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The autophagic machinery ensures nonlytic transmission of mycobacteria.

ABSTRACT: In contrast to mechanisms mediating uptake of intracellular bacterial pathogens, bacterial egress and cell-to-cell transmission are poorly understood. Previously, we showed that the transmission of pathogenic mycobacteria between phagocytic cells also depends on nonlytic ejection through an F-actin based structure, called the ejectosome. How the host cell maintains integrity of its plasma membrane during the ejection process was unknown. Here, we reveal an unexpected function for the autophagic machinery in nonlytic spreading of bacteria. We show that ejecting mycobacteria are escorted by a distinct polar autophagocytic vacuole. If autophagy is impaired, cell-to-cell transmission is inhibited, the host plasma membrane becomes compromised and the host cells die. These findings highlight a previously unidentified, highly ordered interaction between bacteria and the autophagic pathway and might represent the ancient way to ensure nonlytic egress of bacteria.

SUBMITTER: Gerstenmaier L

PROVIDER: S-EPMC4343083 | biostudies-other | 2015 Feb

REPOSITORIES: biostudies-other

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The autophagic machinery ensures nonlytic transmission of mycobacteria.

Gerstenmaier Lilli L Pilla Rachel R Herrmann Lydia L Herrmann Hendrik H Prado Monica M Villafano Geno J GJ Kolonko Margot M Reimer Rudolph R Soldati Thierry T King Jason S JS Hagedorn Monica M

Proceedings of the National Academy of Sciences of the United States of America 20150202 7

In contrast to mechanisms mediating uptake of intracellular bacterial pathogens, bacterial egress and cell-to-cell transmission are poorly understood. Previously, we showed that the transmission of pathogenic mycobacteria between phagocytic cells also depends on nonlytic ejection through an F-actin based structure, called the ejectosome. How the host cell maintains integrity of its plasma membrane during the ejection process was unknown. Here, we reveal an unexpected function for the autophagic ...[more]

PMID: 25646440

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Project description:Simple Summary Tuberculosis is a life-threatening disease caused by infection with mycobacteria. These bacteria can grow inside the cells of our immune system, particularly in macrophages, one of the first cells responding to an infection. Macrophages activate several defense mechanisms to combat the infection, but mycobacteria are notorious for escaping these mechanisms. One of the macrophage defense mechanisms is autophagy, a vital process that keeps cells in a healthy condition by clearing worn-out cell parts or microbial invaders. Stimulating autophagy is explored as a potential strategy to improve the treatment of tuberculosis, but this will require a better understanding of how mycobacteria interact with the autophagy machinery. In this study, we infected zebrafish larvae with a mycobacterial species closely related to the human tuberculosis pathogen. Zebrafish larvae are transparent and ideally suited for microscopic imaging of the early stages of the infection process. Therefore, we could observe that mycobacteria, when taken up by macrophages, reside in vesicles formed by the autophagy machinery. We found that these vesicles dynamically change shapes and that they could serve as a temporary hiding place for mycobacteria, facilitating the spreading of the infection to other tissues. Abstract Existing drug treatment against tuberculosis is no match against the increasing number of multi-drug resistant strains of its causative agent, Mycobacterium tuberculosis (Mtb). A better understanding of how mycobacteria subvert the host immune defenses is crucial for developing novel therapeutic strategies. A potential approach is enhancing the activity of the autophagy machinery, which can direct bacteria to autophagolysosomal degradation. However, the interplay specifics between mycobacteria and the autophagy machinery must be better understood. Here, we analyzed live imaging data from the zebrafish model of tuberculosis to characterize mycobacteria-autophagy interactions during the early stages of infection in vivo. For high-resolution imaging, we microinjected fluorescent Mycobacterium marinum (Mm) into the tail fin tissue of zebrafish larvae carrying the GFP-LC3 autophagy reporter. We detected phagocytosed Mm clusters and LC3-positive Mm-containing vesicles within the first hour of infection. LC3 associations with these vesicles were transient and heterogeneous, ranging from simple vesicles to complex compound structures, dynamically changing shape by fusions between Mm-containing and empty vesicles. LC3-Mm-vesicles could adopt elongated shapes during cell migration or alternate between spacious and compact morphologies. LC3-Mm-vesicles were also observed in cells reverse migrating from the infection site, indicating that the autophagy machinery fails to control infection before tissue dissemination.

