Fig 1.
Comparison of B. melitensis multiplication in lungs of wild-type mice and RAW 264.7 macrophages.
A, B. C57BL/6 mice (n = 7–8) were infected with 5x106 CFU of mCherry-expressing B. melitensis and sacrificed at the indicated time. Lungs were harvested for CFU and fluorescent microscopy analysis. Data represent (A) the CFU count per g of lung from individual mice (n indicates the number of mice per group) and (B) the number of mCherry+ bacteria per cell determined by fluorescent microscopy (n indicates the number of infected cells observed per group). C, D. RAW 264.7 macrophages were infected with an MOI of 50 (50 bacteria per cell on average). Data shown are (C) the CFU count per condition and (D) the number of mCherry+ bacteria per infected cell (n indicates the number of infected cells observed per group). E. Data represent the comparison of the average number of mCherry+ bacteria per infected lung cell and per infected RAW 264.7 macrophage. Significant differences between the indicated groups are marked with asterisks: **p < 0.01, ***p < 0.001, ****p < 0.0001, in a One-Way ANOVA with Kruskal-Wallis post-test. CFU results (A and C) are representative of three independent experiments. Microscopy bacteria count data for lung (B) are pooled from 2 independent in vivo experiment. For each experiment, the lungs of 3 mice were analyzed by fluorescence microscopy. Microscopy bacteria count data for RAW 264.7 (D) are pooled from 2 independent experiments.
Fig 2.
Fluorescent microscopic analysis of B. melitensis multiplication in lungs of wild-type mice and RAW 264.7 macrophages.
A, B. C57BL/6 mice (n = 5) were infected with 5x106 CFU of mCherry-expressing B. melitensis labelled with eFluor670 and sacrificed at the indicated time. Lungs were collected and analyzed by fluorescent and confocal microscopy for the expression of DAPI, phalloidin, mCherry and eFluor670. Data shown are (A) representative confocal images (single z-plane) of infected lungs and (B) the frequency of non-growing mother cells, growing mother cells, daughter cells and presumed dead mother cells estimated using a fluorescent microscope (n indicates the number of infected cells observed per group). C. RAW 264.7 macrophages were infected with an MOI of 50 (50 bacteria per cell on average) and analyzed by fluorescent microscopy at the indicated time. Data shown are (C) the frequency of non-growing mother cells, growing mother cells, daughter cells and presumed dead mother cells (n indicates the number of infected cells observed per group). These results are representative of two independent experiments.
Fig 3.
Transmission electron microscopic analysis of B. melitensis morphology inside alveolar macrophages purified from infected mice.
C57BL/6 mice (n = 10) received intranasally PBS (uninfected mice) or 108 CFU of mCherry-expressing wild-type or ΔvirB strains of B. melitensis and were sacrificed at the indicated time. Lungs were harvested at the indicated time (5, 12, 24, 48 hours post-infection) and alveolar macrophages were purified and analyzed by transmission electron microscopy as described in the Materials and Methods. RAW 264.7 macrophages were infected with an MOI of 50 (50 bacteria per cell on average) of an mCherry-expressing wild-type strain of B. melitensis, harvested at 48 hours post-infection and analyzed by transmission electron microscopy. Data shown are (A) representative images of bacteria from alveolar macrophages displaying live-like, dark, stressed-like, dead-like, degraded and fragmented morphologies, (B) the frequency of each bacterial morphology observed in each condition (n indicates the number of infected cells observed per condition). These results are representative of two independent experiments.
Fig 4.
B. melitensis multiplies exponentially only in a fraction of alveolar macrophages.
C57BL/6 mice (n = 10) received intranasally PBS (uninfected mice) or 5x106 CFU of mCherry-expressing wild-type or ΔvirB strains of B. melitensis labelled with eFluor670. Mice were sacrificed at the indicated time. Lungs were collected and analyzed individually by flow cytometry for the expression of CD11c, Siglec-F, mCherry and eFluor670. Data shown are (A) representative dot plots of total lung cells from control and infected mice analyzed for the expression of CD11c and eFluor670, (B) representative dot plots of total lung cells and eFluor+ cells (R1 gate) analyzed for the expression of CD11c and Siglec-F, (C) representative dot plot of eFluor+ cells analyzed for the expression of CD11c and mCherry, (D) the kinetic percentage of mCherryhigh cells among eFluor+ lung cells per individual mice (n = 10). These results are representative of three independent experiments.
Fig 5.
Comparison of B. melitensis genes required for optimal multiplication in the lung and RAW 264.7 macrophage conditions.
