Article Figures & Data
Figures
- FIG 1
Reduced L. pneumophila infection and increased survival of Acanthamoeba castellanii Neff in the presence of P. amoebophila symbionts. (A) L. pneumophila Lp02-T load in the absence (−) and presence (+) of P. amoebophila (Pam) at 5 weeks postinfection (wpi) was measured as the proportion of amoebae containing L. pneumophila FISH signals (infected), the proportion of amoebae containing >5 L. pneumophila signals (high), and the proportion of amoebae containing only 1 to 5 L. pneumophila signals (low). In addition, growth of Lp02-T was assessed by determining CFU per ml at 5 wpi. The difference between −Pam and +Pam was statistically significant in comparisons of low and high infection levels (*, P < 0.05 by unpaired t test) and CFU/ml (*, P < 0.001 by unpaired t test). (B) L. pneumophila Paris infection levels were also determined via FISH, but here the course of infection over 5 weeks is shown because the endpoints were 0% and 100% infected amoebae. Two-way analysis of variance (ANOVA) combined with Tukey’s test was applied to compare the two curves, and significantly different time points are indicated (box marked by an asterisk, P < 0.001). (C and D) Amoeba growth at 5 weeks after L. pneumophila infection is expressed as the difference between start and endpoint amoeba numbers. In the case of strain Lp02-T (C), symbiont-free and symbiont-containing amoebae not infected with L. pneumophila (−Lpn) served as additional controls. Lowercase letters denote significantly distinct statistical groups (P < 0.01 by one-way ANOVA and Tukey’s test). (D) Statistical testing in the case of strain Paris was done using the unpaired t test (*, P < 0.01). In panels A, C, and D, horizontal lines denote means and error bars show standard deviations from three biological replicates. (E and F) The infection status of L. pneumophila Lp02-T (E) and Paris (F) at 5 wpi was visualized by FISH (red, LEGPNE1 probe specific for L. pneumophila; green, Chls-523 probe specific for chlamydiae) and DAPI staining (gray). Infection experiments were carried out at 30°C for L. pneumophila Lp02-T (MOI of 20) and at 20°C for L. pneumophila Paris (MOI of 0.5). Scale bars, 10 μm.
- FIG 2
Environmental amoeba isolates harboring the symbiont P. amoebophila eliminate different L. pneumophila strains. FISH combined with DAPI staining (blue) was performed at 5 weeks after L. pneumophila infection (MOI of 20, 20°C). Amoeba and L. pneumophila strains used are indicated on the top of each set of images, in which the first row shows infections without the symbiont (−Pam) and the second row with the symbiont (+Pam). Initial infection with L. pneumophila was determined at 2 hpi (diamond symbols in Fig. 4). FISH probes specifically targeted L. pneumophila (LEGPNE1, magenta) and the chlamydial symbiont (Chls-523, green). Amoeba outlines are indicated by white dotted lines. Scale bars, 10 μm.
- FIG 3
Increased fitness of a recovered amoeba isolate in the face of L. pneumophila Lp02-T infection. Acanthamoeba sp. strain ML previously not exposed to L. pneumophila Lp02-T (Lpn; amoebae termed naive) or exposed but cleared from L. pneumophila (termed recovered) were infected with L. pneumophila Lp02-T (MOI of 20, 20°C). Gentamicin treatment was performed to kill extracellular bacteria, and L. pneumophila numbers (left) and net amoeba growth (right) were determined, starting at 2 hpi. The presence and absence of endosymbionts are indicated by +Pam and −Pam, respectively. Error bars show standard deviations from three biological replicates. In total, L. pneumophila growth is only significantly different between symbiont-free amoebae and both conditions with the symbiont present (*, P < 0.05 by one-way ANOVA and Tukey's test). However, L. pneumophila numbers at 48 hpi also significantly vary between recovered and naive amoebae harboring the symbiont (*, P < 0.01 by unpaired t test). Amoeba growth is significantly different between recovered amoebae (+Pam) and naive amoebae with and without symbionts at the last two time points (*, P < 0.001 by two-way ANOVA and Tukey's test).
