Fig 1.
Presence or absence of orthologous genes coding for the capsule in 80 Legionella species/strains.
Colored squares represent orthologous genes. Orthologous gene reconstruction was done with PanOCT: amino acid percentage identity cutoff was 30%, BLAST e-value cutoff 10−5, and minimum percentage match length of subject and query was 65%. For reference, the L. longbeachae NSW150 capsule cluster genes and gene names are shown on the top of the Fig. Color code: blue, pathway genes for nucleotide sugar precursor synthesis; yellow, putative transportation genes; green, putative glycosyltransferases; grey, genes of unknown function.
Fig 2.
The L. longbeachae capsule is expressed in exponential growth phase and contains a highly anionic polysaccharide that cannot be stained by silver staining.
A) L. longbeachae WT, ΔctrC and L. pneumophila WT (negative control) were grown in BYE medium to post-exponential phase. Fixed cells were stained with cationized ferritin. TEM images are representative of n = 3 independent experiments. Scale bar = 200 nm. B) L. longbeachae WT or ΔctrC harboring an empty plasmid (pBCKS) or the complementation plasmid (SSM083) were fixed in post-exponential phase and processed like samples in A). Representative images of n = 3 independent experiments. Scale bar = 500 nm. C) Polysaccharides were extracted from Llo WT, ΔctrC or Lpp WT by enzymatic isolation and subjected to ultracentrifugation. SDS gels were stained with silver nitrate or Stains-all dye. Purified LPS from E. coli O111:B4 (Sigma) was used as a control for silver staining. D) Extracts from Llo WT or ΔctrC harboring the empty plasmid (pBCKS) or the complemented mutant were stained by silver nitrate or Stains-all.
Fig 3.
The L. longbeachae capsule is transcribed in liquid medium and in cellulo, but it is not expressed in a ΔctrC mutant.
A) RNAseq of WT transcripts (n = 4); E vs. PE (E is the reference); consider relevant genes with log2 fold change of ±2 and adjusted p value ≤ 0.05. B) THP-1 cells were infected with wild type L. longbeachae expressing constitutive mKate2 and inducible sfGFP under the control of the capsule promoter (promcap). A control plasmid containing constitutive mKate2 and two copies of sfGFP without the capsule promoter was included. Cells were imaged at 22 hours post infection. Scale bar = 50 μm.
Fig 4.
The capsule mutant is impaired in replication and avirulent in mammalian and protozoan hosts, despite a functional Dot/Icm type IV secretion system.
A) Female C57BL/6 mice were infected with 107 CFU of Llo WT or ΔctrC via the nasal route. Survival was monitored over 15 days. Each group contained 9 mice. B) Infection of female C57BL/6 mice with the capsule mutant harboring an empty plasmid (pBCKS) or the complementation plasmid (SSM083) with 107 CFUs. 10 mice per group. Statistical significance was determined by Log-rank (Mantel-Cox) test. *, p≤0.05; ****, p≤0.0001. C) CFUs from lungs of infected mice were plated at 4, 48, and 96 hours post-infection. Dots represent number of animals per group. D) CFUs from spleens of infected mice were plated at 48 and 96 hours post-infection. Dots represent number of animals per group. Statistical analysis was performed by two-way ANOVA with Tukey’s post-test. E) LDH activity test in converting L-lactate + NAD+ to pyruvate + NADH, measured as absorbance at 490 nm. Lung lavage fluid from mice infected with Llo WT or ΔctrC at 72 hours post-infection. Data points correspond to individual mice and are shown as means ± SD. Statistical significance was tested by unpaired t-test. ns, non-significant; *, p≤0.1; **, p≤0.01; **** p≤0.0001. F) Acanthamoeba castellanii trophozoites were infected at MOI 0.1 at 20°C over 7 days. Samples were taken every 24 hours and CFUs are normalized to the input control. Data show means ± SD for n = 3 independent experiments. G) A. castellanii trophozoites were infected with Llo WT or ΔctrC harboring an empty control plasmid (pBCKS) or the complementation plasmid (SSM083) at an MOI of 1. Samples were taken every 24 hours and CFUs normalized to t = 1. Data show means ± SD for n = 3 independent experiments. H) A. castellanii trophozoites were infected at MOI 0.1 with equal amounts of Llo WT or ΔctrC. Samples were taken at 1, 8, 24, 30, 48, 96, 120, and 144 hours post-infection and normalized to the input control. Data show the competitive index of Llo ΔctrC over Llo WT for n = 3 independent experiments. I) Translocation of the known T4SS effector RomA was tested in THP-1 cells infected with Llo WT, ΔctrC, or a T4SS mutant, ΔdotB. Data represent means ± SD of n = 2 experiments. Statistical significance was tested by two-tailed t-test. J) Bacterial replication of Llo WT, ΔctrC and ΔdotB in hMDMs at 20 hours post-infection. Data show median bacteria area ± SD of infected cells of n = 3 independent experiments. Statistical significance was tested by one-way ANOVA. ns, non-significant; *, p≤0.05, **, p≤0.01, ***, p≤0.001, ****, p≤0.0001.
Fig 5.
The capsule delays phagocytosis of host cells.
A) For phagocytosis, differentiated THP-1 cells were infected with Llo WT or the ctrC mutant at MOI 10. Gentamycin was added at the indicated timepoints for 1 hour to kill extracellular bacteria and cells were lysed in water for CFU plating. Data show means ± SD of n = 3 independent experiments normalized to the input control at t = 0. B) For attachment, THP-1 cells were pre-treated with 2 μM cytochalasin D for two hours before infection with Llo WT or ΔctrC at MOI 10. At the indicated timepoints, gentamycin was added to half of the wells to kill extracellular bacteria. Cells were lysed in water and CFUs of gentamycin-treated cells (intracellular bacteria) and non-treated cells (total bacteria) were plated. Intracellular bacteria were deducted from total CFUs, normalized to the input control at t = 0 and data plotted as means ± SD of n = 3 independent experiments. Statistical significance was tested by two-way ANOVA with Tukey’s post-test. ns, non-significant; *, p≤0.05, **, p≤0.01, ***, p≤0.001.
Fig 6.
The capsule influences the cytokine response of primary macrophages and dendritic cells.
A-C) Human monocyte derived macrophages (hMDMs) were infected at MOI 10 and cell supernatants were collected at 24 hours post-infection. High-sensitivity SP-X array was performed on cell supernatants. Data show means ± SD of fold change over L. longbeachae WT of n = 6 independent experiments. Statistical significance was tested by pairwise t-tests; ns, non-significant; *, p≤0.05. D-F) Murine bone-marrow derived DCs were infected at MOI 10 and cell supernatants collected at 24 hours post-infection. Data show means ± SD of n = 3 independent experiments. G-I) Murine bone-marrow derived macrophages were infected at MOI 10 and cell supernatants collected at 24 hours post-infection. Data show means ± SD of n = 3 independent experiments. Statistical significance was tested by one-way ANOVA with Tukey’s post-test. ns, non-significant; *, p≤0.05; **, p≤0.01; ***, p≤0.001; ****, p≤0.0001.
Table 1.
Bacteria and plasmids used in this study.
Table 2.
Primers used in this study.