Diverse protist grazers select for virulence-related traits in Legionella

Abstract

It is generally accepted that selection for resistance to grazing by protists has contributed to the evolution of Legionella pneumophila as a pathogen. Grazing resistance is becoming more generally recognized as having an important role in the ecology and evolution of bacterial pathogenesis. However, selection for grazing resistance presupposes the existence of protist grazers that provide the selective pressure. To determine whether there are protists that graze on pathogenic Legionella species, we investigated the existence of such organisms in a variety of environmental samples. We isolated and characterized diverse protists that graze on L. pneumophila and determined the effects of adding L. pneumophila on the protist community structures in microcosms made from these environmental samples. Several unrelated organisms were able to graze efficiently on L. pneumophila. The community structures of all samples were markedly altered by the addition of L. pneumophila. Surprisingly, some of the Legionella grazers were closely related to species that are known hosts for L. pneumophila, indicating the presence of unknown specificity determinants for this interaction. These results provide the first direct support for the hypothesis that protist grazers exert selective pressure on Legionella to acquire and retain adaptations that contribute to survival, and that these properties are relevant to the ability of the bacteria to cause disease in people. We also report a novel mechanism of killing of amoebae by one Legionella species that requires an intact Type IV secretion system but does not involve intracellular replication. We refer to this phenomenon as 'food poisoning'.

Figure 1

Impact of L. pneumophila on the structure of eukaryotic microbial communities in laboratory microcosms. (a): Taxonomic affiliation of protist OTUs retrieved from different environmental samples incubated in the presence of E. coli (EC) or L. pneumophila str. Philadelphia-1 (PHI). Bars represent the relative abundance of protist taxa after rarefaction to correct for uneven sampling as described in the Materials and methods section. Sample identities are given on the x axis. MIC: fresh-water sample from Lake Michigan. ONT: fresh-water sample from Lake Ontario. SUP: fresh-water sample from Lake Superior. PWP: Pond-Washington Park (Chicago, IL, USA). U2: soil from Aguascalientes (Mexico). U8: soil from Neyaldi (India). WPD and WPR: soil samples from Washington Park (Chicago, IL, USA). MG: soil from Hyde Park (Chicago, IL, USA). C37B4: Core sample from ocean subsurface, Expedition IODP 311. SWG: Sewage (WWTP, Chicago, USA). AZ: soil from Tucson, AZ, USA. Sample-name_0: microcosm analyzed before the addition of E. coli or L. pneumophila. Sample-name_EC: microcosm incubated with 10⁷ cells of E. coli. Sample-name_PHI: microcosm incubated with 10⁷ cells of L. pneumophila Philadelphia 1. Relative abundance values (%) of Cercozoa, Heterolobosea and Amoebozoa are highlighted in orange, purple and blue color, respectively. See Microcosm design in Materials and methods Section for details.

Figure 2

Protist morphotypes that resist L. pneumophila infection fall into three different groups. Combined DIC and epifluorescence images of protozoan isolates recovered from microcosms incubated with L. pneumophila Philadelphia-1. Pure cultures of each protist isolate (established as described in Materials and methods) were incubated with mCherry-expressing L. pneumophila at MOI of 1000. Uptake and survival of Legionella was followed from 2–72 h after the addition of the bacteria. Images showed correspond to 24 h time point. Similar results were observed for 2 and 48 h. Group 1: protists that avoid taking up Legionella (pictures a and e). Group 2: protists that expel Legionella packaged into pellets (pictures b and f). Group 3: protists that consume Legionella. The digestion of the bacteria (pictures c, d, g and h) imparted a homogeneous red fluorescence on the digestive vacuoles, presumably caused by the release of mCherry from the lysed Legionella cells. Scale bar represents 10 μm.

Figure 3

Protist grazers on L. pneumophila. Transmission electron micrographs of isolates S. palustris (a–c), Paracercomonas CWPL (d–f) and Cercomonas MG33 (g–i) showing control preparations with ingested E. coli (a, d, g) and those fed with L. pneumophila JR32 (b, e, h) or L. steelei IMVS3376 (c, f, i). TEM micrographs were taken 48 h after the addition of Legionella to the protist. b: bacteria; DV: digestive vacuole; e: extrusomes; m: mitochondria; N: nucleus. Black arrow indicates bacterial cell being digested. White arrow highlights an autophagosome. See Supplementary Figure S5 for more evidence of cellular damage in S. palustris trophozoites grazing on L. steelei.

Figure 4

Growth of protozoan isolates S. palustris (a), Paracercomonas CWPL (b), Cercomonas MG33 (c) on L. pneumophila or L. steelei as food source. Protist growth (a–c) and Legionella consumption (d–f) was quantified by real-time PCR by targeting 18 S and 16 S rDNA genes, respectively. In total, 10⁴ trophozoites were cultivated in 96-well plates with 10⁷ cells of either L. pneumophila JR32 or L. steelei IMVS3376 wild-type or dotA- mutant in pond water at room temperature. On successive days, the number of Genome equivalents (GE) of protist trophozoites and Legionella was quantified by real-time PCR as described in Methods. Results shown correspond to the average of three independent experiments. Error bars indicate s.d. ***Indicates significant (P<0.0001, ANOVA and Bonferroni) decline in the number of trophozoites when compared with the control group (no bacteria).

Figure 5

Translocation of hybrid protein TEM-1-LegC5 by wild-type L. pneumophila strain JR32; L. steelei and dotA mutant into A. castellanii (a) and S. palustris (b). Five hours after infection, amoebae were harvested and lysed. Cell lysates were centrifuged to separate the soluble fraction (S), consisting of the amoeba cytosol and translocated effector proteins; from the insoluble fraction (P), containing the internalized bacteria. Samples from both fractions were analyzed by western blot using anti-TEM-1 antibody.