- FIG 4
The symbiont P. amoebophila does not affect L. pneumophila uptake and host cell invasion. (A) To assess susceptibility of amoebae to L. pneumophila infection, we measured the number of viable L. pneumophila cells per amoeba cell (upper) and percentage of L. pneumophila-infected amoebae (lower), both at 2 hpi. CFU/amoeba were determined not only for naive amoebae with (+Pam) and without the symbiont (−Pam) but also for fully recovered symbiont-harboring amoebae (+Pam-recov.). Color groups denote separate experiments, with each data point representing a biological replicate. Circles show results from experiments using A. castellanii Neff, whereas experiments conducted with Acanthamoeba sp. strain ML are represented by diamonds. Data points filled with color were obtained using L. pneumophila Paris, and data points with black dots were obtained using L. pneumophila Lp02-T. Horizontal solid lines indicate medians, taking into account all data points. Dotted lines show medians only considering the infections with symbionts from Fig. 3 (pink and orange diamond shapes). Error bars denote interquartile ranges. No individual experiment yielded a statistically significant difference between the presence and absence of the symbiont or between naive and recovered amoebae (P > 0.05 by unpaired t test). (B) The role of extracellularly present symbionts in invasion by L. pneumophila was assessed by adding viable or heat-inactivated (hi) P. amoebophila (Pam) to uninfected A. castellanii Neff together with L. pneumophila Lp02-T (Lpn) but at different ratios. At 2 hpi, L. pneumophila infection levels were determined and compared to the respective levels for L. pneumophila only (control). A fold difference around 1 indicates that L. pneumophila infection levels were similar between treatment (viable or heat-inactivated P. amoebophila) and the control. The experiment was conducted in three biological replicates. Error bars indicate the 95% confidence intervals of a ratio of two means. Infections of A. castellanii Neff with L. pneumophila Lp02-T were carried out at 30°C; all other infections shown were conducted at 20°C.
- FIG 5
L. pneumophila released from symbiont-harboring amoebae are reduced in number and infectivity. L. pneumophila strain Paris was used to infect Acanthamoeba sp. strain ML (MOI of 5, 20°C). At 96 hpi, the supernatants containing the released L. pneumophila cells were harvested, and L. pneumophila cells were counted (A) and used to infect symbiont-free amoebae at an MOI of 30 (−Pam) or 23 (+Pam) (B). Both the CFU/ml released at 96 hpi and the percentage of amoebae at 2 hpi infected with L. pneumophila (DAPI counts) were significantly different from those of the control, even when normalized for the slight difference of a factor of 1.3 in MOI (*, P < 0.001 by unpaired t test). Error bars indicate standard deviations, and horizontal lines in panel B show the means from three biological replicates. –Pam, P. amoebophila absent; +Pam, P. amoebophila present.
- FIG 6
Altered L. pneumophila gene expression in the presence of the symbiont suggests impaired transition from replicative to transmissive phase. Transcriptomes of L. pneumophila Paris infecting A. castellanii Neff with (+Pam) and without (−Pam) the P. amoebophila symbiont at 24 h, at 96 h within amoebae, and after host cell release (extracell) were determined by RNA-Seq (20°C; for MOIs see Materials and Methods). Processes and functional categories that were significantly enriched among differentially expressed genes include flagellum biosynthesis, PHB metabolism, and genes involved in growth phase regulation (Fig. S6). Heatmaps show log2 fold changes (logFC) of all genes that were differentially expressed between at least one pair of conditions (false discovery rate of <0.05). A logFC below 1 (no differential expression) is shown in white, significant downregulation is shown in blue, and significant upregulation is shown in red. The stronger the color, the stronger the gene expression change between two conditions. Flagellar genes are ordered by genetic locus, and the other genes are ordered by processes. Note that fleQ, phbC-1, letS, lqsS, lqsT, rpoS, cpxA, and cpxR were not differentially expressed. 24→96hpi, expression at 96 hpi compared to that at 24 hpi; 96hpi→extracell., extracellular expression (96 hpi) compared to intracellular expression.
- FIG 7
Symbiont-mediated defense against L. pneumophila. In the absence of chlamydial endosymbionts, L. pneumophila undergoes a characteristic intra-amoeba life cycle involving entry, replication within a Legionella-containing vacuole (LCV), transition to the transmissive form, amoeba lysis, and bacterial escape. The transmissive form can subsequently infect other host cells (left). The present study, together with that of Maita et al. (47), demonstrates that chlamydial endosymbionts of Acanthamoeba spp. provide the host with protection against different strains of L. pneumophila, although the modes of protection are different. While Neochlamydia species-harboring amoebae block the uptake of L. pneumophila (right) (47), P. amoebophila-containing amoebae interfere with the intracellular L. pneumophila life cycle, resulting in a significantly reduced number of released bacteria that are less virulent (center) (this study). Steps 3 and 4 in the Neochlamydia sp. strain S13 model have not been demonstrated but would be expected to be a consequence of impaired L. pneumophila uptake (in parentheses). Note that the two types of endosymbionts differ in that P. amoebophila is enclosed within an inclusion membrane, whereas Neochlamydia sp. strain S13 is found directly in the host cytoplasm. Replicative-phase L. pneumophila is shown in dark violet, whereas transmissive forms are depicted in pink.
