A lung-on-chip model of early M. tuberculosis infection reveals an essential role for alveolar epithelial cells in controlling bacterial growth
- Swiss Federal Institute of Technology in Lausanne (EPFL), Switzerland
- University of Cincinnati, United States
- Emulate Inc, United States
Abstract
We establish a murine lung-on-chip infection model and use time-lapse imaging to reveal the dynamics of host-Mycobacterium tuberculosis interactions at an air-liquid interface with a spatiotemporal resolution unattainable in animal models and to probe the direct role of pulmonary surfactant in early infection. Surfactant deficiency results in rapid and uncontrolled bacterial growth in both macrophages and alveolar epithelial cells. In contrast, under normal surfactant levels, a significant fraction of intracellular bacteria are non-growing. The surfactant-deficient phenotype is rescued by exogenous addition of surfactant replacement formulations, which have no effect on bacterial viability in the absence of host cells. Surfactant partially removes virulence-associated lipids and proteins from the bacterial cell surface. Consistent with this mechanism, the attenuation of bacteria lacking the ESX-1 secretion system is independent of surfactant levels. These findings may partly explain why smokers and elderly persons with compromised surfactant function are at increased risk of developing active tuberculosis.
Data availability
Figures in the main text include all the data for bacterial growth rates within the scatter plots, and all the data for qRT-PCR measurements and quantification of lamellar body size, number, and volume. A summary of the code used to calculate growth rates is included in the Materials and Methods. Annotated code used for data analysis written in Matlab, raw data for bacterial fluorescence intensity over time that was used to calculate growth rates, and image stacks related to Fig. 1, Fig. 1 - figure supplement 3 and Fig. 2 - figure supplement 1is available on Zenodo under DOI 10.5281/zenodo.4266198.
Article and author information
Author details
Funding
Human Frontier Science Program (Long-Term Fellowship,LT000231/2016-L)
- Vivek V Thacker
European Molecular Biology Organization (Long-Term Fellowship,921-2015)
- Vivek V Thacker
Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Project Funding,310030B_176397)
- John D McKinney
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: Animal protocols were reviewed and approved by EPFL's Chief Veterinarian, by the Service de la Consommation et des Affaires Vétérinaires of the Canton of Vaud, and by the Swiss Office Vétérinaire Fédéral (License Number VD 3434 for experiments involving organ collection and License Number VD 3472 for experiments involving infection with Mycobacterium tuberculosis)
Copyright
© 2020, Thacker et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 7,886
- views
-
- 1,092
- downloads
-
- 101
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
A lung-on-chip model of early M. tuberculosis infection reveals an essential role for alveolar epithelial cells in controlling bacterial growth
eLife 9:e59961.
https://doi.org/10.7554/eLife.59961
Further reading
-
- Biochemistry and Chemical Biology
- Microbiology and Infectious Disease
Mycofactocin is a redox cofactor essential for the alcohol metabolism of mycobacteria. While the biosynthesis of mycofactocin is well established, the gene mftG, which encodes an oxidoreductase of the glucose-methanol-choline superfamily, remained functionally uncharacterized. Here, we show that MftG enzymes are almost exclusively found in genomes containing mycofactocin biosynthetic genes and are present in 75% of organisms harboring these genes. Gene deletion experiments in Mycolicibacterium smegmatis demonstrated a growth defect of the ∆mftG mutant on ethanol as a carbon source, accompanied by an arrest of cell division reminiscent of mild starvation. Investigation of carbon and cofactor metabolism implied a defect in mycofactocin reoxidation. Cell-free enzyme assays and respirometry using isolated cell membranes indicated that MftG acts as a mycofactocin dehydrogenase shuttling electrons toward the respiratory chain. Transcriptomics studies also indicated remodeling of redox metabolism to compensate for a shortage of redox equivalents. In conclusion, this work closes an important knowledge gap concerning the mycofactocin system and adds a new pathway to the intricate web of redox reactions governing the metabolism of mycobacteria.
-
- Microbiology and Infectious Disease
Chlamydia trachomatis is an obligate intracellular bacterial pathogen with a unique developmental cycle. It differentiates between two functional and morphological forms: the elementary body (EB) and the reticulate body (RB). The signals that trigger differentiation from one form to the other are unknown. EBs and RBs have distinctive characteristics that distinguish them, including their size, infectivity, proteome, and transcriptome. Intriguingly, they also differ in their overall redox status as EBs are oxidized and RBs are reduced. We hypothesize that alterations in redox may serve as a trigger for secondary differentiation. To test this, we examined the function of the primary antioxidant enzyme alkyl hydroperoxide reductase subunit C (AhpC), a well-known member of the peroxiredoxins family, in chlamydial growth and development. Based on our hypothesis, we predicted that altering the expression of ahpC would modulate chlamydial redox status and trigger earlier or delayed secondary differentiation. Therefore, we created ahpC overexpression and knockdown strains. During ahpC knockdown, ROS levels were elevated, and the bacteria were sensitive to a broad set of peroxide stresses. Interestingly, we observed increased expression of EB-associated genes and concurrent higher production of EBs at an earlier time in the developmental cycle, indicating earlier secondary differentiation occurs under elevated oxidation conditions. In contrast, overexpression of AhpC created a resistant phenotype against oxidizing agents and delayed secondary differentiation. Together, these results indicate that redox potential is a critical factor in developmental cycle progression. For the first time, our study provides a mechanism of chlamydial secondary differentiation dependent on redox status.
