Structural analysis of the Legionella pneumophila Dot/Icm Type IV Secretion System Core Complex

  1. Clarissa L Durie
  2. Michael J Sheedlo
  3. Jeong Min Chung
  4. Brenda G Byrne
  5. Min Su
  6. Thomas Knight
  7. Michele Swanson  Is a corresponding author
  8. D Borden Lacy  Is a corresponding author
  9. Melanie D Ohi  Is a corresponding author
  1. University Of Michigan, United States
  2. Vanderbilt University Medical Center, United States
  3. University of Michigan, United States

Abstract

Legionella pneumophila is an opportunistic pathogen that causes the potentially fatal pneumonia Legionnaires' Disease. This infection and subsequent pathology require the Dot/Icm Type IV Secretion System (T4SS) to deliver effector proteins into host cells. Compared to prototypical T4SSs, the Dot/Icm assembly is much larger, containing ~27 different components including a core complex reported to be composed of five proteins: DotC, DotD, DotF, DotG, and DotH. Using single particle cryo-electron microscopy (cryo-EM), we report reconstructions of the core complex of the Dot/Icm T4SS that includes a symmetry mismatch between distinct structural features of the outer membrane cap (OMC) and periplasmic ring (PR). We present models of known core complex proteins, DotC, DotD, and DotH, and two structurally similar proteins within the core complex, DotK and Lpg0657. This analysis reveals the stoichiometry and contact interfaces between the key proteins of the Dot/Icm T4SS core complex and provides a framework for understanding a complex molecular machine.

Data availability

All cryo-EM data included in this manuscript are available through the Electron Microscopy Data Bank (EMD-22068, EMD-22069, EMD-22070 and EMD-22071). All models that were constructed from these data are available via the Protein Data Bank (PDB 6x62, 6x64, 6x65, and 6x66).

The following data sets were generated

Article and author information

Author details

  1. Clarissa L Durie

    Life Sciences Institute, University Of Michigan, Ann Arbor, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4027-4386
  2. Michael J Sheedlo

    Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3185-1727
  3. Jeong Min Chung

    Life Sciences Institute, University of Michigan, Ann Arbor, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4285-8764
  4. Brenda G Byrne

    Department of Microbiology & Immunology, University of Michigan, Ann Arbor, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Min Su

    Life Sciences Institute, University of Michigan, Ann Arbor, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Thomas Knight

    Department of Microbiology & Immunology, University of Michigan, Ann Arbor, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Michele Swanson

    Department of Microbiology & Immunology, University of Michigan, Ann Arbor, United States
    For correspondence
    mswanson@umich.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2542-0266
  8. D Borden Lacy

    Pathology, Microbiology and Immunology; Biochemistry, Vanderbilt University Medical Center, Nashville, United States
    For correspondence
    borden.lacy@vanderbilt.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2273-8121
  9. Melanie D Ohi

    Life Sciences Institute, University of Michigan, Ann Arbor, United States
    For correspondence
    mohi@umich.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1750-4793

Funding

National Institute of Allergy and Infectious Diseases (F32 AI150027-01)

  • Clarissa L Durie

National Institute of General Medical Sciences (S10OD020011)

  • Melanie D Ohi

National Institute of Allergy and Infectious Diseases (2T32DK007673)

  • Michele Swanson

National Institute of Allergy and Infectious Diseases (R01AI118932)

  • Melanie D Ohi

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

Copyright

© 2020, Durie 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

  • 4,898
    views
  • 471
    downloads
  • 52
    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. Clarissa L Durie
  2. Michael J Sheedlo
  3. Jeong Min Chung
  4. Brenda G Byrne
  5. Min Su
  6. Thomas Knight
  7. Michele Swanson
  8. D Borden Lacy
  9. Melanie D Ohi
(2020)
Structural analysis of the Legionella pneumophila Dot/Icm Type IV Secretion System Core Complex
eLife 9:e59530.
https://doi.org/10.7554/eLife.59530

Share this article

https://doi.org/10.7554/eLife.59530

Further reading

    1. Microbiology and Infectious Disease
    Tao Tang, Weiming Zhong ... Zhipeng Gao
    Research Article

    Saprolegnia parasitica is one of the most virulent oomycete species in freshwater aquatic environments, causing severe saprolegniasis and leading to significant economic losses in the aquaculture industry. Thus far, the prevention and control of saprolegniasis face a shortage of medications. Linalool, a natural antibiotic alternative found in various essential oils, exhibits promising antimicrobial activity against a wide range of pathogens. In this study, the specific role of linalool in protecting S. parasitica infection at both in vitro and in vivo levels was investigated. Linalool showed multifaceted anti-oomycetes potential by both of antimicrobial efficacy and immunomodulatory efficacy. For in vitro test, linalool exhibited strong anti-oomycetes activity and mode of action included: (1) Linalool disrupted the cell membrane of the mycelium, causing the intracellular components leak out; (2) Linalool prohibited ribosome function, thereby inhibiting protein synthesis and ultimately affecting mycelium growth. Surprisingly, meanwhile we found the potential immune protective mechanism of linalool in the in vivo test: (1) Linalool enhanced the complement and coagulation system which in turn activated host immune defense and lysate S. parasitica cells; (2) Linalool promoted wound healing, tissue repair, and phagocytosis to cope with S. parasitica infection; (3) Linalool positively modulated the immune response by increasing the abundance of beneficial Actinobacteriota; (4) Linalool stimulated the production of inflammatory cytokines and chemokines to lyse S. parasitica cells. In all, our findings showed that linalool possessed multifaceted anti-oomycetes potential which would be a promising natural antibiotic alternative to cope with S. parasitica infection in the aquaculture industry.

    1. Genetics and Genomics
    2. Microbiology and Infectious Disease
    Iti Mehta, Jacob B Hogins ... Larry Reitzer
    Research Article

    Polyamines are biologically ubiquitous cations that bind to nucleic acids, ribosomes, and phospholipids and, thereby, modulate numerous processes, including surface motility in Escherichia coli. We characterized the metabolic pathways that contribute to polyamine-dependent control of surface motility in the commonly used strain W3110 and the transcriptome of a mutant lacking a putrescine synthetic pathway that was required for surface motility. Genetic analysis showed that surface motility required type 1 pili, the simultaneous presence of two independent putrescine anabolic pathways, and modulation by putrescine transport and catabolism. An immunological assay for FimA—the major pili subunit, reverse transcription quantitative PCR of fimA, and transmission electron microscopy confirmed that pili synthesis required putrescine. Comparative RNAseq analysis of a wild type and ΔspeB mutant which exhibits impaired pili synthesis showed that the latter had fewer transcripts for pili structural genes and for fimB which codes for the phase variation recombinase that orients the fim operon promoter in the ON phase, although loss of speB did not affect the promoter orientation. Results from the RNAseq analysis also suggested (a) changes in transcripts for several transcription factor genes that affect fim operon expression, (b) compensatory mechanisms for low putrescine which implies a putrescine homeostatic network, and (c) decreased transcripts of genes for oxidative energy metabolism and iron transport which a previous genetic analysis suggests may be sufficient to account for the pili defect in putrescine synthesis mutants. We conclude that pili synthesis requires putrescine and putrescine concentration is controlled by a complex homeostatic network that includes the genes of oxidative energy metabolism.