Transcriptional Profiling of Mycobacterium tuberculosis Replicating in the Human Type II Alveolar Epithelial Cell Line, A549.
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ABSTRACT:
SUBMITTER: Ryndak MB
PROVIDER: S-EPMC4527333 | biostudies-literature | 2015 Sep
REPOSITORIES: biostudies-literature
Publications
Transcriptional Profiling of <i>Mycobacterium tuberculosis</i> Replicating in the Human Type II Alveolar Epithelial Cell Line, A549.
Ryndak Michelle B MB Singh Krishna K KK Peng Zhengyu Z Laal Suman S
Genomics data 20150901
Alveolar epithelial cells outnumber alveolar macrophages by ~500 fold and increasing evidence suggests <i>Mycobacterium tuberculosis</i> may replicate dramatically in these cells during the initial weeks of infecting the lung [1,2]. Here, we report in experimental detail the transcriptional profiling of <i>Mycobacterium tuberculosis</i> replicating at 72 hr post-infection in the human type II alveolar epithelial cell line, A549, as compared to <i>Mycobacterium tuberculosis</i> growing logarithmi ...[more]
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Project description:Mycobacterium tuberculosis (M. tb) infection is initiated by the few bacilli inhaled into the alveolus. Studies in lungs of aerosol-infected mice provided evidence for extensive replication of M. tb in non-migrating, non-antigen-presenting cells in the alveoli during the first 2-3 weeks post-infection. Alveoli are lined by type II and type I alveolar epithelial cells (AEC) which outnumber alveolar macrophages by several hundred-fold. M. tb DNA and viable M. tb have been demonstrated in AEC and other non-macrophage cells of the kidney, liver, and spleen in autopsied tissues from latently-infected subjects from TB-endemic regions indicating systemic bacterial dissemination during primary infection. M. tb have also been demonstrated to replicate rapidly in A549 cells (type II AEC line) and acquire increased invasiveness for endothelial cells. Together, these results suggest that AEC could provide an important niche for bacterial expansion and development of a phenotype that promotes dissemination during primary infection. In the current studies, we have compared the transcriptional profile of M. tb replicating intracellularly in A549 cells to that of M. tb replicating in laboratory broth, by microarray analysis. Genes significantly upregulated during intracellular residence were consistent with an active, replicative, metabolic, and aerobic state, as were genes for tryptophan synthesis and for increased virulence (ESAT-6, and ESAT-6-like genes, esxH, esxJ, esxK, esxP, and esxW). In contrast, significant downregulation of the DevR (DosR) regulon and several hypoxia-induced genes was observed. Stress response genes were either not differentially expressed or were downregulated with the exception of the heat shock response and those induced by low pH. The intra-type II AEC M. tb transcriptome strongly suggests that AEC could provide a safe haven in which M. tb can expand dramatically and disseminate from the lung prior to the elicitation of adaptive immune responses.
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Project description:Infection with Mycobacterium tuberculosis (M. tb) is initiated when an aerosol droplet carrying a few bacilli is inhaled into an alveolus. Alveolar epithelial cells (AEC) (type II and type I) are among the first cells encountered by the infecting bacteria and greatly outnumber macrophages in the alveolus. M. tb replicates dramatically (>20,000 fold) in a “non-migrating” compartment in the lung prior to the development of the cell-mediated immune response in aerosol-infected mice (Wolf AJ, 2008). M. tb DNA has been found in type II AEC in autopsied lung tissue of latently infected individuals (Hernandez-Pando R et. al, 2000; Barrios-Payan J et. al 2012), and M. tb-infected AEC in broncho-alveolar lavage (BAL) and in sputum samples from TB patients indicates the infection of these cells in active as well as latent human TB (Eum SY et. al, 2010). M. tb has been shown to infect and replicate in the human type II AEC line, A549, and passaging of M. tb through A549 increases M. tb invasiveness (Bermudez LE et.al, 2002). In this work, we have used DNA microarray analysis to investigate the transcriptome of M. tb replicating in type II AEC (A549) compared to M. tb grown logarithmically in Middlebrook 7H9 broth media in order to identify M. tb adaptations to this intracellular environment as well as M. tb mechanisms/factors contributing to M. tb replication and increased invasiveness in primary infection.