Supplemental Material
FIG S1
Amoeba growth and spread of P. amoebophila infection. (A) A. castellanii Neff amoebae with and without the endosymbiont P. amoebophila (Pam) were seeded at low densities to avoid contact-dependent inhibition of growth (Text S1), and growth was monitored over 4 days at two different temperatures. Data points from all three replicates are plotted (filled circles), and exponential functions were applied to model growth (lines). Mean doubling times (td [h]) under each condition are depicted on the right. Independent of the temperature, symbiont-free amoebae replicated faster than amoebae with the symbiont, indicating that harboring the symbiont impairs host fitness. Furthermore, as symbiont-harboring amoebae replicated continuously without notable lysis over the time examined, the symbiont is transmitted vertically, and the association is stable. (B) Amoebae with and without the symbiont were mixed (1:10), and the proportion of P. amoebophila-containing amoebae was monitored over three days (30°C) (Text S1). Data points from all three replicates are plotted (filled circles), and logistic curves (lines) best represent the course of increasing proportion of symbiont-containing amoebae and decreasing proportion of uninfected amoebae. All or nearly all amoebae harbored symbionts after 69 h of cocultivation in all replicates, demonstrating horizontal (in addition to vertical) transmission of P. amoebophila. Download FIG S1, PDF file, 0.05 MB.
Copyright © 2019 König et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
TEXT S1
Supplemental materials and methods. Download Text S1, DOCX file, 0.01 MB.
Copyright © 2019 König et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
TABLE S1
Summary of infection experiments. Download Table S1, DOCX file, 0.02 MB.
Copyright © 2019 König et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
FIG S2
Decrease of L. pneumophila cell numbers in the presence of the symbiont. L. pneumophila numbers are shown as CFU/ml. (A) L. pneumophila numbers are drastically reduced already one week postinfection when A. castellanii Neff harbors symbionts. The difference is statistically significant for both L. pneumophila strains measured (*, P < 0.05, unpaired t test). (B) L. pneumophila Lp02-T within A. castellanii Neff initially grows intracellularly before numbers decrease when the endosymbiont is present. (A and B) Error bars show the standard deviations from three biological replicates. Infections of A. castellanii Neff with L. pneumophila Lp02-T were carried out at 30°C (MOI of 20); infections with L. pneumophila Paris were conducted at 20°C (MOI of 10). –Pam, P. amoebophila absent; +Pam, P. amoebophila present. Download FIG S2, PDF file, 0.05 MB.
Copyright © 2019 König et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
FIG S3
Course of L. pneumophila infection in the presence and absence of P. amoebophila monitored by FISH. (A) Amoebae harboring the symbiont do not block L. pneumophila infection at 2 hpi and still support L. pneumophila growth. A. castellanii Neff amoebae with (+Pam) and without (−Pam) the symbiont were infected with L. pneumophila Lp02-T (upper; MOI of 20, 30°C) or Paris (lower; MOI of 10, 20°C), and the course of infection was monitored targeting L. pneumophila (red/pink, LEGPNE1 probe) and P. amoebophila (green, Chls523 probe). Amoeba outlines (white lines) are based on corresponding phase contrast images (upper) or FISH staining of the amoebae (lower). Note that amoeba cell sizes differed at 48 and 96 hpi (unpaired t test, P < 0.05), with infected amoeba carrying the symbiont being smaller on average (13 to 17 μm versus 19 to 26 μm for strain Lp02-T and 10 to 11 μm versus 12 to 17 μm for strain Paris). Scale bars, 20 μm. (B) L. pneumophila and P. amoebophila always appear spatially well separated within amoeba trophozoites. L. pneumophila Paris (pink) and P. amoebophila (green) at 48 hpi are visualized as described above. The lack of overlap between L. pneumophila and P. amoebophila-specific probes indicates that both bacteria are located in distinct vacuoles. Note that P. amoebophila cells are enclosed individually in host-derived inclusions. Blue color indicates DNA (DAPI staining); scale bar, 5 μm. Download FIG S3, PDF file, 2.6 MB.
