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Cited by 131Tuberculosis Research and EpidemiologyBacterial Genetics and BiotechnologyMycobacterium research and diagnosis

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The ESX-3 Secretion System Is Necessary for Iron and Zinc Homeostasis in Mycobacterium tuberculosis

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Serafini, Agnese, Pisu, Davide, Palù, Giorgio, Rodriguez, G. Marcela, Manganelli, Riccardo·2013·PLoS ONE
DOI 10.1371/journal.pone.0078351PMC PMC3796483
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Abstract

ESX-3 is one of the five type VII secretion systems encoded by the Mycobacterium tuberculosis genome. We recently showed the essentiality of ESX-3 for M. tuberculosis viability and proposed its involvement in iron and zinc metabolism. In this study we confirmed the role of ESX-3 in iron uptake and its involvement in the adaptation to low zinc environment in M. tuberculosis. Moreover, we unveiled functional differences between the ESX-3 roles in M. tuberculosis and M. smegmatis showing that in the latter ESX-3 is only involved in the adaptation to iron and not to zinc restriction. Finally, we also showed that in M. tuberculosis this secretion system is essential for iron and zinc homeostasis not only in conditions in which the concentrations of these metals are limiting but also in metal sufficient conditions.

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Figures

Figure 1 Growth of ESX-3 depleted M. tuberculosis. Strains were grown in rolling bottles with or without ATc 200 ng/ml in A) CMM (50μM FeCl3 and 3,67 μM ZnSO4) first dilution; B) CMM second dilution; C) LIMM (2μM FeCl3 and 3,67μM ZnSO4) first dilution; D) LZMM (50μM FeCl3 and no ZnSO4) first dilution; E) Middlebrook 7H9 (≈150μM ferric ammonium citrate and ≈3,67μM ZnSO4) first dilution. The arrow indicates the time point at which the second dilution was performed. Growth of the parental strain in all tested conditions was the same with or without ATc: only growth curve of cultures with ATc are shown. Values represent the average of three independent experiments with relative standard errors.
Figure 1 Growth of ESX-3 depleted M. tuberculosis. Strains were grown in rolling bottles with or without ATc 200 ng/ml in A) CMM (50μM FeCl3 and 3,67 μM ZnSO4) first dilution; B) CMM second dilution; C) LIMM (2μM FeCl3 and 3,67μM ZnSO4) first dilution; D) LZMM (50μM FeCl3 and no ZnSO4) first dilution; E) Middlebrook 7H9 (≈150μM ferric ammonium citrate and ≈3,67μM ZnSO4) first dilution. The arrow indicates the time point at which the second dilution was performed. Growth of the parental strain in all tested conditions was the same with or without ATc: only growth curve of cultures with ATc are shown. Values represent the average of three independent experiments with relative standard errors.CC BY · PubMed Central
Figure 2 Expression analysis in ESX-3 depleted M. tuberculosis in defined medium. Quantitative real-time RT-PCR performed on total RNA extracted from cells grown in CMM (50μM FeCl3 and 3,67 μM ZnSO4) and in LIMM (2μM FeCl3 and 3,67μM ZnSO4). The graphs represent the ratio between expression levels (normalized to sigA mRNA) in the conditional esx-3 mutant (TB79) and its parental strain (TB38) grown with or without ATc (+ symbol) in A) CMM and B) LIMM. Values represent the average of three independent experiments with the relative standard errors.
Figure 2 Expression analysis in ESX-3 depleted M. tuberculosis in defined medium. Quantitative real-time RT-PCR performed on total RNA extracted from cells grown in CMM (50μM FeCl3 and 3,67 μM ZnSO4) and in LIMM (2μM FeCl3 and 3,67μM ZnSO4). The graphs represent the ratio between expression levels (normalized to sigA mRNA) in the conditional esx-3 mutant (TB79) and its parental strain (TB38) grown with or without ATc (+ symbol) in A) CMM and B) LIMM. Values represent the average of three independent experiments with the relative standard errors.CC BY · PubMed Central
Figure 3 Streptonigrin sensitivity of the M. tuberculosis esx-3 conditional mutant. Cells in exponential phase were treated with 5μg/ml STN (5X MIC) for 48h. The graphs show the survival rate in cultures treated 48 h with STN in respect to untreated cultures (see methods). The values represent the average of three independent experiments with relative standard errors. Asterisks represent statistically significant differences (p<0.05) respect to the conditional mutant (TB79) grown with ATc.
Figure 3 Streptonigrin sensitivity of the M. tuberculosis esx-3 conditional mutant. Cells in exponential phase were treated with 5μg/ml STN (5X MIC) for 48h. The graphs show the survival rate in cultures treated 48 h with STN in respect to untreated cultures (see methods). The values represent the average of three independent experiments with relative standard errors. Asterisks represent statistically significant differences (p<0.05) respect to the conditional mutant (TB79) grown with ATc.CC BY · PubMed Central
Figure 4 Fe3 carboxymycobactin (CM) uptake in ESX-3-depleted M. tuberculosis. Incorporation of Fe55-CM in TB79 cultured in the presence or absence of ATc was measured as described in materials and methods. The results are the average of duplicates in a representative experiment that was repeated 4 times.
Figure 4 Fe3 carboxymycobactin (CM) uptake in ESX-3-depleted M. tuberculosis. Incorporation of Fe55-CM in TB79 cultured in the presence or absence of ATc was measured as described in materials and methods. The results are the average of duplicates in a representative experiment that was repeated 4 times.CC BY · PubMed Central
Figure 5 Growth in different metals concentrations and streptonigrin sensitivity of the M. smegmatis esx-3 null mutants. The wild type strain mc2155 and its derivatives mutants esx-3/fxbA, esx-3, and fxbA were grown in shaking cultures in MM with different iron and zinc concentrations: (A) CMM (50μM FeCl3 and 3,67 μM ZnSO4); (B) LIMM (2μM FeCl3 and 3,67μM ZnSO4), (C) LZMM (50μM FeCl3 and no ZnSO4 added). Cells were pre-grown in CMM. The experiment was repeated three times. Data from one representative experiment are shown. D) Sensitivity to STN: cells in exponential phase were treated with 5μg/ml STN (5X MIC) for 30’; the graphs shows the survival rate in cultures treated 30 min with STN compared with untreated cultures (see methods). The values represent the average of three independent experiments with relative standard errors. Asterisks represent statistically significant differences (p<0.05) respect to the wild type strain.
Figure 5 Growth in different metals concentrations and streptonigrin sensitivity of the M. smegmatis esx-3 null mutants. The wild type strain mc2155 and its derivatives mutants esx-3/fxbA, esx-3, and fxbA were grown in shaking cultures in MM with different iron and zinc concentrations: (A) CMM (50μM FeCl3 and 3,67 μM ZnSO4); (B) LIMM (2μM FeCl3 and 3,67μM ZnSO4), (C) LZMM (50μM FeCl3 and no ZnSO4 added). Cells were pre-grown in CMM. The experiment was repeated three times. Data from one representative experiment are shown. D) Sensitivity to STN: cells in exponential phase were treated with 5μg/ml STN (5X MIC) for 30’; the graphs shows the survival rate in cultures treated 30 min with STN compared with untreated cultures (see methods). The values represent the average of three independent experiments with relative standard errors. Asterisks represent statistically significant differences (p<0.05) respect to the wild type strain.CC BY · PubMed Central
Figure 6 Growth in hemin-supplemented medium. The strains were grown in NIMM (3,67μM ZnSO4 and no FeCl3) with or without 50 µM DFO and 5 or 10 µM hemin. (A) M. tuberculosis esx-3 conditional mutant (TB79) and its parental strain (TB38) grown with or without 200ng/ml of ATc. The growth of the parental strain in all tested conditions was the same with or without ATc and only growth curve of cultures with ATc are shown. (B) M. smegmatis esx-3/fxbA mutant and its parental wild type strain. Values represent one representative experiment out of three performed.
Figure 6 Growth in hemin-supplemented medium. The strains were grown in NIMM (3,67μM ZnSO4 and no FeCl3) with or without 50 µM DFO and 5 or 10 µM hemin. (A) M. tuberculosis esx-3 conditional mutant (TB79) and its parental strain (TB38) grown with or without 200ng/ml of ATc. The growth of the parental strain in all tested conditions was the same with or without ATc and only growth curve of cultures with ATc are shown. (B) M. smegmatis esx-3/fxbA mutant and its parental wild type strain. Values represent one representative experiment out of three performed.CC BY · PubMed Central
Figure 7 Infection of THP1-derived macrophages. THP1-derived macrophages were infected with conditional mutant (TB79) and parental strain (TB38) at an MOI of 1:20 in presence or absence of 200 ng/ml of ATc. The growth curve was measured as CFU/ml after release of intracellular bacteria in 100 µl of 0.05% SDS . The values represent the average of two independent experiments with relative standard error.
Figure 7 Infection of THP1-derived macrophages. THP1-derived macrophages were infected with conditional mutant (TB79) and parental strain (TB38) at an MOI of 1:20 in presence or absence of 200 ng/ml of ATc. The growth curve was measured as CFU/ml after release of intracellular bacteria in 100 µl of 0.05% SDS . The values represent the average of two independent experiments with relative standard error.CC BY · PubMed Central

