Fig 1.
Infection of different A549 cells with SARS-CoV-2.
(A) Extracts from ACE2 expressing or parental A549 cells (20 µg protein per lane) were separated by SDS-PAGE and transferred for western blotting against ACE2 and beta-actin, respectively. (B) ACE2-A549 and A549 cells were infected at MOI 10 with SARS-CoV-2/WA01. Parallel infections were fixed at 6 and 16 hpi and stained with SARS-CoV-2 anti-nucleocapsid antibody (white) with Dapi (blue, nuclei). Scale bar is 100 μm. (C) Co-staining of the SARS-CoV-2 anti nucleocapsid antibody (red) with phalloidin (green) with Dapi (blue, nuclei) in ACE2-A549 cells at 16 hpi at MOI of 10. (D) Titers from ACE2-A549 and A549 cells infected with SARS-CoV-2 at an MOI 10. Both cell extracts (Total) and supernatants (Extracellular) were collected and titered at 0, 6, and 16 hpi. All data represented as the mean ± SD of three independently infected samples.
Fig 2.
Metabolic profiling analysis pipeline for infected cells.
(A) ACE2-A549 and A549 cells were infected with SARS-CoV-2 Isolate USA-WA1/2020 at an MOI of 10. Metabolites were extracted at multiple time points post infection. (B) Metabolic profiles are generated following LC-MS detection in the Mass Spectrometry Core Facility. (C) Data processing and analysis allows for both global metabolite profiling and pathway enrichment analysis. Image generated with Biorender.
Fig 3.
Global metabolite profiling of SARS-CoV-2 infected ACE2-A549 cells.
A total of 1085 metabolites were analyzed by (A) principal component analysis (PCA) and (B) supervised partial least-squares discriminant analysis (PLSDA). (C) Heatmap analysis of significant metabolites (n = 152) reveals temporal changes in metabolite phenotypes from 0 to 16 hpi. Mean intensities of each metabolite were clustered into 3 groups: 0 hpi (n = 6), 6 hpi (n = 5), and 16 hpi (n = 6). Normalized fold change of specific metabolites is relative to peak concentration across 0, 6, and 16 hpi.
Fig 4.
Putative metabolites identified from each of the four classes.
The normalized fold change value for 4 separate metabolites are presented within box plots. Individual replicates within each time point are represented as black spots. Average value is the yellow diamond. Each plot is labeled for the individual metabolite(s) based on m/z value identification.
Fig 5.
Global metabolite profiling of SARS-CoV-2 inoculated A549 cells.
Similar to ACE2-A549 cells, metabolite profiles from A549 cells were compared by (A) PCA and (B) PLSDA. (C) Heatmap analysis of significant metabolites (n = 377) reveals a different temporal phenotype in cells that are exposed to SARS-CoV-2 but remain uninfected. Mean intensities of each metabolite were clustered into 3 groups: 0 hpi (n = 6), 6 hpi (n = 6), and 16 hpi (n = 6). Normalized fold change of specific metabolites is relative to peak concentration across 0, 6, and 16 hpi.
Fig 6.
A549 cells have quantifiably different metabolic responses to SARS-CoV-2.
(A) Significant metabolites (FC > 1.5) from ACE2-A549 and A549 cells were compared to identify similar and different metabolites that change in response to SARS-CoV-2. (B) Pathway analysis on significant metabolites from ACE2-A549 and A549 cells. (C) Evaluating metabolic pathway changes relative to energy generation. Amino acids that were downregulated in ACE2-A549 are shown within the pathway highlighted in purple. Fatty acid metabolism was upregulated in A549 cells exposed to SARS-CoV-2, highlighted separately in blue. Both pathways directly or indirectly connect with components of the TCA cycle and glycolysis.
Table 1.
Altered metabolic pathways during SARS-CoV-2 infection of ACE2-A549 cells.
Table 2.
Altered metabolic pathways following exposure of A549 cells to SARS-CoV-2.