Copyright © 2019 König et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
FIG S4
Clearance of L. pneumophila in symbiont-containing amoebae as demonstrated by PCR. At 5 weeks post-L. pneumophila infection, DNA was extracted from replicate cultures with (+Pam) or without (−Pam) the chlamydial symbiont, and L. pneumophila was targeted by PCR primers specific for the L. pneumophila mip gene. In the case of infection of A. castellanii Neff with L. pneumophila Paris and Acanthamoeba sp. strain 2HH with L. pneumophila Lp02-T (left and center gel images), primers Legmip_f/Legmip_r were used (R. M. Ratcliff, J. A. Lanser, P. A. Manning, and M. W. Heuzenroeder, J Clin Microbiol 36:1560–1567, 1998), whereas L. pneumophila Lp02-T infecting Acanthamoeba sp. strain ML was targeted using primers designed in this study (Lp02mipF/R, right gel image) (Text S1). All infection experiments were conducted at 20°C, with an MOI of 0.5 for Paris infecting Neff and an MOI of 20 for Lp02-T infecting 2HH and ML. M, marker; numbers 1 to 3, replicates; −, negative control; +, positive control. Download FIG S4, PDF file, 1.1 MB.
Copyright © 2019 König et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
FIG S5
Effect of L. pneumophila MOI and incubation temperature. (A) L. pneumophila MOI affects amoeba recovery. A. castellanii Neff cells with (+Pam) and without (−Pam) the symbiont were infected with L. pneumophila Paris using different MOIs (20°C), and amoeba numbers were determined one week postinfection. Note that in all experiments, amoeba numbers were reduced compared to starting numbers (658.7 ± 113.3 amoebae/μl), but the reduction occurred to various degrees depending on MOI and presence of the symbiont. Importantly, in all cases L. pneumophila numbers were lower when the symbiont was present (Table S1). At low (0.5) and high (10) L. pneumophila MOI, cell numbers of amoebae without the symbiont did not decline as much as those of amoebae with the symbiont, indicating that infection load (MOI) plays a role. In contrast, when MOIs of 2, 5, and 8 were used, amoebae without the symbiont declined to a greater extent than amoebae with the symbiont. The difference between amoebae with or without Pam was statistically significant for MOIs of 0.5, 5, 8, and 10 (*, P < 0.01, unpaired t test). Error bars mark the standard deviations from three biological replicates. (B) Temperature affects L. pneumophila infection level but not amoeba recovery. Acanthamoeba sp. strain ML amoebae with (+Pam) and without (−Pam) the symbiont were infected with L. pneumophila Lp02-T (MOI of 20). After 5 weeks of incubation at either 20°C or 30°C, the percentage of L. pneumophila-infected amoebae (upper) and amoeba growth (lower) were determined. L. pneumophila was only cleared at 20°C (*, P < 0.05 at 20°C, P = 0.14 at 30°C). As for the laboratory strain A. castellanii Neff, Acanthamoeba sp. strain ML harboring the symbiont showed growth over 5 weeks postinfection. Controls without the symbiont exhibited zero net growth, indicating a certain level of resilience even without the symbiont (*, P < 0.01 at both temperatures). Statistically significant differences were determined using the unpaired t test. (A and B) Error bars mark the standard deviations from three biological replicates. Download FIG S5, PDF file, 0.04 MB.
Copyright © 2019 König et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
TABLE S2
RNA-Seq read and mapping statistics. Download Table S2, DOCX file, 0.02 MB.
Copyright © 2019 König et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
FIG S6
Enriched functional categories and processes among differentially expressed P. amoebophila and L. pneumophila Paris genes. Gene expression was determined by RNA-Seq. All functional categories that were significantly overrepresented (false discovery rate, <0.05) among up (↑)- and downregulated (↓) genes are shown (for details see Data set S1). Numbers of up- and downregulated genes are depicted. Long arrows point out the direction of comparison, e.g., 450 symbiont genes were significantly upregulated at 24 h post-L. pneumophila infection (24 hpi) compared to the uninfected control (A), and 685 L. pneumophila genes were significantly downregulated at 96 hpi compared to levels at 24 hpi, when the amoebae did not harbor symbionts (B). (A) Terms in grey were found by trend to be enriched, as evaluated by manual inspection. Note that some genes encoding a putative type IV secretion system (tra genes) were also detected to be differentially expressed. (B) Terms in black are shared between the two conditions (amoebae with and without symbionts), and terms in blue are unique for the respective condition. The putative DNA transfer island known as lvr genes (grey) was significantly differentially expressed where indicated but failed to show up as overrepresented due to missing automatic functional annotation. T3SS, type 3 secretion system; LPS, lipopolysaccharide; Pro, proline; Met, methionine; Ala, alanine; Tyr, tyrosine; Lpn, L. pneumophila Paris; Pam, P. amoebophila. Download FIG S6, PDF file, 0.05 MB.
Copyright © 2019 König et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
DATA SET S1
Gene expression values, gene annotations, and enrichment analyses of P. amoebophila and L. pneumophila Paris. Download Data Set S1, XLSX file, 1.5 MB.
Copyright © 2019 König et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
