title: Infection of A549 human type II epithelial cells with Mycobacterium tuberculosis induces changes in mitochondrial morphology, distribution and mass that are dependent on the early secreted antigen, ESAT-6.
abstract: Pulmonary infection by Mycobacterium tuberculosis (Mtb) involves the invasion of alveolar epithelial cells (AECs). We used Mitotracker Red(®) to assess changes in mitochondrial morphology/distribution and mass from 6 to 48 h post infection (hpi) by confocal microscopy and flow cytometry in Mtb-infected A549 type II AECs. During early infection there was no effect on mitochondrial morphology, however, by 48 hpi mitochondria appeared fragmented and concentrated around the nucleus. In flow cytometry experiments, the median fluorescence intensity (MFI) decreased by 44% at 48 hpi; double-labelling using antibodies to the integral membrane protein COXIV revealed that these changes were due to a decrease in mitochondrial mass. These changes did not occur with the apathogenic strain, Mycobacterium bovis BCG. ESAT-6 is a virulence factor present in Mtb Erdman but lacking in M. bovis BCG. We performed similar experiments using Mtb Erdman, an ESAT-6 deletion mutant and its complement. MFI decreased at 48 hpi in the parent and complemented strains versus uninfected controls by 52% and 36% respectively; no decrease was detected in the deletion mutant. These results indicate an involvement of ESAT-6 in the perturbation of mitochondria induced by virulent Mtb in AECs and suggest mitophagy may play a role in the infection process.
authors.affiliation: Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, GA, USA.
authors.forename: Kari
authors.initials: K
authors.lastname: Fine-Coulson
authors.affiliation: Department of Large Animal Medicine, College of Veterinary Medicine, University of Georgia, Athens, GA, USA.
authors.forename: Steeve
authors.initials: S
authors.lastname: Giguère
authors.affiliation: Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, GA, USA.
authors.forename: Frederick D
authors.initials: FD
authors.lastname: Quinn
authors.affiliation: Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, GA, USA. Electronic address: bjreaves@uga.edu.
authors.forename: Barbara J
authors.initials: BJ
authors.lastname: Reaves
mesh_terms: D000942:Antigens, Bacterial / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D004847:Epithelial Cells / Q000382:microbiology*; D005434:Flow Cytometry; D017353:Gene Deletion; D005816:Genetic Complementation Test; D054884:Host-Pathogen Interactions; D006801:Humans; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000502:physiology* / Q000648:ultrastructure*; D009163:Mycobacterium bovis / Q000502:physiology; D009169:Mycobacterium tuberculosis / Q000502:physiology*; D013194:Staining and Labeling; D013997:Time Factors; D037521:Virulence Factors / Q000235:genetics / Q000378:metabolism
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; C093276:ESAT-6 protein, Mycobacterium tuberculosis; D037521:Virulence Factors
keywords: Alveolar epithelial cells; ESAT-6; Inflammasome; Mitochondria; Mitophagy; Mycobacterium tuberculosis