Impact of the elderly lung mucosa on Mycobacterium tuberculosis transcriptional adaptation during infection of alveolar epithelial cells

Abstract

Tuberculosis is one of the leading causes of death due to a single infectious agent. Upon infection, Mycobacterium tuberculosis (M.tb) is deposited in the alveoli and encounters the lung mucosa or alveolar lining fluid (ALF). We previously showed that, as we age, ALF presents a higher degree of oxidation and inflammatory mediators, which favors M.tb replication in human macrophages and alveolar epithelial cells (ATs). Here, we define the transcriptional profile of M.tb when exposed to healthy ALF from adult (A-ALF) or elderly (E-ALF) humans before and during infection of ATs. Prior to infection, M.tb exposure to E-ALF upregulated genes essential for bacterial host adaptation directly involved in M.tb pathogenesis. During infection of ATs, E-ALF exposed M.tb further upregulated genes involved in its ability to escape into the AT cytosol bypassing critical host defense mechanisms, as well as genes associated with defense against oxidative stress. These findings demonstrate how alterations in human ALF during the aging process can impact the metabolic status of M.tb, potentially enabling a greater adaptation and survival within host cells. Importantly, we present the first transcriptomic analysis on the impact of the elderly lung mucosa on M.tb pathogenesis during intracellular replication in ATs.IMPORTANCETuberculosis is one of the leading causes of death due to a single infectious agent. Upon infection, Mycobacterium tuberculosis (M.tb) is deposited in the alveoli and comes in contact with the alveolar lining fluid (ALF). We previously showed that elderly ALF favors M.tb replication in human macrophages and alveolar epithelial cells (ATs). Here we define the transcriptional profile of when exposed to healthy ALF from adult (A-ALF) or elderly (E-ALF) humans before and during infection of ATs. Prior to infection, exposure to E-ALF upregulates genes essential for bacterial host adaptation and pathogenesis. During infection of ATs, E-ALF further upregulates M.tb genes involved in its ability to escape into the AT cytosol, as well as genes for defense against oxidative stress. These findings demonstrate how alterations in human ALF during the aging process can impact the metabolic status of M.tb, potentially enabling a greater adaptation and survival within host cells.

Keywords: Mycobacterium tuberculosis; aging; alveolar epithelial cells; alveolar lining fluid; transcriptomics.

Fig 1

Illustration of the experimental conditions and data comparisons performed in this study. (A) Prior to and (B) during AT infection. (C) Experimental strategy for RNA-seq and data analyses. Figure created using BioRender (https://biorender.com/).

Fig 2

Differential expression analysis of ALF-exposed M.tb prior to AT infection. (A) The number of differentially expressed genes in ALF-exposed M.tb vs unexposed M.tb prior to infection obtained with the DESeq2 method in the iDEP software, with the following settings: Log₂ FC equal or greater than an absolute value of 1, FDR < 0.1. Upregulated genes are shown in white bars and downregulated genes in black bars. (B) Venn diagram of DEGs of ALF-exposed M.tb vs unexposed M.tb prior to infection showing unique and shared DEGs in ALF-exposed M.tb prior to ATs infection. (C) Total number of DEGs distributed by functional categories for A-ALF-exposed vs unexposed M.tb (A vs UE) and (D) for E-ALF exposed vs Unexposed M.tb (E vs UE) prior to ATs infection (represented as a percentage). Graphs were generated in GraphPad Prism v9.1.1. A, A-ALF-exposed M.tb; E, E-ALF-exposed M.tb; UE, unexposed M.tb.

Fig 3

Heatmaps of M.tb genes associated with ESX-secretion systems prior to AT infection. (A) ESX-1 and ESAT-6-like proteins; (B) ESX-2, ESX-3, and ESX-4; and (C) ESX-5 secretion system after A- or E-ALF exposure, prior to infection. Cells depict Log₂ FC values in ALF-exposed M.tb (A, Adult ALF; E, Elderly ALF) vs unexposed bacteria (UE), upregulated: red, downregulated: blue. Genes in bold indicate significant DEGs (Log₂ FC equal or greater than an absolute value of 1, and FDR < 0.1) for both conditions. Genes with an asterisk indicate significance in only one of the comparisons, highlighting differences between conditions. Notice the different scales used in A, B, and C, for better visualization of the results. Heatmaps were generated in GraphPad Prism v9.1.1.

Fig 4

Differential expression analysis of ALF-exposed M.tb during AT infection. (A) Number of differentially expressed genes during AT infection (ALF-exposed M.tb in ATs vs ALF-exposed M.tb before infection) obtained with the DESeq2 method in the iDEP software, with the following settings: Log₂ FC equal or greater than an absolute value of 1, and FDR < 0.1. Upregulated genes are shown in white bars and downregulated genes in black bars. (B) Venn diagram of DEGs during AT infection (ALF-exposed M.tb in ATs vs ALF-exposed M.tb before infection) showing unique and shared DEGs in ALF-exposed M.tb during AT infection. (C) Total number of DEGs distributed by functional categories during AT infection for A-ALF-exposed M.tb in ATs vs A-ALF-exposed M.tb prior infection (A_i vs A) and (D) for E-ALF-exposed M.tb in ATs vs E-ALF-exposed M.tb prior infection (E_i vs E) during ATs infection (represented as a percentage). Graphs were generated in GraphPad Prism v9.1.1. A, A-ALF-exposed M.tb; E, E-ALF-exposed M.tb; A_i, A-ALF-exposed M.tb during AT infection; E_i, E-ALF-exposed M.tb during AT infection.

Fig 5

M.tb genes associated with ESX-secretion systems and ROS defense mechanisms at 72 h post-infection (hpi) in ATs. (A) ESX-1 and ESAT-6-like proteins; (B) ESX-2, ESX-3, and ESX-4 secretion systems; (C) ESX-5 secretion system, and (D) ROS defense mechanisms, after infection of ATs with A-ALF or E-ALF-exposed M.tb for 72 h. Heatmap cells depict Log₂ FC values in ALF-exposed M.tb in ATs (A_i, Adult ALF-exposed M.tb during infection; E_i, Elderly ALF-exposed during infection) vs ALF-exposed M.tb before infection, upregulated: red, downregulated: blue. Genes in bold indicate significant DEGs (Log₂ FC equal greater than an absolute value of 1, and FDR < 0.1) for both conditions. Genes with an asterisk indicate significance in only one of the comparisons, highlighting differences between conditions. Notice the different scales used in A, B, C, and D for better visualization of the results. Heatmaps were generated in GraphPad Prism v9.1.1.

Fig 6

Schematic overview of the main findings in this study. Transcriptional profile of ALF-exposed-M.tb prior to and during intracellular replication in ATs. Figure created using BioRender (https://biorender.com/).