Abstract

The Gram-negative bacterium, Legionella pneumophila, is a protozoan parasite and accidental intracellular pathogen of humans. We propose a model in which cycling through multiple protozoan hosts in the environment holds L. pneumophila in a state of evolutionary stasis as a broad host-range pathogen. Using an experimental evolution approach, we tested this hypothesis by restricting L. pneumophila to growth within mouse macrophages for hundreds of generations. Whole-genome resequencing and high-throughput genotyping identified several parallel adaptive mutations and population dynamics that led to improved replication within macrophages. Based on these results, we provide a detailed view of the population dynamics of an experimentally evolving bacterial population, punctuated by frequent instances of transient clonal interference and selective sweeps. Non-synonymous point mutations in the flagellar regulator, fleN, resulted in increased uptake and broadly increased replication in both macrophages and amoebae. Mutations in multiple steps of the lysine biosynthesis pathway were also independently isolated, resulting in lysine auxotrophy and reduced replication in amoebae. These results demonstrate that under laboratory conditions, host restriction is sufficient to rapidly modify L. pneumophila fitness and host range. We hypothesize that, in the environment, host cycling prevents L. pneumophila host-specialization by maintaining pathways that are deleterious for growth in macrophages and other hosts.

Highlights

  • L. pneumophila is a Gram-negative intracellular pathogen with a broad host range that extends from unicellular protozoa to alveolar macrophages of the human lung [1]

  • Recent evidence supports a model in which the large repertoire of Dot/Icm translocated substrates is essential to the broad host range of L. pneumophila, with different subsets of these proteins contributing to optimal replication in distinct protozoan hosts [17]

  • Experimental evolution of Legionella pneumophila leads to strains with improved replication in mouse macrophages

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Summary

Introduction

L. pneumophila is a Gram-negative intracellular pathogen with a broad host range that extends from unicellular protozoa to alveolar macrophages of the human lung [1]. L. pneumophila has been shown to replicate in over 15 species of protozoa [8,9,10], consistent with the bacterium being a generalist in that it shows little evidence of species specificity After uptake by these natural protozoan hosts, the L. pneumophila type IVB Dot/Icm translocation system translocates a large cadre of proteins across host membranes [11,12,13], remodeling the Legionellacontaining vacuole (LCV) into a non-acidified compartment supportive of intracellular replication [14,15,16]. Recent evidence supports a model in which the large repertoire of Dot/Icm translocated substrates is essential to the broad host range of L. pneumophila, with different subsets of these proteins contributing to optimal replication in distinct protozoan hosts [17]. We hypothesize that host cycling in the environment maintains L. pneumophila as a generalist, presumably through purifying selection against mutations that diminish fitness in any of several naturally encountered protozoan hosts

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