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Diverse type VI secretion phospholipases are functionally plastic antibacterial effectors

Nature volume 496, pages 508–512 (2013)Cite this article

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Abstract

Membranes allow the compartmentalization of biochemical processes and are therefore fundamental to life. The conservation of the cellular membrane, combined with its accessibility to secreted proteins, has made it a common target of factors mediating antagonistic interactions between diverse organisms. Here we report the discovery of a diverse superfamily of bacterial phospholipase enzymes. Within this superfamily, we defined enzymes with phospholipase A1 and A2 activity, which are common in host-cell-targeting bacterial toxins and the venoms of certain insects and reptiles1,2. However, we find that the fundamental role of the superfamily is to mediate antagonistic bacterial interactions as effectors of the type VI secretion system (T6SS) translocation apparatus; accordingly, we name these proteins type VI lipase effectors. Our analyses indicate that PldA of Pseudomonas aeruginosa, a eukaryotic-like phospholipase D3, is a member of the type VI lipase effector superfamily and the founding substrate of the haemolysin co-regulated protein secretion island II T6SS (H2-T6SS). Although previous studies have specifically implicated PldA and the H2-T6SS in pathogenesis3,4,5, we uncovered a specific role for the effector and its secretory machinery in intra- and interspecies bacterial interactions. Furthermore, we find that this effector achieves its antibacterial activity by degrading phosphatidylethanolamine, the major component of bacterial membranes. The surprising finding that virulence-associated phospholipases can serve as specific antibacterial effectors suggests that interbacterial interactions are a relevant factor driving the continuing evolution of pathogenesis.

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Figure 1: Overview of the Tle superfamily.
Figure 2: Tle GXSXG-type proteins are antibacterial phospholipase effectors delivered by the T6SS.
Figure 3: Tle5PA is an HXKXXXXD-type interspecies antibacterial phospholipase effector delivered by the H2-T6SS of P. aeruginosa.

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Accession codes

Data deposits

GenBank accession numbers for all Tle proteins identified in this study are found in Supplementary Figs 1–5.

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Acknowledgements

We thank H. Kulasekara, B. E. Uhlin and members of the Mougous and Wai laboratories for discussions, J. Woodward for sharing chemistry expertise, the Manoil laboratory for sharing B. thailandensis transposon mutants, and the Parsek laboratory and K. Korotkov for sharing reagents. This work was supported by grants from the National Institutes of Health (NIH) (AI080609, AI057141 and AI105268), Cystic Fibrosis Foundation (CFR565-CR07), the National Science Foundation (PHY-084845 and MCB-1151043), the Swedish Research Council (2010-3073, 2007-8673 UCMR Linnaeus and 2006-7431 MIMS), and the Faculty of Medicine, Umeå University. A.B.R. was supported by a Graduate Research Fellowship from the National Science Foundation (DGE-0718124), M.L. was supported by the NIH Cellular and Molecular Training Grant (GM07270), P.A.W. received support from the University of Washington Royalty Research Fund and the Sloan Foundation, and J.D.M. holds an Investigator in the Pathogenesis of Infectious Disease Award from the Burroughs Wellcome Fund.

Author information

Authors and Affiliations

  1. Department of Microbiology, University of Washington, Seattle, 98195, Washington, USA

    Alistair B. Russell, Michele LeRoux, Danielle M. Agnello & Joseph D. Mougous

  2. Molecular and Cellular Biology Program, University of Washington, Seattle, 98195, Washington, USA

    Michele LeRoux & Joseph D. Mougous

  3. Department of Molecular Biology, Umeå University, SE-90187 Umeå, Sweden,

    Krisztina Hathazi, Takahiko Ishikawa & Sun Nyunt Wai

  4. The Laboratory for Molecular Infection Medicine Sweden (MIMS), Umeå University, SE-90187 Umeå, Sweden,

    Krisztina Hathazi, Takahiko Ishikawa & Sun Nyunt Wai

  5. Department of Physics, University of Washington, Seattle, 98195, Washington, USA

    Paul A. Wiggins

  6. Department of Bioengineering, University of Washington, Seattle, 98195, Washington, USA

    Paul A. Wiggins

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Contributions

A.B.R., M.L., P.A.W., S.N.W. and J.D.M. conceived and designed experiments. A.B.R., M.L., K.H., D.M.A., T.I. and J.D.M. conducted experiments. A.B.R. and J.D.M. wrote the paper.

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Correspondence to Joseph D. Mougous.

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Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-15 and Supplementary Table 1. (PDF 1614 kb)

Tle1BT-sensitive strains exhibit increased PI uptake in competition with wild-type B. thailandensis

This video shows wild-type B. thailandensis co-cultured with a GFP-labeled Tle1BT-sensitive B. thailandensis strain (∆I2698-2703). Phase, PI, and GFP images were acquired at 5-min intervals (overlays depicted in movie sequence). The Tle1BT-sensitive B. thailandensis strain appears in the green channel and PI staining in the red channel. (MOV 13098 kb)

Tle1BT-sensitive strains do not exhibit increased PI uptake in competition with B. thailandensis lacking clpV1

This video shows B. thailandensis lacking clpV1 (∆I2958) co-cultured with a GFP-labeled Tle1BT-sensitive B. thailandensis strain (∆I2698-2703). Phase, PI, and GFP images were acquired at 5-min intervals (overlays depicted in movie sequence). The Tle1BT-sensitive B. thailandensis strain appears in the green channel and PI staining in the red channel. (MOV 11786 kb)

Tle1BT-sensitive strains do not exhibit increased PI uptake in competition with B. thailandensis lacking Tle1BT and its upstream homolog

This video shows B. thailandensis lacking Tle1BT and its upstream homolog (∆I2698 ∆I2701-2703) co-cultured with a GFP-labeled Tle1BT-sensitive B. thailandensis strain (∆I2698-2703). Phase, PI, and GFP images were acquired at 5-min intervals (overlays depicted in movie sequence). The Tle1BT-sensitive B. thailandensis strain appears in the green channel and PI staining in the red channel. (MOV 8763 kb)

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Russell, A., LeRoux, M., Hathazi, K. et al. Diverse type VI secretion phospholipases are functionally plastic antibacterial effectors. Nature 496, 508–512 (2013). https://doi.org/10.1038/nature12074

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