(C) PLOS One This story was originally published by PLOS One and is unaltered. . . . . . . . . . . Genome-wide analysis of Brucella melitensis genes required throughout intranasal infection in mice [1] ['Georges Potemberg', 'Unité De Recherche En Biologie Des Microorganismes', 'Urbm', 'Laboratoire D Immunologie Et De Microbiologie', 'Narilis', 'University Of Namur', 'Namur', 'Laboratoire De Parasitologie', 'Ulb Center For Research In Immunology', 'U-Cri'] Date: 2022-08 Brucellae are facultative intracellular Gram-negative coccobacilli that chronically infect various mammals and cause brucellosis. Human brucellosis is among the most common bacterial zoonoses and the vast majority of cases are attributed to B. melitensis. Using transposon sequencing (Tn-seq) analysis, we showed that among 3369 predicted genes of the B. melitensis genome, 861 are required for optimal growth in rich medium and 186 additional genes appeared necessary for survival of B. melitensis in RAW 264.7 macrophages in vitro. As the mucosal immune system represents the first defense against Brucella infection, we investigated the early phase of pulmonary infection in mice. In situ analysis at the single cell level indicates a succession of killing and growth phases, followed by heterogenous proliferation of B. melitensis in alveolar macrophages during the first 48 hours of infection. Tn-seq analysis identified 94 additional genes that are required for survival in the lung at 48 hours post infection. Among them, 42 genes are common to RAW 264.7 macrophages and the lung conditions, including the T4SS and purine synthesis genes. But 52 genes are not identified in RAW 264.7 macrophages, including genes implicated in lipopolysaccharide (LPS) biosynthesis, methionine transport, tryptophan synthesis as well as fatty acid and carbohydrate metabolism. Interestingly, genes implicated in LPS synthesis and β oxidation of fatty acids are no longer required in Interleukin (IL)-17RA -/- mice and asthmatic mice, respectively. This demonstrates that the immune status determines which genes are required for optimal survival and growth of B. melitensis in vivo. Brucellosis is one of the most widespread bacterial zoonoses worldwide. Using transposon sequencing (Tn-seq) analysis, we showed that among 3369 predicted genes of the Brucella melitensis genome, 861 are required for optimal growth in rich medium and 186 additional genes appeared necessary for survival of B. melitensis in RAW 264.7 macrophages in vitro. We also investigated the early phase of pulmonary infection in mice and identified 94 additional genes that are required for survival in the lung at 48 hours post infection. Among them, 42 genes are common to RAW 264.7 macrophages and the lung conditions, including the T4SS and purine synthesis genes. But 52 genes are not identified in RAW 264.7 macrophages, including genes implicated in lipopolysaccharide (LPS) biosynthesis, methionine transport, tryptophan synthesis as well as fatty acid and carbohydrate metabolism. Interestingly, genes implicated in LPS synthesis and β oxidation of fatty acids are no longer required in Interleukin (IL)-17RA -/- mice and asthmatic mice, respectively. Our work demonstrates that both the immune status and the nature of the infected cell type determines which genes are required for optimal survival and growth of B. melitensis in vivo. Funding: This work was supported by grants from the Fonds National de la Recherche Scientifique (FNRS) (Fonds De La Recherche Scientifique - FNRS 1.4.013.16.F and 3.4.600.06.F to E.M. and Fonds De La Recherche Scientifique - FNRS T.0060.15 and T.0058.20 to X.D.B.). E.M. is a Senior Research Associate from the FRS-FNRS (Belgium). G.P., A.D., E.B. and F-X.S. hold FRIA PhD grants from the FRS-FNRS (Belgium). G.P. and A.D. were supported by Fonds Spécial de Recherche (FSR) PhD grants from the UNamur (Belgium). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Copyright: © 2022 Potemberg et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. In the current study, we characterized the initial growth of B. melitensis in the lungs of intranasally-infected C57BL/6 mice in order to identify the early selection pressures affecting its multiplication. Then, we used Tn-seq screens to compare genes contributing to the fitness of Brucella melitensis in 2YT nutrient rich media, in RAW 264.7 macrophages and in lungs from wild-type C57BL/6 mice. To identify genes specifically implicated in the escape from the immune response, we also performed Tn-seq screens on infected mice deficient for the control of Brucella growth, such as IL-17RA -/- [ 9 ] and asthmatic mice [ 11 ]. Signature-tagged mutagenesis (STM)[ 12 ] or more classical