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Identification of genes required for long-term survival of Legionella Pneumophila in water
Department of Enteropathogenic Bacteria and Legionella, Robert Koch Institute, Wernigerode, Germany; Department of Molecular Biology and Microbiology, Tufts University School of Medicine, MA, Boston, United States.
Department of Molecular Biology and Microbiology, Tufts University School of Medicine, MA, Boston, United States.
Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).ORCID iD: 0000-0001-5995-718x
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2023 (English)In: mSphere, E-ISSN 2379-5042, Vol. 8, no 2, article id e0045422Article in journal (Refereed) Published
Abstract [en]

Long-term survival of Legionella pneumophila in aquatic environments is thought to be important for facilitating epidemic outbreaks. Eliminating bacterial colonization in plumbing systems is the primary strategy that depletes this reservoir and prevents disease. To uncover L. pneumophila determinants facilitating survival in water, a Tn-seq strategy was used to identify survival-defective mutants during 50-day starvation in tap water at 42°C. The mutants with the most drastic survival defects carried insertions in electron transport chain genes, indicating that membrane energy charge and/or ATP synthesis requires the generation of a proton gradient by the respiratory chain to maintain survival in the presence of water stress. In addition, periplasmically localized proteins that are known (EnhC) or hypothesized (lpg1697) to stabilize the cell wall against turnover were essential for water survival. To test that the identified mutations disrupted water survival, candidate genes were knocked down by CRISPRi. The vast majority of knockdown strains with verified transcript depletion showed remarkably low viability after 50-day incubations. To demonstrate that maintenance of cell wall integrity was an important survival determinant, a deletion mutation in lpg1697, in a gene encoding a predicted l,d-transpeptidase domain, was analyzed. The loss of this gene resulted in increased osmolar sensitivity and carbenicillin hypersensitivity relative to the wild type, as predicted for loss of an l,d-transpeptidase. These results indicate that the L. pneumophila envelope has been evolutionarily selected to allow survival under conditions in which the bacteria are subjected to long-term exposure to starvation and low osmolar conditions. IMPORTANCE Water is the primary vector for transmission of L. pneumophila to humans, and the pathogen is adapted to persist in this environment for extended periods of time. Preventing survival of L. pneumophila in water is therefore critical for prevention of Legionnaires' disease. We analyzed dense transposon mutation pools for strains with severe survival defects during a 50-day water incubation at 42°C. By tracking the associated transposon insertion sites in the genome, we defined a distinct essential gene set for water survival and demonstrate that a predicted peptidoglycan cross-linking enzyme, lpg1697, and components of the electron transport chain are required to ensure survival of the pathogen. Our results indicate that select characteristics of the cell wall and components of the respiratory chain of L. pneumophila are primary evolutionary targets being shaped to promote its survival in water.

Place, publisher, year, edition, pages
Washington: American Society for Microbiology, 2023. Vol. 8, no 2, article id e0045422
Keywords [en]
CRISPRi, Legionella, persistence, starvation, Tn-seq, virulence, water
National Category
Microbiology in the medical area Microbiology
Identifiers
URN: urn:nbn:se:umu:diva-208070DOI: 10.1128/msphere.00454-22ISI: 000960147700001PubMedID: 36988466Scopus ID: 2-s2.0-85153414758OAI: oai:DiVA.org:umu-208070DiVA, id: diva2:1757761
Funder
The Kempe FoundationsKnut and Alice Wallenberg FoundationSwedish Research CouncilAvailable from: 2023-05-17 Created: 2023-05-17 Last updated: 2023-05-17Bibliographically approved

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Pinedo, VictorCava, Felipe

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Molecular Infection Medicine Sweden (MIMS)Umeå Centre for Microbial Research (UCMR)Department of Molecular Biology (Faculty of Medicine)
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