Live attenuated vaccines (LAVs) whose virulence would be controlled at the tissue level could be a crucial tool to effectively fight intracellular bacterial pathogens, because they would optimize the ...induction of protective immune memory while avoiding the long-term persistence of vaccine strains in the host. Rational development of these new LAVs implies developing an exhaustive map of the bacterial virulence genes according to the host organs implicated. We report here the use of transposon sequencing to compare the bacterial genes involved in the multiplication of Brucella melitensis, a major causative agent of brucellosis, in the lungs and spleens of C57BL/6 infected mice. We found 257 and 135 genes predicted to be essential for B. melitensis multiplication in the spleen and lung, respectively, with 87 genes common to both organs. We selected genes whose deletion is predicted to produce moderate or severe attenuation in the spleen, the main known reservoir of Brucella, and compared deletion mutants for these genes for their ability to protect mice against challenge with a virulent strain of B. melitensis. The protective efficacy of a deletion mutant for the plsC gene, implicated in phospholipid biosynthesis, is similar to that of the reference Rev.1 vaccine but with a shorter persistence in the spleen. Our results demonstrate that B. melitensis faces different selective pressures depending on the organ and underscore the effectiveness of functional genome mapping for the design of new safer LAV candidates.Live attenuated vaccines (LAVs) whose virulence would be controlled at the tissue level could be a crucial tool to effectively fight intracellular bacterial pathogens, because they would optimize the induction of protective immune memory while avoiding the long-term persistence of vaccine strains in the host. Rational development of these new LAVs implies developing an exhaustive map of the bacterial virulence genes according to the host organs implicated. We report here the use of transposon sequencing to compare the bacterial genes involved in the multiplication of Brucella melitensis, a major causative agent of brucellosis, in the lungs and spleens of C57BL/6 infected mice. We found 257 and 135 genes predicted to be essential for B. melitensis multiplication in the spleen and lung, respectively, with 87 genes common to both organs. We selected genes whose deletion is predicted to produce moderate or severe attenuation in the spleen, the main known reservoir of Brucella, and compared deletion mutants for these genes for their ability to protect mice against challenge with a virulent strain of B. melitensis. The protective efficacy of a deletion mutant for the plsC gene, implicated in phospholipid biosynthesis, is similar to that of the reference Rev.1 vaccine but with a shorter persistence in the spleen. Our results demonstrate that B. melitensis faces different selective pressures depending on the organ and underscore the effectiveness of functional genome mapping for the design of new safer LAV candidates.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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
.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract
Understanding how specific cancer mutations modulate the tumor microenvironment is essential for developing new precision cancer therapies. The histone methyltransferase MLL3 is a tumor ...suppressor that is frequently mutated together with p53 and PI3-kinase in breast cancer. Herein, using a mouse mammary-stem-cell-based tumor model and CRISPR gene editing, we recapitulated this genetic setting and revealed that Mll3 loss promotes tumorigenesis through enhancing tumor-intrinsic aggressiveness and establishing an immunosuppressive microenvironment. Specifically, HIF1a was upregulated in Mll3-mutant tumors and controlled proliferation and stem cell activity of the tumor cells. HIF1a also transcriptionally upregulated expression of the chemokine CCL2 by tumor cells. CCL2 further contributed to rapid CCR2-dependent accumulation of activated, proliferative KLRG1 +Foxp3 +regulatory T (T reg) cells at very early stages of tumor initiation, promoting faster tumor onset. These results link MLL3 loss to HIF signaling, CCL2 and Treg cells, and the establishment of an early immune-suppressive microenvironment, a finding that may open new therapeutic opportunities for MLL3-mutant cancers.
Department of Defense Breakthrough awards BC161696 and BC200410
Live attenuated vaccines (LAVs) whose virulence would be controlled at the tissue level could be a crucial tool to effectively fight intracellular bacterial pathogens, because they would optimize the ...induction of protective immune memory while avoiding the long-term persistence of vaccine strains in the host. Rational development of these new LAVs implies developing an exhaustive map of the bacterial virulence genes according to the host organs implicated. We report here the use of transposon sequencing to compare the bacterial genes involved in the multiplication of Brucella melitensis, a major causative agent of brucellosis, in the lungs and spleens of C57BL/6 infected mice. We found 257 and 135 genes predicted to be essential for B. melitensis multiplication in the spleen and lung, respectively, with 87 genes common to both organs. We selected genes whose deletion is predicted to produce moderate or severe attenuation in the spleen, the main known reservoir of Brucella, and compared deletion mutants for these genes for their ability to protect mice against challenge with a virulent strain of B. melitensis. The protective efficacy of a deletion mutant for the plsC gene, implicated in phospholipid biosynthesis, is similar to that of the reference Rev.1 vaccine but with a shorter persistence in the spleen. Our results demonstrate that B. melitensis faces different selective pressures depending on the organ and underscore the effectiveness of functional genome mapping for the design of new safer LAV candidates.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Cyclic‐di‐GMP plays crucial role in the cell cycle regulation of the α‐Proteobacterium Caulobacter crescentus. Here we investigated its role in the α‐Proteobacterium Brucella abortus, a zoonotic ...intracellular pathogen. Surprisingly, deletion of all predicted cyclic‐di‐GMP synthesizing or degrading enzymes did not drastically impair the growth of B. abortus, nor its ability to grow inside cell lines. As other Rhizobiales, B. abortus displays unipolar growth from the new cell pole generated by cell division. We found that the phosphodiesterase PdeA, the ortholog of the essential polar growth factor RgsP of the Rhizobiale Sinorhizobium meliloti, is required for rod shape integrity but is not essential for B. abortus growth. Indeed, the radius of the pole is increased by 31 ± 1.7% in a ΔpdeA mutant, generating a coccoid morphology. A mutation in the cyclic‐di‐GMP phosphodiesterase catalytic site of PdeA does not generate the coccoid morphology and the ΔpdeA mutant kept the ability to recruit markers of new and old poles. However, the presence of PdeA is required in an intra‐nasal mouse model of infection. In conclusion, we propose that PdeA contributes to bacterial morphology and virulence in B. abortus, but it is not crucial for polarity and asymmetric growth.
Asymmetric growth and polarity are key features of the Rhizobiales, to which Brucella abortus belongs. We identified the polar protein PdeA as an important factor to maintain a proper rod‐shaped morphology. In a pdeA mutant, pole radius is enlarged, but asymmetric growth and cell polarity are preserved. The pdeA mutant is not affected for growth in cultured macrophages but the protein is required for successful infection in a mice model.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Perturbation of the endoplasmic reticulum (ER), a central organelle of the cell, can have critical consequences for cellular homeostasis. An elaborate surveillance system known as ER quality control ...ensures that cells can respond and adapt to stress via the unfolded protein response (UPR) and that only correctly assembled proteins reach their destination. Interestingly, several bacterial pathogens hijack the ER to establish an infection. However, it remains poorly understood how bacterial pathogens exploit ER quality-control functions to complete their intracellular cycle.
spp. replicate extensively within an ER-derived niche, which evolves into specialized vacuoles suited for exit from infected cells. Here we present
secreted protein L (BspL), a
effector that interacts with Herp, a central component of the ER-associated degradation (ERAD) machinery. We found that BspL enhances ERAD at the late stages of the infection. BspL targeting of Herp and ERAD allows tight control of the kinetics of autophagic
-containing vacuole formation, delaying the last step of its intracellular cycle and cell-to-cell spread. This study highlights a mechanism by which a bacterial pathogen hijacks ERAD components for fine regulation of its intracellular trafficking.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
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