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Metadata

Authors
Serafini, A., Pisu, D., Palù, G., Rodriguez, G. M., & Manganelli, R.
Affiliations
University of Padua (IT)
Corresponding author
Manganelli, Riccardo
ORCID
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2013-10-14
Volume
8
Issue
10
Pages
e78351
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0078351
PMC
PMC3796483
OpenAlex
W2088316319
Cited by
131
References
71
Citations by year
2026: 12 · 2025: 13 · 2024: 6 · 2023: 8 · 2022: 11 · 2021: 10 · 2020: 10 · 2019: 13 · 2018: 12 · 2017: 11 · 2016: 11 · 2015: 9 · 2014: 5
FWCI
6.08
Type
journal article
Language
en
Update notices
correction (2014-01-06)
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, other-oa)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Figshare (submittedVersion, cc-by)
  • Padua Research Archive (University of Padova) (submittedVersion, cc-by)
Funders
  • European Commission (201762)
  • National Institutes of Health (FP7/2007-2013)
  • National Institute of Allergy and Infectious Diseases (R01AI044856, R56AI044856, AI044856)
Topics
  • Health Sciences › Medicine › Infectious Diseases › Tuberculosis Research and Epidemiology
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
  • Health Sciences › Medicine › Epidemiology › Mycobacterium research and diagnosis
Keywords
type VII secretion system, Mycobacterium tuberculosis, Mycobacterium smegmatis, iron homeostasis, zinc homeostasis, iron absorption, metal metabolism, bacterial virulence
MeSH terms
Bacterial Proteins, Cell Line, Cell Wall, Genes, Bacterial, Hemin, Homeostasis, Humans, Iron, Macrophages, Mutation, Mycobacterium tuberculosis, Oxazoles, Permeability, Streptonigrin, Transcription, Genetic, Zinc, Gene Expression Regulation, Bacterial, Mycobacterium smegmatis, Intracellular Space, Bacterial Secretion Systems

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