Project description:Mitophagy is a conserved intracellular catabolic process responsible for the selective removal of dysfunctional or superfluous mitochondria to maintain mitochondrial quality and need in cells. Here, we examine the mechanisms of receptor-mediated mitophagy activation, with the focus on BNIP3L/NIX mitophagy receptor, proven to be indispensable for selective removal of mitochondria during the terminal differentiation of reticulocytes. The molecular mechanisms of selecting damaged mitochondria from healthy ones are still very obscure. We investigated BNIP3L dimerization as a potentially novel molecular mechanism underlying BNIP3L-dependent mitophagy. Forming stable homodimers, BNIP3L recruits autophagosomes more robustly than its monomeric form. Amino acid substitutions of key transmembrane residues of BNIP3L, BNIP3LG204A or BNIP3LG208V, led to the abolishment of dimer formation, resulting in the lower LC3A-BNIP3L recognition and subsequently lower mitophagy induction. Moreover, we identified the serine 212 as the main amino acid residue at the C-terminal of BNIP3L, which extends to the intermembrane space, responsible for dimerization. In accordance, the phosphomimetic mutation BNIP3LS212E leads to a complete loss of BNIP3L dimerization. Thus, the interplay between BNIP3L phosphorylation and dimerization indicates that the combined mechanism of LIR phosphorylation and receptor dimerization is needed for proper BNIP3L-dependent mitophagy initiation and progression.Abbreviations: AMBRA1: autophagy and beclin 1 regulator 1; Baf A1: bafilomycin A1; BH3: BCL2 homology 3; BNIP3: BCL2 interacting protein 3; BNIP3L/NIX: BCL2 interacting protein 3 like; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; CoCl2: cobalt (II) chloride; FKBP8: FKBP prolyl isomerase 8; FUNDC1: FUN14 domain containing 1; GABARAP: GABA type A receptor-associated protein; GST: glutathione S-transferase; IMM: inner mitochondrial membrane; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; OMM: outer mitochondrial membrane; PHB2: prohibitin 2; PI: propidium iodide; PINK1: PTEN induced kinase 1; TM: transmembrane domain; TOMM20: translocase of outer mitochondrial membrane 20.

Project description:Rationale: Healthcare-associated transmission of nontuberculous mycobacteria (NTM) among people with cystic fibrosis (pwCF) has been investigated at CF centers worldwide, with conflicting conclusions. We investigated transmission at the Colorado Adult CF Program. Objectives: To systematically investigate healthcare-associated transmission and/or acquisition of NTM to determine similarity among respiratory and environmental isolates, and to compare home residence watershed mapping among pwCF having genetically similar NTM isolates. Methods: Whole-genome sequencing of NTM isolates from 80 pwCF was conducted to identify genetically similar isolate clusters (⩽30 SNP differences). Epidemiology, comparison of respiratory and environmental isolates, and home residence watershed mapping were analyzed. Measurements and Main Results: Whole-genome sequencing analysis revealed 11 clusters of NTM [6 Mycobacterium abscessus subspecies (ssp.) abscessus, 1 M. abscessus ssp. massiliense, 2 Mycobacterium avium, and 2 Mycobacterium intracellulare] among pwCF. Epidemiologic investigation demonstrated opportunities for healthcare-associated transmission in two M. abscessus and two M. avium clusters. Respiratory and healthcare environmental isolate comparisons revealed no genetic similarity. Individuals comprising one M. abscessus cluster, with no plausible healthcare-associated transmission, resided in the same watershed. Conclusions: This study suggests healthcare-associated transmission of M. abscessus is rare and includes a report of potential healthcare-associated transmission of M. avium among pwCF. One M. abscessus cluster possibly had common acquisition arising from residing in the same watershed. The presence of genetically similar isolates is insufficient to demonstrate healthcare-associated NTM transmission. Standardizing epidemiologic investigation, combined with environmental sampling and watershed analysis, will improve understanding of the frequency and nature of healthcare-associated NTM transmission among pwCF.

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The autophagic machinery ensures nonlytic transmission of mycobacteria.

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The autophagic machinery ensures nonlytic transmission of mycobacteria.

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