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Updated: Mar 14, 2026
Quantifying Yersinia pseudotuberculosis Type III Secretion System Activity Following Iron Starvation and Anaerobic Growth
Published on: May 31, 2024
Fis Is Essential for Yersinia pseudotuberculosis Virulence and Protects against Reactive Oxygen Species Produced by
Erin R Green1, Stacie Clark1, Gregory T Crimmins2
1Graduate Program in Molecular Microbiology, Sackler School of Graduate Biomedical Sciences, Tufts University School of Medicine, Boston, Massachusetts, United States of America.
Abstract:
All three pathogenic Yersinia species share a conserved virulence plasmid that encodes a Type 3 Secretion System (T3SS) and its associated effector proteins. During mammalian infection, these effectors are injected into innate immune cells, where they block many bactericidal functions, including the production of reactive oxygen species (ROS). However, Y. pseudotuberculosis (Yptb) lacking the T3SS retains the ability to colonize host organs, demonstrating that chromosome-encoded factors are sufficient for growth within mammalian tissue sites. Previously we uncovered more than 30 chromosomal factors that contribute to growth of T3SS-deficient Yptb in livers. Here, a deep sequencing-based approach was used to validate and characterize the phenotype of 18 of these genes during infection by both WT and plasmid-deficient Yptb. Additionally, the fitness of these mutants was evaluated in immunocompromised mice to determine whether any genes contributed to defense against phagocytic cell restriction. Mutants containing deletions of the dusB-fis operon, which encodes the nucleoid associated protein Fis, were markedly attenuated in immunocompetent mice, but were restored for growth in mice lacking neutrophils and inflammatory monocytes, two of the major cell types responsible for restricting Yersinia infection. We determined that Fis was dispensable for secretion of T3SS effectors, but was essential for resisting ROS and regulated the transcription of several ROS-responsive genes. Strikingly, this protection was critical for virulence, as growth of ΔdusB-fis was restored in mice unable to produce ROS. These data support a model in which ROS generated by neutrophils and inflammatory monocytes that have not been translocated with T3SS effectors enter bacterial cells during infection, where their bactericidal effects are resisted in a Fis-dependent manner. This is the first report of the requirement for Fis during Yersinia infection and also highlights a novel mechanism by which Yptb defends against ROS in mammalian tissues.
Insights
Yersinia pseudotuberculosis uses the Fis protein to resist reactive oxygen species (ROS) produced by immune cells. This defense mechanism is crucial for bacterial survival and colonization in mammalian tissues, especially when the Type 3 Secretion System is absent.
Area of Science:
- Microbiology
- Immunology
- Bacterial Pathogenesis
Background:
- Pathogenic Yersinia species possess a virulence plasmid encoding a Type 3 Secretion System (T3SS) that delivers effectors into host cells, inhibiting immune responses like reactive oxygen species (ROS) production.
- Yersinia pseudotuberculosis (Yptb) lacking T3SS can still colonize organs, indicating the importance of chromosomally encoded factors for virulence.
- Over 30 chromosomal factors aiding T3SS-deficient Yptb growth in livers were previously identified.
Purpose of the Study:
- To validate and characterize the role of 18 previously identified chromosomal genes in Yptb virulence.
- To assess the fitness of Yptb mutants in immunocompromised mice to understand defense against phagocytic cell restriction.
- To elucidate the specific function of the nucleoid-associated protein Fis in Yptb pathogenesis.
Main Methods:
- Deep sequencing was employed to analyze the phenotypes of 18 Yptb chromosomal mutants during infection.
- Mutant fitness was evaluated in both immunocompetent and immunocompromised mouse models, including those lacking neutrophils and inflammatory monocytes.
- The role of Fis in T3SS effector secretion, ROS resistance, and gene transcription was investigated.
Main Results:
- Mutants lacking the dusB-fis operon, encoding Fis, showed significant attenuation in immunocompetent mice but recovered in mice lacking neutrophils and monocytes.
- Fis was found to be dispensable for T3SS effector secretion but essential for resisting ROS and regulating ROS-responsive genes.
- The virulence defect of the ΔdusB-fis mutant was fully restored in mice unable to produce ROS, highlighting Fis-dependent ROS resistance.
Conclusions:
- Fis is essential for Yersinia pseudotuberculosis virulence by enabling resistance to ROS generated by phagocytic immune cells.
- This study reveals a novel mechanism of bacterial defense against ROS in mammalian tissues, independent of T3SS effectors.
- The findings underscore the critical role of chromosomally encoded factors in bacterial survival against host immune defenses.
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