2015-03-31 | GSE58466 | GEO
Project description:Infection with Mycobacterium tuberculosis (M. tb) is initiated when an aerosol droplet carrying a few bacilli is inhaled into an alveolus. Alveolar epithelial cells (AEC) (type II and type I) are among the first cells encountered by the infecting bacteria and greatly outnumber macrophages in the alveolus. M. tb replicates dramatically (>20,000 fold) in a “non-migrating” compartment in the lung prior to the development of the cell-mediated immune response in aerosol-infected mice (Wolf AJ, 2008). M. tb DNA has been found in type II AEC in autopsied lung tissue of latently infected individuals (Hernandez-Pando R et. al, 2000; Barrios-Payan J et. al 2012), and M. tb-infected AEC in broncho-alveolar lavage (BAL) and in sputum samples from TB patients indicates the infection of these cells in active as well as latent human TB (Eum SY et. al, 2010). M. tb has been shown to infect and replicate in the human type II AEC line, A549, and passaging of M. tb through A549 increases M. tb invasiveness (Bermudez LE et.al, 2002). In this work, we have used DNA microarray analysis to investigate the transcriptome of M. tb replicating in type II AEC (A549) compared to M. tb grown logarithmically in Middlebrook 7H9 broth media in order to identify M. tb adaptations to this intracellular environment as well as M. tb mechanisms/factors contributing to M. tb replication and increased invasiveness in primary infection. The global gene expression of M. tb H37Rv replicating in A549 cells at 72 hr was compared to that of M. tb grown to log phase in Middlebrook 7H9 media.
2015-03-31 | E-GEOD-58466 | biostudies-arrayexpress
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Project description:Mycobacterium tuberculosis can persist for years in the hostile environment of the host in a non-replicating or slowly replicating state. While active disease predominantly results from reactivation of a latent infection, the molecular mechanisms of M. tuberculosis reactivation are still poorly understood. We characterized the physiology and global transcriptomic profiles of M. tuberculosis during reactivation from hypoxia-induced non-replicating persistence. We found that M. tuberculosis reactivation upon reaeration was associated with a lag phase, in which the recovery of cellular physiological and metabolic functions preceded the resumption of cell replication. Enrichment analysis of the transcriptomic dynamics revealed changes to many metabolic pathways and transcription regulons/subnetworks that orchestrated the metabolic and physiological transformation in preparation for cell division. In particular, we found that M. tuberculosis reaeration lag phase is associated with down-regulation of persistence-associated regulons/subnetworks, including DosR, MprA, SigH, SigE, and ClgR, as well as metabolic pathways including those involved in the uptake of lipids and their catabolism. More importantly, we identified a number of up-regulated transcription regulons and metabolic pathways, including those involved in metal transport and remobilization, second messenger-mediated responses, DNA repair and recombination, and synthesis of major cell wall components. We also found that inactivation of the major alternative sigma factors SigE or SigH disrupted exit from persistence, underscoring the importance of the global transcriptional reprogramming during M. tuberculosis reactivation. Our observations suggest that M. tuberculosis lag phase is associated with a global gene expression reprogramming that defines the initiation of a reactivation process.
