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Multiple Legionella pneumophila effector virulence phenotypes revealed through high-throughput analysis of targeted mutant libraries

  1. Craig R. Roya,2
  1. aDepartment of Microbial Pathogenesis, Yale University School of Medicine, New Haven, CT 06519;
  2. bMicrobial Sciences Institute, Yale University School of Medicine, New Haven, CT 06519
  1. Edited by Ralph R. Isberg, Howard Hughes Medical Institute/Tufts University School of Medicine, Boston, MA, and approved October 20, 2017 (received for review May 23, 2017)

Significance

The contribution of individual effectors to Legionella pneumophila virulence has not been systematically examined. This study employed a parallel high-throughput transposon insertion sequencing technique called INSeq to probe the L. pneumophila effector repertoire and identified multiple effectors that contribute to virulence in several host organisms, including an animal model of Legionnaires’ disease. Importantly, this study demonstrates that effector proteins contribute to host virulence both positively and negatively by controlling intracellular replication and influencing host immune responses, which demonstrates that the subtle alterations in the effector repertoire of a single L. pneumophila strain can greatly impact host pathogenicity.

Abstract

Legionella pneumophila is the causative agent of a severe pneumonia called Legionnaires’ disease. A single strain of L. pneumophila encodes a repertoire of over 300 different effector proteins that are delivered into host cells by the Dot/Icm type IV secretion system during infection. The large number of L. pneumophila effectors has been a limiting factor in assessing the importance of individual effectors for virulence. Here, a transposon insertion sequencing technology called INSeq was used to analyze replication of a pool of effector mutants in parallel both in a mouse model of infection and in cultured host cells. Loss-of-function mutations in genes encoding effector proteins resulted in host-specific or broad virulence phenotypes. Screen results were validated for several effector mutants displaying different virulence phenotypes using genetic complementation studies and infection assays. Specifically, loss-of-function mutations in the gene encoding LegC4 resulted in enhanced L. pneumophila in the lungs of infected mice but not within cultured host cells, which indicates LegC4 augments bacterial clearance by the host immune system. The effector proteins RavY and Lpg2505 were important for efficient replication within both mammalian and protozoan hosts. Further analysis of Lpg2505 revealed that this protein functions as a metaeffector that counteracts host cytotoxicity displayed by the effector protein SidI. Thus, this study identified a large cohort of effectors that contribute to L. pneumophila virulence positively or negatively and has demonstrated regulation of effector protein activities by cognate metaeffectors as being critical for host pathogenesis.

Footnotes

  • ?1Present address: Division of Biology, Kansas State University, Manhattan, KS 66506.

  • ?2To whom correspondence should be addressed. Email: craig.roy{at}yale.edu.
  • Author contributions: S.R.S., A.L.G., and C.R.R. designed research; S.R.S., L.L., and J.C.H. performed research; W.B.S. and A.L.G. contributed new reagents/analytic tools; S.R.S., L.L., A.L.G., and C.R.R. analyzed data; and S.R.S. and C.R.R. wrote the paper.

  • The authors declare no conflict of interest.

  • This article is a PNAS Direct Submission.

  • This article contains supporting information online at www.danielhellerman.com/lookup/suppl/doi:10.1073/pnas.1708553114/-/DCSupplemental.

Published under the PNAS license.

Online Impact

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