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 |
REPOSITORIES: biostudies-literature
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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.
| S-EPMC4386821 | biostudies-literature
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
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:Hematogenous dissemination of Mycobacterium tuberculosis (M. tb) occurs during both primary and reactivated tuberculosis (TB). Although hematogenous dissemination occurs in non-HIV TB patients, in ∼80% of these patients, TB manifests exclusively as pulmonary disease. In contrast, extrapulmonary, disseminated, and/or miliary TB is seen in 60-70% of HIV-infected TB patients, suggesting that hematogenous dissemination is likely more common in HIV+ patients. To understand M. tb adaptation to the blood environment during bacteremia, we have studied the transcriptome of M. tb replicating in human whole blood. To investigate if M. tb discriminates between the hematogenous environments of immunocompetent and immunodeficient individuals, we compared the M. tb transcriptional profiles during replication in blood from HIV- and HIV+ donors. Our results demonstrate that M. tb survives and replicates in blood from both HIV- and HIV+ donors and enhances its virulence/pathogenic potential in the hematogenous environment. The M. tb blood-specific transcriptome reflects suppression of dormancy, induction of cell-wall remodeling, alteration in mode of iron acquisition, potential evasion of immune surveillance, and enhanced expression of important virulence factors that drive active M. tb infection and dissemination. These changes are accentuated during bacterial replication in blood from HIV+ patients. Furthermore, the expression of ESAT-6, which participates in dissemination of M. tb from the lungs, is upregulated in M. tb growing in blood, especially during growth in blood from HIV+ patients. Preliminary experiments also demonstrate that ESAT-6 promotes HIV replication in U1 cells. These studies provide evidence, for the first time, that during bacteremia, M. tb can adapt to the blood environment by modifying its transcriptome in a manner indicative of an enhanced-virulence phenotype that favors active infection. Additionally, transcriptional modifications in HIV+ blood may further accentuate M. tb virulence and drive both M. tb and HIV infection.
| S-EPMC3995690 | biostudies-literature
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.
| S-EPMC5005354 | biostudies-literature
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.
| S-ECPF-GEOD-52105 | biostudies-other
Project description:Alveolar macrophages (AMs) are the first cells to be infected during Mycobacterium tuberculosis (M.tb.) infection. Thus, the AM response to infection is the first of many steps leading to initiation of the adaptive immune response required for efficient control of infection. A hallmark of M.tb. infection is the slow initiation of the adaptive response, yet the mechanisms responsible for this are largely unknown. To study the initial AM response to infection, we developed a system to identify, sort, and analyze M.tb.-infected AMs from the lung within the first 10 days of infection. In contrast to what has been previously described using in vitro systems, M.tb.-infected AMs up-regulate a cell-protective antioxidant transcriptional signature that is dependent on the lung environment but not bacterial virulence. Computational approaches including pathway analysis and transcription factor motif enrichment analysis identify NRF2 as a master regulator of the response. Using knockout mouse models, we demonstrate that NRF2 drives expression of the cell-protective signature in AMs and impairs the control of early bacterial growth. AMs up-regulate a substantial pro-inflammatory response to M.tb. infection only 10 days after infection, yet comparisons with bystander AMs from the same infected animals demonstrate that M.tb.-infected AMs generate a less robust inflammatory response than the uninfected cells around them. Our findings demonstrate that the initial macrophage response to M.tb. in the lung is far less inflammatory than has previously been described by in vitro systems and may impede the overall host response to infection.
| S-EPMC6910245 | biostudies-literature
Project description:The respiratory tract is considered the main port of entry of Mycobacterium leprae, the causative agent of leprosy. However, the great majority of individuals exposed to the leprosy bacillus will never manifest the disease due to their capacity to develop protective immunity. Besides acting as a physical barrier, airway epithelium cells are recognized as key players by initiating a local innate immune response that orchestrates subsequent adaptive immunity to control airborne infections. However, to date, studies exploring the interaction of M. leprae with the respiratory epithelium have been scarce. In this work, the capacity of M. leprae to immune activate human alveolar epithelial cells was investigated, demonstrating that M. leprae-infected A549 cells secrete significantly increased IL-8 that is dependent on NF-κB activation. M. leprae was also able to induce IL-8 production in human primary nasal epithelial cells. M. leprae-treated A549 cells also showed higher expression levels of human β-defensin-2 (hβD-2), MCP-1, MHC-II and the co-stimulatory molecule CD80. Furthermore, the TLR-9 antagonist inhibited both the secretion of IL-8 and NF-κB activation in response to M. leprae, indicating that bacterial DNA sensing by this Toll-like receptor constitutes an important innate immune pathway activated by the pathogen. Finally, evidence is presented suggesting that extracellular DNA molecules anchored to Hlp, a histone-like protein present on the M. leprae surface, constitute major TLR-9 ligands triggering this pathway. The ability of M. leprae to immune activate respiratory epithelial cells herein demonstrated may represent a very early event during infection that could possibly be essential to the generation of a protective response.
| S-EPMC8397448 | biostudies-literature
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.
| S-EPMC3784436 | biostudies-literature
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.
| S-EPMC4995911 | biostudies-literature