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Project description:A/Zhejiang/DTID-ZJU02/2009(H1N1) is a strain of the swine-origin influenza A(H1N1) virus isolated during the human swine flu outbreak of 2009. To analyze the miRNA expression profiles of A549 cells infected with A/Zhejiang/DTID-ZJU02/2009(H1N1) at 0, 24, 48, and 72 h post-infection (hpi) and investigate the relation between the miRNA expression profile and its pathogenesis, Human MicroRNA Array v2.0 was applied. At 24 hpi, 174 miRNAs were detected to change their expression compared with 0 hpi, 28 of them increased and 146 decreased. At 48 hpi, 214 changed miRNAs were detected, 21 of them increased and 193 decreased. At 72 hpi, 282 changed miRNAs were detected, 19 of them increased and 263 decreased. Targets of the 21 significantly differentially expressed miRNAs were analyzed by bioinformatics technology. The function categories of the predicted targets were analyzed by GO(Gene ontology) annotation. The signaling pathways involving the changed miRNAs were analyzed by KEGG(Kyoto Encyclopedia of Genes and Genomes) and GO annotation. Four key signaling pathways were identified, namely, the MAPK, apoptosis, JAK_STAT, and toll-like receptor signaling pathways. The apoptosis and MAPK signaling pathways were activated by all miRNAs, whereas the JAK_STAT and toll-like receptor signaling pathways were activated by some miRNAs but inhibited by the others, suggesting balance in the host–virus interaction. We also constructed and analyzed the protein-protein interaction network of all the predicted targets and found some key nodes. This finding provides a picture of miRNA expression in A549 cells infected with A/Zhejiang/DTID-ZJU02/2009(H1N1) as complete as possible, which may provide important information for investigation of H1N1 pathogenesis and therapeutic method. the miRNA expression profiles of A549 cells infected with A/Zhejiang/DTID-ZJU02/2009(H1N1) at 0, 24, 48, and 72 h post-infection (hpi) were analyzed and the relation between the miRNA expression profile and its pathogenesis was investigated.
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Project description:Increasing experimental evidence supports the idea that Mycobacterium tuberculosis has evolved strategies to survive within lysosomes of activated macrophages. To further our knowledge of M. tuberculosis response to the hostile lysosomal environment, we profiled the global transcriptional activity of M. tuberculosis when exposed to the lysosomal soluble fraction (SF) prepared from activated macrophages. Transcriptome sequencing (RNA-seq) analysis was performed using various incubation conditions, ranging from noninhibitory to cidal based on the mycobacterial replication or killing profile. Under inhibitory conditions that led to the absence of apparent mycobacterial replication, M. tuberculosis expressed a unique transcriptome with modulation of genes involved in general stress response, metabolic reprogramming, respiration, oxidative stress, dormancy response, and virulence. The transcription pattern also indicates characteristic cell wall remodeling with the possible outcomes of increased infectivity, intrinsic resistance to antibiotics, and subversion of the host immune system. Among the lysosome-specific responses, we identified the glgE-mediated 1,4 α-glucan synthesis pathway and a defined group of VapBC toxin/anti-toxin systems, both of which represent toxicity mechanisms that potentially can be exploited for killing intracellular mycobacteria. A meta-analysis including previously reported transcriptomic studies in macrophage infection and in vitro stress models was conducted to identify overlapping and nonoverlapping pathways. Finally, the Tap efflux pump-encoding gene Rv1258c was selected for validation. An M. tuberculosis ΔRv1258c mutant was constructed and displayed increased susceptibility to killing by lysosomal SF and the antimicrobial peptide LL-37, as well as attenuated survival in primary murine macrophages and human macrophage cell line THP-1.
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Project description:Macrophages and dendritic cells are recognized as key players in the defense against mycobacterial infection. Recent research has confirmed that alveolar epithelial cells (AECs) also play important roles against mycobacterium infections. Thus, establishing a stable cattle AEC line for future endogenous immune research on bacterial invasion is necessary. In the present study, we first purified and immortalized type II AECs (AEC II cells) by transfecting them with a plasmid containing the human telomerase reverse trancriptase gene. We then tested whether or not the immortalized cells retained the basic physiological properties of primary AECs by reverse-transcription polymerase chain reaction and Western blot. Finally, we tested the secretion capacity of immortalized AEC II cells upon stimulation by bacterial invasion. The cattle type II alveolar epithelial cell line (HTERT-AEC II) that we established retained lung epithelial cell characteristics: the cells were positive for surfactants A and B, and they secreted tumor necrosis factor-? and interleukin-6 in response to bacterial invasion. Thus, the cell line we established is a potential tool for research on the relationship between AECs and Mycobacterium tuberculosis.
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Project description: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.
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