The figure shows the distribution of the ΔTnIF values of all B. melitensis genes (n = 2508) which are predicted not to induce an attenuation of fitness in rich medium (CTRL). Each gene is defined by two ΔTnIF values. The x-axis indicates the ΔTnIF value of the lung (TnIFlung—TnIFCTRL) at 24 (A), 48 (B) and 120 (C) hours post infection. The y-axis indicates the ΔTnIF value in macrophage RAW 264.7 (TnIFRAW 264.7—TnIFCTRL) at 24 hours post infection. These ΔTnIF values comparisons allow to visualize all the genes associated with a drop in fitness both in the lung and macrophages (purple area), in the lung specifically (pink area) or in RAW 264.7 macrophages specifically (blue area). Numbers indicate the number of LF or VLF genes (ΔTnIF<-0.5) in each area.
Fig 6.
Clustering analysis of very low fitness genes in the lung condition at 48 hours post-infection.
The diagram shows the potential interactions between the 58 genes displaying a ΔTnIF < -1.0 identified in lungs of wild-type mice at 48 hours post-infection. The color code of the legend indicates whether the genes are specifically attenuated in the lung or if they are also attenuated in the RAW 264.7 cell line condition at 24 hours post-infection. The legend also indicates which genes are predicted to be partially restored in IL-17RA-/- mice and asthmatic mice conditions. This clustering analysis was carried out with STRING (https://string-db.org).
Fig 7.
List of 14 Very Low Fitness (VLF) genes specific to the lung condition (ΔTnIF lung 48 hours < -1.5, ΔTnIF RAW 264.7 > -0.5) with TnIF values in the 2YT, RAW 264.7 and lung conditions, as well as a predicted function.
Color scale shows the score of ΔTnIF of each gene.
Fig 8.
Functional confirmation of the prediction derived from the Tn-seq analysis of RAW 264.7 and lung conditions.
Data shown are bacterial count (CFU) at the indicated time post-infection in RAW 264.7 (A) and lung from wild-type mice infected intranasally (B) with wild-type (wt), ΔglpX, ΔvirB, ΔwbkF, Δgmd, Δper, ΔfadA, ΔfadJ and ΔtrpD strains of B. melitensis at a dose of 5x106 CFU and a MOI of 1:50, respectively. Significant differences between wt and the indicated groups are marked with asterisks: *p < 0.1, **p < 0.01, ***p < 0.001, ****p < 0.0001, in a One-Way ANOVA with Kruskal-Wallis post-test. These results are representative of three independent experiments.
Fig 9.
IL-17RA deficiency and asthma induce distinct patterns of infection in the lung.
Control and asthmatic wild-type C57BL/6 mice and IL-17RA-/- mice were infected intranasally with a dose of 5x107 CFU of mCherry-B. melitensis labelled with eFluor670. Mice were sacrificed at 48 hours post-infection and the lungs were collected and analyzed by confocal and fluorescent microscopy for the expression of DAPI, phalloidin, mCherry and eFluor670 markers. Data shown are (A) representative confocal images (single z-plane) of infected cells, (B) the number of mCherry+ bacteria / alveolar macrophages and (C) the number of mCherry+ bacteria per lung surface unit determined by fluorescent microscopy. In order to avoid biases in the analysis, an automatic acquisition of the entire surface of the tissue section is carried out using MosaiX module from AxioVision program (Zeiss). We exclude from this analysis the edges of the organ as well as the damaged areas. n indicates the number of cells analyzed (B) or the number of surfaces analyzed (C) for each condition. Significant differences between control wt and the indicated groups are marked with asterisks: ****p < 0.0001, in a One-Way ANOVA with Kruskal-Wallis post-test. The data are representative of two independent experiments.
Fig 10.
Comparison of B. melitensis genes required for optimal multiplication in lungs of wild-type and IL-17RA-/- mice.
A. List of VLF genes in lungs of wild-type mice (< -1.0 ΔTnIF) that are recovered (> 1.0) in lungs of IL-17RA-/- mice associated with their TnIF values in the CTRL 2YT and lung from wild type and IL-17RA-/- mice conditions, as well as predicted function. B. Schematic representation of the lipopolysaccharide biosynthesis pathway of Brucella specifying the genes that become partially dispensable in the IL-17RA-/- mice condition (adapted from the KEGG PATHWAY database, https://www.genome.jp/kegg/pathway.html).
Fig 11.
Functional confirmation of the prediction derived from the Tn-seq analysis of the IL-17RA-/- and asthmatic conditions.
A. Data shown are bacterial count (CFU) at 5 and 48 hours post-infection in wild-type and IL-17RA-/- mice infected intranasally with wild-type (wt), ΔwbkF and Δper strains of B. melitensis at a dose of 5x106 CFU. B. Data shown are bacterial count (CFU) at 5 and 48 hours post-infection in control and asthmatic wild-type mice intranasally infected with wild-type, ΔfadA and ΔfadJ strains of B. melitensis at a dose of 5x106 CFU. Significant differences between wt and the indicated groups are marked with asterisks: *p < 0.1, **p < 0.01, ****p < 0.0001, in a One-Way ANOVA with Kruskal-Wallis post-test. These results are representative of three independent experiments.