screening of transposon mutants has been employed to identify genes that are essential for Brucella growth and survival in a macrophage cell line in vitro [ 13 ][ 14 ], in mice [ 15 ][ 16 ][ 17 ] and in natural hosts such as the goat [ 18 ]. However, STM uses a limited number of transposon mutants for screening, thus failing to truly saturate the entire genome. Transposon sequencing ( Tn-seq ) is a recent powerful approach to rapidly and comprehensively determine an organism’s minimal genetic requirements for growth and survival under a variety of different conditions [ 19 ][ 20 ]. A high-density transposon insertion library is exposed to a condition of interest, and then subjected to high-throughput sequencing to map the transposon insertion site for each mutant in the library. The number of reads detected for each insertion mutant is proportional to the fitness of that mutant under the selected growth condition. Recently, a Tn-seq screen of B. abortus identified many genes important for growth in a nutrient rich media and in vitro in RAW 264.7 macrophages [ 21 ]. However, much remains to be learned regarding genes required for the survival of Brucella in a well characterized in vivo host infection model. During the host infection, B. melitensis mainly leads a stealthy intracellular lifestyle [ 6 ]. The type IV secretion system (T4SS), which is encoded by the virB operon, is required for the establishment of intracellular replicative niches [ 7 ]. B. melitensis strains lacking a functional T4SS appear to be highly attenuated in mice and in their natural host, the goat [ 8 ]. Over the last decade, our group has characterized the protective response against B. melitensis in an intranasal (i.n.)[ 9 ][ 10 ] murine infection model and demonstrated that the early phase of lung infection is controlled by an Interleukin (IL)-17RA-dependent Th17 response and the late phase by an IFN-γR-dependent Th1 response. We also demonstrated that the asthma-induced Th2 response can greatly increase the Brucella load in the lungs [ 11 ]. Brucellae are small Gram-negative facultative intracellular bacteria which belong to the Rhizobiales order within the α2-proteobacteria subgroup. They are the causative agent of brucellosis, one of the most common bacterial anthropozoonoses that generates major economic losses and public health issues. B. melitensis is the species most often involved in ovine and caprine brucellosis and is also the most pathogenic species for humans [ 1 ]. Human brucellosis primarily occurs following ingestion of contaminated foods or mucosal exposure to contaminated aerosols [ 2 ][ 3 ][ 4 ]. It is characterized by nonspecific flu-like symptoms during the early acute phase, and is followed by a chronic infection with debilitating consequences in the absence of prolonged antibiotic treatment [ 1 ][ 5 ]. Results Comparison of B. melitensis multiplication in a RAW 264.7 cell line and the lung Our main objective was to use a Tn-seq approach to identify bacterial genes essential for the initiation of B. melitensis infection in vivo. To produce infection by a natural route that confronts B. melitensis with mucosal immunity, we chose a well characterized model of intranasal (i.n.) infection in the mouse model [9] that mimics aerosol infection. As shown previously [10], i.n. infection produces a specific pattern of bacterial dissemination, limited to a small number of tissues. B. melitensis must colonize the lung and the mediastinal lymph node before spreading and establishing in the spleen. To characterize the infected cells in the i.n. model, we used a mCherry-expressing stain of B. melitensis (mCherry-Br) stained with eFluor670 (eFluor), which is a dye used to monitor polar growth [10]. We observed that, during the first 48 hours post-infection, the eFluor+ lung cells from intranasally infected wild-type C57BL/6 mice are FSChigh (S1A Fig) MHCIImed F4/80med CD11bmed CD11chigh Ly6Clow Ly6Glow Siglec-Fhigh (S1B Fig), thus demonstrating that the cells infected in the lung are mainly alveolar macrophages (AMs). We hypothesize that alveolar macrophages constitute one of the first lines of defense against pulmonary B. melitensis infection in our experimental model. The Tn-seq approach is known to be highly sensitive to bottleneck effects [22]. Many false positive classifications are observed when stochastic loss of transposon mutants due to bottlenecks overshadows the effects of fitness defects. The risk of trans-complementation, where a strain with a transpositional (Tn) mutation is able to survive or multiply thanks to the presence of strains with a wild-type allele, resulting from coinfection of a single host cell with several independent Tn mutants, must