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Identification of Type 4B Secretion System Substrates That Are Conserved among Coxiella burnetii Genomes and Promote

Charles L Larson1,2, Willis Pullman1, Paul A Beare1,3

  • 1Coxiella Pathogenesis Section, Laboratory of Bacteriology, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, USA.

Microbiology Spectrum
|May 18, 2023
PubMed

Insights

This study identifies key Coxiella burnetii type 4B secretion system (T4BSS) substrates essential for bacterial replication and vacuole formation. Researchers validated several T4BSS substrates, refining criteria for identifying bona fide effectors.

Area of Science:

  • Microbiology
  • Cell Biology
  • Pathogen-Host Interactions

Background:

  • Coxiella burnetii is a Gram-negative bacterium causing Q fever in humans and abortion in livestock.
  • Pathogen survival depends on replication within a specialized lysosomal Coxiella-containing vacuole (CCV).
  • A type 4B secretion system (T4BSS) is crucial for delivering effector proteins into host cells, facilitating CCV biogenesis and bacterial replication.

Purpose of the Study:

  • To investigate the function of 32 conserved proteins previously identified as putative T4BSS substrates in C. burnetii.
  • To validate and identify bona fide C. burnetii T4BSS substrates essential for intracellular replication and CCV formation.
  • To refine the criteria for designating T4BSS substrates, addressing discrepancies from heterologous secretion assays.

Main Methods:

  • Utilized reporter fusions (CyaA, BlaM) to assess protein translocation by C. burnetii's T4BSS.
  • Employed CRISPR interference (CRISPRi) to evaluate the role of validated T4BSS substrates in bacterial replication and CCV biogenesis.
  • Used fluorescently tagged proteins (mCherry) to determine subcellular localization within host cells (HeLa, THP-1, Vero).

Main Results:

  • Most previously reported T4BSS substrates were not translocated by C. burnetii.
  • CRISPRi identified four validated T4BSS substrates (CBU0122, CBU1752, CBU1825, CBU2007) that promote C. burnetii replication and CCV biogenesis.
  • CBU0122 localized to the CCV membrane and mitochondria, suggesting effector function.

Conclusions:

  • Identified a refined set of bona fide C. burnetii T4BSS substrates crucial for pathogen virulence.
  • Demonstrated that many previously designated T4BSS substrates lack functional export by C. burnetii.
  • Highlighted the importance of validated secretion assays and conserved genomic presence for identifying true effector proteins.
Keywords:
CellProfilerDot/Icmmacrophages

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