 CFU/ml A. baumannii ATCC 17978 for 2, 4, or 8 h. An assay of adherence and invasion of A. baumannii ATCC 17978 into A549 cells was performed as described in Materials and Methods. The effect of TFEB siRNA and pEGFP-N1-TFEB mediated TFEB depletion and overexpression, respectively, on adherence or invasion of A. baumannii ATCC 17978. The percentages of total nontransfected A549 cells and A549 cells incubated with A. baumannii ATCC 17978 are shown for both adhesion and invasion. Results are from three independent experiments, and data are the means plus SEM (error bars). Values for untransfected and transfected groups in panels B and E that are significantly different (P < 0.05) are indicated by an asterisk. Values in panel E that are significantly different (P < 0.05) are indicated by bars and asterisks as follows: **, ATCC 17978 cells transfected with siRNA TFEB and ATCC 17978 cells or ATCC 17978 cells transfected with pEGFP-N1-TFEB; ***, ATCC 17978 cells and ATCC 17978 cells transfected with pEGFP-N1-TFEB.
- FIG 3
Evaluation of the role of the autophagosome-lysosome system in A. baumannii intracellular trafficking. (A) The lysosomes in A549 cells were incubated with A. baumannii ATCC 17978 for 2 h, immunostained, and imaged by immunofluorescence microscopy. Acidic organelles were detected with LysoTracker red (75 nM), and mitochondria were detected with MitoTracker green (250 nM). The values for labeling of lysosomes in infected A549 cells in the bar graph are percentages compared to the value for noninfected cells. Values that are significantly different (P < 0.05) are indicated by bars and asterisks as follows: *, ATCC 17978 and control (CTL) cells; **, ATCC 17978 and heat-killed (HK) ATCC 17978 cells. (B) A. baumannii ATCC 17978 and ATCC 17978 HK invasion into A549 cells for up to 8 h of infection. (C) Western blot analysis of cathepsin D in A549 cells infected with A. baumannii ATCC 17978 for 2 h. Blots were part of the same internally controlled experiment in Fig. 5B. Values are expressed as the percentage of cathepsin D expression level in control and infected A549 cells. Values that are significantly different (P < 0.05) are indicated by an asterisk. (D) A. baumannii ATCC 17978 invasion into A549 cells pretreated for 30 min with NH4Cl or KCl for various lengths of time up to 8 h of infection. Values that are significantly different (P < 0.05) are indicated by asterisks as follows: *, ATCC 17978 cells and ATCC 17978 cells treated with NH4Cl; **, ATCC 17978 and ATCC 17978 treated with KCl.
- FIG 4
Bacterial acid resistance. (A) Bacterial growth in LB medium during 24 h under acidic or neutral conditions. (B) pH determination during 24 h of LB medium in the presence of A. baumannii ATCC 17978.
- FIG 5
A. baumannii stimulates the autophagy. (A) Expression of human autophagy genes after A. baumannii infection. Total RNA was isolated from A549 cells infected with A. baumannii ATCC 17978 and uninfected cells. cDNAs were synthesized by reverse transcription of the total RNA. A real-time PCR analysis was performed by using the Stratagene Mx3005p system. Samples were normalized to beta-2-microglobulin. Human autophagy gene expression after infection is represented by the heat map. Results are representative of two independent experiments. (B) Western blot analysis of LC3B in A549 cells infected with A. baumannii ATCC 17978 for 2 h. The blots were part of the same internally controlled experiment in Fig. 3C. Results are representative of three independent experiments. The solid white line separates the spliced portions between control and infected cells.
- FIG 6
A. baumannii activates the autophagosome-lysosome system. (A and B) Additive effect of autophagy and TFEB on A. baumannii internalization by A549 cells. A549 cells were transfected with scrambled (SC) or TFEB siRNA or pEGFP-N1-TFEB and treated with pepstatin (20 µg/ml), bafilomycin (0.8 µM), or wortmannin (1 µM), and infected with 108 CFU/ml A. baumannii ATCC 17978 for 2 h to study bacterial adherence and invasion to host cells. Results are representative of three independent experiments, and data are the means plus SEM. Values for treated and untreated groups that are significantly different (P < 0.05): are indicated by an asterisk. CTL, control.
- FIG 7
HLH-30 is required for C. elegans survival against A. baumannii infection. (A) Longevity assay of hlh-30(tm1978) mutant worms compared to wild-type control worms growing on A. baumannii at 20°C. Survival of wild-type (n = 140) and hlh-30(tm1978) mutants (n = 134) of C. elegans when growing at the same temperature in the nonpathogenic E. coli OP50 or in the pathogenic A. baumannii ATCC 17978. Two independent experiments were performed, and the data for both experiments are shown (***, P < 0.001). (B) Brood size quantification of wild-type and hlh-30(tm1978) mutants growing from eggs on E. coli OP50 and A. baumannii. Values are means plus SEM for 30 individuals (***, P < 0.001; ns, not significantly different). (C to H) Differential interference contrast (DIC) micrographs of wild-type and hlh-30(tm1978) mutants growing from eggs on E. coli OP50 and A. baumannii showing alterations in worm germline like enlarged oocytes and embryos (white asterisks in panels F, G, and H), binucleated oocytes (white arrowhead in panel F), vulva extrusion (white arrowhead in panel G), and extensive blebbing in the intestine, vulva, and head regions (H). The solid white line in panel D separates the spliced portions. (I) Fluorescence micrographs (left) and quantification (right) of HLH-30::GFP nuclear translocation in intestinal cells of transgenic worms expressing the integrated array sqIs17 [Phlh-30::hlh-30::GFP; rol-6(su1006)] when grown in S. aureus 29213 or A. baumannii ATCC 17978. Bars, 100 µm (C, D, E, and I) and 50 µm (F, G, and H). D, dorsal; V, ventral; A, anterior; P, posterior.
Supplemental Material
FIG S1
Effect of TFEB siRNA on the cellular viability of A549 cells. A549 cells were transfected with siRNA TFEB for 24 h. An assay of A549 cell viability was determined by using the MTT assay. Results are representative of three independent experiments, and values are means plus SEM (error bars). CTL, control; SC, scrambled. Download FIG S1, PDF file, 0.1 MB.
Copyright © 2018 Parra-Millán et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
FIG S2
Effects of inhibitors of the autophagosome-lysosome system on the cellular viability of A549 cells. A549 cells were treated with pepstatin (20 µg/ml), bafilomycin (0.8 µM), or wortmannin (1 µM) for 24 h. An assay of the viability of A549 cells was determined by using the MTT assay. Results are representative of three independent experiments, and values are means plus SEM. CTL, control. Download FIG S2, PDF file, 0.1 MB.
Copyright © 2018 Parra-Millán et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
