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Peer-reviewedCC BY 4.0PLOS Pathogens

Multi-Dot/Icm effector mutants reveal distinct host responses in Legionella infected macrophages

  • Raissa Wibawa1
  • Pengfei Li1
  • Linden J. Gearing1
  • Sacha Pidot2
  • Yuliia Faidiuk1
  • Wilson Wong1,3
  • Garrett Z. Ng1,3
  • Kathleen McCaffrey1
  • Paul J. Hertzog1,3
  • Shivani Pasricha4
  • Elizabeth L. Hartland1,3,4
4 affiliations
  1. 1Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, Clayton, Australia
  2. 2Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, Australia
  3. 3Department of Molecular and Translational Science, Monash University, Clayton, Australia
  4. 4Department of Infectious Diseases, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, Australia
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Abstract

Legionella pneumophila is the causative agent of Legionnaires’ disease, an accidental and severe form of pneumonia. The replication of L. pneumophila within alveolar macrophages requires the Dot/Icm type IV secretion system that translocates more than 330 bacterial effector proteins into the infected host cell. Many Dot/Icm effectors possess eukaryotic-like domains and manipulate host processes by functional mimicry. However, only a minority of effectors have been fully characterized. Redundancy among effector protein activities and host targets has complicated functional characterization of individual effector proteins. To explore the collective impact of Dot/Icm effectors on host cell processes, we created a library of 14 L. pneumophila genomic mutant strains lacking effector-rich regions (a total of 85 effector genes deleted) and used the multi-deletion mutants to interrogate host-pathogen interactions. While none of the mutants exhibited a defect in intracellular replication in THP-1 macrophages, several were attenuated in amoebae, including a mutant lacking a predicted amino acid transporter. In THP-1 cells, we observed that wild-type L. pneumophila 130b triggered a strong inflammatory and cellular stress transcriptional response in macrophages, whereas six mutants displayed reduced interferon signalling and one mutant displayed a marked decrease in the global expression of host heat shock response (HSR) genes. Through the creation of further sub-deletions, we found that the Dot/Icm effector kinase LegK4 was responsible for induction of the host HSR via the canonical transcription factor heat shock factor 1 (HSF1). Infection of THP-1 macrophages deficient in HSF1 revealed a potential role for the host HSR in the early stages of L. pneumophila infection. Together, these results reinforce the utility of multi-gene deletions to identify Dot/Icm effector phenotypes and the importance of the host HSR to L. pneumophila infection. Author summary: The bacterial pathogen Legionella pneumophila modulates host cell processes to replicate in human macrophages. Intracellular replication requires the Legionella Dot/Icm type IV secretion system that translocates more than 330 effector proteins into the infected macrophage that collectively enable intracellular replication. Here, we created a library of multi-effector deletion mutants to study the role of Dot/Icm effectors in the modulation of macrophage transcriptional responses during infection. While none of the mutants exhibited a defect in intracellular replication in THP-1 macrophages, several were attenuated in amoebae. Wild type L. pneumophila induced a strong host inflammatory and cell stress response in macrophages while some mutants produced a weaker response, despite similar levels of bacterial replication. We found that the effector protein LegK4 induced a global heat shock transcriptional response that was dependent on the host heat shock factor 1 (HSF1). Infection of HSF1-deficient macrophages with L. pneumophila suggested a potential role for HSF1 in the early stages of L. pneumophila infection.

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Cite

BibTeXRIS
BibTeX
@article{wibawa2026multidoticm,
  title = {{Multi-Dot/Icm effector mutants reveal distinct host responses in Legionella infected macrophages}},
  author = {Raissa Wibawa and Pengfei Li and Linden J. Gearing and Sacha Pidot and Yuliia Faidiuk and Wilson Wong and Garrett Z. Ng and Kathleen McCaffrey and Paul J. Hertzog and Shivani Pasricha and Elizabeth L. Hartland},
  journal = {PLOS Pathogens},
  year = {2026},
  month = oct,
  doi = {10.1371/journal.ppat.1014083},
  url = {https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1014083},
  note = {CC BY 4.0},
}
RIS
TY  - JOUR
TI  - Multi-Dot/Icm effector mutants reveal distinct host responses in Legionella infected macrophages
AU  - Raissa Wibawa
AU  - Pengfei Li
AU  - Linden J. Gearing
AU  - Sacha Pidot
AU  - Yuliia Faidiuk
AU  - Wilson Wong
AU  - Garrett Z. Ng
AU  - Kathleen McCaffrey
AU  - Paul J. Hertzog
AU  - Shivani Pasricha
AU  - Elizabeth L. Hartland
PY  - 2026
DA  - 2026/10/22
JO  - PLOS Pathogens
DO  - 10.1371/journal.ppat.1014083
UR  - https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1014083
AB  - Legionella pneumophila is the causative agent of Legionnaires’ disease, an accidental and severe form of pneumonia. The replication of L. pneumophila within alveolar macrophages requires the Dot/Icm type IV secretion system that translocates more than 330 bacterial effector proteins into the infected host cell. Many Dot/Icm effectors possess eukaryotic-like domains and manipulate host processes by functional mimicry. However, only a minority of effectors have been fully characterized. Redundancy among effector protein activities and host targets has complicated functional characterization of individual effector proteins. To explore the collective impact of Dot/Icm effectors on host cell processes, we created a library of 14 L. pneumophila genomic mutant strains lacking effector-rich regions (a total of 85 effector genes deleted) and used the multi-deletion mutants to interrogate host-pathogen interactions. While none of the mutants exhibited a defect in intracellular replication in THP-1 macrophages, several were attenuated in amoebae, including a mutant lacking a predicted amino acid transporter. In THP-1 cells, we observed that wild-type L. pneumophila 130b triggered a strong inflammatory and cellular stress transcriptional response in macrophages, whereas six mutants displayed reduced interferon signalling and one mutant displayed a marked decrease in the global expression of host heat shock response (HSR) genes. Through the creation of further sub-deletions, we found that the Dot/Icm effector kinase LegK4 was responsible for induction of the host HSR via the canonical transcription factor heat shock factor 1 (HSF1). Infection of THP-1 macrophages deficient in HSF1 revealed a potential role for the host HSR in the early stages of L. pneumophila infection. Together, these results reinforce the utility of multi-gene deletions to identify Dot/Icm effector phenotypes and the importance of the host HSR to L. pneumophila infection. Author summary: The bacterial pathogen Legionella pneumophila modulates host cell processes to replicate in human macrophages. Intracellular replication requires the Legionella Dot/Icm type IV secretion system that translocates more than 330 effector proteins into the infected macrophage that collectively enable intracellular replication. Here, we created a library of multi-effector deletion mutants to study the role of Dot/Icm effectors in the modulation of macrophage transcriptional responses during infection. While none of the mutants exhibited a defect in intracellular replication in THP-1 macrophages, several were attenuated in amoebae. Wild type L. pneumophila induced a strong host inflammatory and cell stress response in macrophages while some mutants produced a weaker response, despite similar levels of bacterial replication. We found that the effector protein LegK4 induced a global heat shock transcriptional response that was dependent on the host heat shock factor 1 (HSF1). Infection of HSF1-deficient macrophages with L. pneumophila suggested a potential role for HSF1 in the early stages of L. pneumophila infection.
SN  - 1553-7374
SN  - 1553-7366
ER  -