also be reduced [23]. To avoid these problems, we used an infection dose that reduces the risk of bottleneck effects while limiting the number of bacteria initially phagocytosed per cell to one on average. Following infection with 5x106 colony forming units (CFU) of wild-type mCherry-Br, the CFU count in the lungs of wild-type C57BL/6 mice remained stable at 107 CFU/g, which is approximately half the infectious dose, between 5 and 24 hours post-infection, and then reaches 108 CFU at 48 hours post-infection (Fig 1A). Fluorescent microscopic analysis of lung sections showed that the average number of mCherry+ bacteria per cell at 5 hours post-infection was 1.93 (Fig 1B). Similar experiments with ΔvirB mCherry-Br confirmed that B. melitensis persistence in the lungs (Fig 1A) and B. melitensis multiplication in AMs (Fig 1B) require a functional T4SS, like in RAW 264.7 macrophages, a cell line frequently used for the in vitro study of Brucella infection (Fig 1C and 1D). PPT PowerPoint slide PNG larger image TIFF original image Download: 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. https://doi.org/10.1371/journal.ppat.1010621.g001 Interestingly, although the number of CFU in the lung remained stable between 5 and 24 hours post-infection (Fig 1A), we observed a first peak in the number of bacteria per cell at 12 hours and a second at 48 hours post-infection (Fig 1B), which suggests that there may be a selection step between 12 and 24 hours post-infection. This phenomenon was not observed in infected RAW 264.7 macrophages, where the number of CFUs (Fig 1C) as well as the number of bacteria per cell (Fig 1D) increased steadily between 5 and 48 hours post-infection (see Fig 1E for a comparison between the lung and RAW 264.7 macrophages). B. melitensis multiplies exponentially in only a fraction of alveolar macrophages Confocal microscopy of lungs infected for 48 hours (Fig 2A) showed that only a small fraction of infected cells was “permissive” to Brucella multiplication and contained a high number of daughter bacteria. At that point, most infected cells contain only dead bacteria and were considered as “resistant cells”. Flow cytometry analysis of lung cells from wild-type C57BL/6 mice infected i.n. with 5x106 CFU of wild-type or ΔvirB mCherry-Br, both stained with eFLuor, confirmed the presence of two well distinct populations of infected AMs at 48 hours post-infection (Fig 4). As AMs are auto-fluorescent at most wavelengths used in common fluorescence reporter systems, we chose to identify infected cells on the basis of the eFluor signal, which is particularly intense and easily detectable, as shown in Fig 4A. At 48 hours, >90% of eFluor+ cells appeared as CD11chigh Siglec-Fhigh AMs (Fig 4B). These cells can be divided into two distinct populations based on the mCherry-Br count measured by the intensity of the mCherry signal (Fig 4C). Kinetic analysis showed that the frequency of mCherryhigh cells strongly increases between 12 and 48 hours post-infection (Fig 4D). At 48 hours post-infection, 10.6 ± 2.5% of eFluor670+ cells appeared to be mCherryhigh permissive cells. Permissive and resistant cells were not distinguishable based on CD11b, CD11c, Ly6C, Ly6G, F4/80 and MHCII expression (S1B Fig). As expected, only a negligible frequency of mCherryhigh cells were detected at 48 hours post-infection in the lungs of mice infected with the ΔvirB mCherry-Br strain (Fig 4C and 4D). PPT PowerPoint slide PNG larger image TIFF original image Download: 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. https://doi.org/10.1371/journal.ppat.1010621.g004 On the whole, flow cytometry analysis of lung cells from infected mice demonstrated that growth of Brucella during pulmonary infection is observed only in a small fraction of infected alveolar macrophages. These permissive cells are thus thought to be responsible for establishing the infection and disseminating B. melitensis to the spleen. At this stage of our study, we can therefore affirm that B. melitensis, despite a constant level of CFU in the lung of infected mice, is exposed to strong and complex selection pressures. The effect of the latter on B. melitensis is observable very early and allows the elimination of B. melitensis in the majority of infected alveolar macrophages. This model therefore seems to us to be optimal for identifying the bacterial genes allowing B. melitensis to escape the immune response. [END] --- [1] Url: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1010621 Published and (C) by PLOS One Content appears here under this condition or license: Creative Commons - Attribution BY 4.0. via Magical.Fish Gopher News Feeds: gopher://magical.fish/1/feeds/news/plosone/