Functional membrane microdomains and the hydroxamate siderophore transporter ATPase FhuC govern Isd-dependent heme acquisition in Staphylococcus aureus

  1. Lea Antje Adolf
  2. Angelika Müller-Jochim
  3. Lara Kricks
  4. Jan-Samuel Puls
  5. Daniel Lopez
  6. Fabian Grein
  7. Simon Heilbronner  Is a corresponding author
  1. University of Tübingen, Germany
  2. Spanish National Research Council, Spain
  3. University of Bonn, Germany

Abstract

Sufficient access to transition metals such as iron is essential for bacterial proliferation and their active limitation within host tissues effectively restricts infection. To overcome iron limitation, the invasive pathogen Staphylococcus aureus uses the iron-regulated surface determinant (Isd) system to acquire hemoglobin-derived heme. While heme transport over the cell wall is well understood, its transport over the membrane is hardly investigated. In this study, we show the heme-specific permease IsdF to be energized by the general ATPase FhuC. Additionally, we show that IsdF needs appropriate location within the membrane for functionality. The membrane of S. aureus possesses special compartments (functional membrane microdomains - FMMs) to organize membrane complexes. We show IsdF to be associated with FMMs, to directly interact with the FMM scaffolding protein flotillin A (FloA) and to co-localize with the latter on intact bacterial cells. Additionally, Isd-dependent bacterial growth required FMMs and FloA. Our study shows that Isd-dependent heme acquisition requires a highly structured cell envelope to allow coordinated transport over the cell wall and membrane and it gives the first example of a bacterial nutrient acquisition system that depends on FMMs.

Data availability

All datasets underlying the diagrams are provided as Source Data

Article and author information

Author details

  1. Lea Antje Adolf

    Department of Infection Biology, University of Tübingen, Tübingen, Germany
    Competing interests
    The authors declare that no competing interests exist.
  2. Angelika Müller-Jochim

    Department of Infection Biology, University of Tübingen, Tübingen, Germany
    Competing interests
    The authors declare that no competing interests exist.
  3. Lara Kricks

    National Centre for Biotechnology, Spanish National Research Council, Madrid, Spain
    Competing interests
    The authors declare that no competing interests exist.
  4. Jan-Samuel Puls

    Institute for Pharmaceutical Microbiology, University of Bonn, Bonn, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8130-7375
  5. Daniel Lopez

    National Centre for Biotechnology, Spanish National Research Council, Madrid, Spain
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8627-3813
  6. Fabian Grein

    Institute for Pharmaceutical Microbiology, University of Bonn, Bonn, Germany
    Competing interests
    The authors declare that no competing interests exist.
  7. Simon Heilbronner

    Department of Infection Biology, University of Tübingen, Tübingen, Germany
    For correspondence
    simon.heilbronner@uni-tuebingen.de
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6774-2311

Funding

German Center of Infection Research (TTU 08.708_00)

  • Simon Heilbronner

Deutsche Forschungsgemeinschaft (EXC 2124 - 390838134)

  • Simon Heilbronner

Fortuene Program University Hospital Tuebingen (2507-0-0)

  • Simon Heilbronner

Spanish Ministry of Science (PID2020-115699GB-100)

  • Daniel Lopez

Deutsche Forschungsgemeinschaft (398967434 -TRR261)

  • Simon Heilbronner

Deutsche Forschungsgemeinschaft (398967434 -TRR261)

  • Fabian Grein

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Bavesh D Kana, University of the Witwatersrand, South Africa

Version history

  1. Received: December 1, 2022
  2. Preprint posted: January 13, 2023 (view preprint)
  3. Accepted: April 11, 2023
  4. Accepted Manuscript published: April 12, 2023 (version 1)
  5. Version of Record published: April 28, 2023 (version 2)

Copyright

© 2023, Adolf 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

  • 700
    views
  • 128
    downloads
  • 2
    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)

  1. Lea Antje Adolf
  2. Angelika Müller-Jochim
  3. Lara Kricks
  4. Jan-Samuel Puls
  5. Daniel Lopez
  6. Fabian Grein
  7. Simon Heilbronner
(2023)
Functional membrane microdomains and the hydroxamate siderophore transporter ATPase FhuC govern Isd-dependent heme acquisition in Staphylococcus aureus
eLife 12:e85304.
https://doi.org/10.7554/eLife.85304

Share this article

https://doi.org/10.7554/eLife.85304

Further reading

    1. Microbiology and Infectious Disease
    Moagi Tube Shaku, Peter K Um ... Bavesh D Kana
    Research Article

    Mechanisms by which Mycobacterium tuberculosis (Mtb) evades pathogen recognition receptor activation during infection may offer insights for the development of improved tuberculosis (TB) vaccines. Whilst Mtb elicits NOD-2 activation through host recognition of its peptidoglycan-derived muramyl dipeptide (MDP), it masks the endogenous NOD-1 ligand through amidation of glutamate at the second position in peptidoglycan side-chains. As the current BCG vaccine is derived from pathogenic mycobacteria, a similar situation prevails. To alleviate this masking ability and to potentially improve efficacy of the BCG vaccine, we used CRISPRi to inhibit expression of the essential enzyme pair, MurT-GatD, implicated in amidation of peptidoglycan side-chains. We demonstrate that depletion of these enzymes results in reduced growth, cell wall defects, increased susceptibility to antibiotics, altered spatial localization of new peptidoglycan and increased NOD-1 expression in macrophages. In cell culture experiments, training of a human monocyte cell line with this recombinant BCG yielded improved control of Mtb growth. In the murine model of TB infection, we demonstrate that depletion of MurT-GatD in BCG, which is expected to unmask the D-glutamate diaminopimelate (iE-DAP) NOD-1 ligand, yields superior prevention of TB disease compared to the standard BCG vaccine. In vitro and in vivo experiments in this study demonstrate the feasibility of gene regulation platforms such as CRISPRi to alter antigen presentation in BCG in a bespoke manner that tunes immunity towards more effective protection against TB disease.

    1. Microbiology and Infectious Disease
    Ryan Thiermann, Michael Sandler ... Suckjoon Jun
    Tools and Resources

    Despite much progress, image processing remains a significant bottleneck for high-throughput analysis of microscopy data. One popular platform for single-cell time-lapse imaging is the mother machine, which enables long-term tracking of microbial cells under precisely controlled growth conditions. While several mother machine image analysis pipelines have been developed in the past several years, adoption by a non-expert audience remains a challenge. To fill this gap, we implemented our own software, MM3, as a plugin for the multidimensional image viewer napari. napari-MM3 is a complete and modular image analysis pipeline for mother machine data, which takes advantage of the high-level interactivity of napari. Here, we give an overview of napari-MM3 and test it against several well-designed and widely used image analysis pipelines, including BACMMAN and DeLTA. Researchers often analyze mother machine data with custom scripts using varied image analysis methods, but a quantitative comparison of the output of different pipelines has been lacking. To this end, we show that key single-cell physiological parameter correlations and distributions are robust to the choice of analysis method. However, we also find that small changes in thresholding parameters can systematically alter parameters extracted from single-cell imaging experiments. Moreover, we explicitly show that in deep learning-based segmentation, ‘what you put is what you get’ (WYPIWYG) – that is, pixel-level variation in training data for cell segmentation can propagate to the model output and bias spatial and temporal measurements. Finally, while the primary purpose of this work is to introduce the image analysis software that we have developed over the last decade in our lab, we also provide information for those who want to implement mother machine-based high-throughput imaging and analysis methods in their research.