Variability in bacterial sterilization is a key feature of Mycobacterium tuberculosis (Mtb) disease. In a population of human macrophages, there are macrophages that restrict Mtb growth and those ...that do not. However, the sources of heterogeneity in macrophage state during Mtb infection are poorly understood. Here, we perform RNAseq on restrictive and permissive macrophages and reveal that the expression of genes involved in GM-CSF signaling discriminates between the two subpopulations. We demonstrate that blocking GM-CSF makes macrophages more permissive of Mtb growth while addition of GM-CSF increases bacterial control. In parallel, we find that the loss of bacterial control that occurs in HIV-Mtb coinfected macrophages correlates with reduced GM-CSF secretion. Treatment of coinfected cells with GM-CSF restores bacterial control. Thus, we leverage the natural variation in macrophage control of Mtb to identify a critical cytokine response for regulating Mtb survival and identify components of the antimicrobial response induced by GM-CSF.
The widespread use of antibiotics has placed bacterial pathogens under intense pressure to evolve new survival mechanisms. Genomic analysis of 51,229
(
)clinical isolates has identified an essential ...transcriptional regulator,
, herein called
for resilience regulator, as a frequent target of positive (adaptive) selection.
mutants do not show canonical drug resistance or drug tolerance but instead shorten the post-antibiotic effect, meaning that they enable
to resume growth after drug exposure substantially faster than wild-type strains. We refer to this phenotype as antibiotic resilience. ResR acts in a regulatory cascade with other transcription factors controlling cell growth and division, which are also under positive selection in clinical isolates of
. Mutations of these genes are associated with treatment failure and the acquisition of canonical drug resistance.
Macrophages are a protective replicative niche for Mycobacterium tuberculosis (Mtb) but can kill the infecting bacterium when appropriately activated. To identify mechanisms of clearance, we compared ...levels of bacterial restriction by human macrophages after treatment with 26 compounds, including some currently in clinical trials for tuberculosis. All-
-retinoic acid (ATRA), an active metabolite of vitamin A, drove the greatest increase in Mtb control. Bacterial clearance was transcriptionally and functionally associated with changes in macrophage cholesterol trafficking and lipid metabolism. To determine how these macrophage changes affected bacterial control, we performed the first Mtb CRISPR interference screen in an infection model, identifying Mtb genes specifically required to survive in ATRA-activated macrophages. These data showed that ATRA treatment starves Mtb of cholesterol and the downstream metabolite propionyl coenzyme A (propionyl-CoA). Supplementation with sources of propionyl-CoA, including cholesterol, abrogated the restrictive effect of ATRA. This work demonstrates that targeting the coupled metabolism of Mtb and the macrophage improves control of infection and that it is possible to genetically map the mode of bacterial death using CRISPR interference.
Tuberculosis, caused by the bacterium Mycobacterium tuberculosis, is a leading cause of death due to infectious disease. Improving the immune response to tuberculosis holds promise for fighting the disease but is limited by our lack of knowledge as to how the immune system kills M. tuberculosis. Our research identifies a potent way to make relevant immune cells more effective at fighting M. tuberculosis and then uses paired human and bacterial genomic methods to determine the mechanism of that improved bacterial clearance.
Tuberculosis (TB) remains a leading cause of morbidity and mortality worldwide. To date, the mainstay of vaccination involves the use of Mycobacterium bovis bacillus Calmette-Guérin (BCG), a ...live-attenuated vaccine that confers protection against extrapulmonary disease in infants and children but not against lung disease. Thus, there is an urgent need for novel vaccines. Here, we show that a multicomponent acellular vaccine (TB-MAPS) induces robust antibody responses and long-lived systemic and tissue-resident memory Th1, Th17, and cytotoxic CD4
and CD8
T cells, and promotes trained innate immunity mediated by γδT and NKT cells in mice. When tested in a mouse aerosol infection model, TB-MAPS significantly reduced bacterial loads in the lungs and spleens to the same extent as BCG. When used in conjunction with BCG, TB-MAPS further enhanced BCG-mediated protection, especially in the lungs, further supporting this construct as a promising TB vaccine candidate.
Tuberculosis (TB) remains a leading cause of morbidity and mortality worldwide. Here, we evaluate a novel vaccine which induces a broad immune response to Mycobacterium tuberculosis including robust antibody responses and long-lived systemic and tissue-resident memory Th1, Th17, and cytotoxic CD4
and CD8
T cells. When tested in a mouse aerosol infection model, this vaccine significantly reduced bacterial loads in the lungs and spleens to the same extent as BCG. When used in conjunction with BCG, TB-MAPS further enhanced BCG-mediated protection, especially in the lungs, further supporting this construct as a promising TB vaccine candidate.
Shigellosis causes most diarrheal deaths worldwide, particularly affecting children.
invades and replicates in the epithelium of the large intestine, eliciting inflammation and tissue destruction. To ...understand how
rewires macrophages prior to epithelium invasion, we performed genome-wide and focused secondary CRISPR knockout and CRISPR interference (CRISPRi) screens in Shigella flexneri-infected human monocytic THP-1 cells. Knockdown of the Toll-like receptor 1/2 signaling pathway significantly reduced proinflammatory cytokine and chemokine production, enhanced host cell survival, and controlled intracellular pathogen growth. Knockdown of the enzymatic component of the mitochondrial pyruvate dehydrogenase complex enhanced THP-1 cell survival. Small-molecule inhibitors blocking key components of these pathways had similar effects; these were validated with human monocyte-derived macrophages, which closely mimic the
physiological state of macrophages postinfection. High-throughput CRISPR screens can elucidate how S. flexneri triggers inflammation and redirects host pyruvate catabolism for energy acquisition before killing macrophages, pointing to new shigellosis therapies.
Treatment for shigellosis is becoming increasingly difficult as resistance to antibiotics becomes more prevalent. One way to prevent this significant public health problem from developing into a full-blown crisis is to approach shigellosis intervention from the point of view of the host. So far, little is known about the specific biological pathways that might be modulated in macrophages, sentinel cells of the innate immune system, to strengthen the response to
infection. In this work, we conducted CRISPR screens to comprehensively decipher the complexity of macrophage-
interactions and to discover new potential therapeutic interventions against Shigella flexneri infection. Our work highlights systematic genetic perturbation strategies to provide direct causal evidence showing how intracellular pathogens manipulate innate immune cells.
There is increasing evidence that phenotypically drug-resistant bacteria may be important determinants of antibiotic treatment failure. Using high-throughput imaging, we defined distinct ...subpopulations of mycobacterial cells that exhibit heritable but semi-stable drug resistance. These subpopulations have distinct transcriptional signatures and growth characteristics at both bulk and single-cell levels, which are also heritable and semi-stable. We find that the mycobacterial histone-like protein HupB is required for the formation of these subpopulations. Using proteomic approaches, we further demonstrate that HupB is posttranslationally modified by lysine acetylation and lysine methylation. Mutation of a single posttranslational modification site specifically abolishes the formation of one of the drug-resistant subpopulations of cells, providing the first evidence in prokaryotes that posttranslational modification of a bacterial nucleoid-associated protein may epigenetically regulate cell state.
Existing antibiotics are inadequate to defeat tuberculosis (TB), a leading cause of death worldwide. We sought potential targets for host-directed therapies (HDTs) by investigating the host immune ...response to mycobacterial infection. We used high-throughput CRISPR knockout and CRISPR interference (CRISPRi) screens to identify perturbations that improve the survival of human phagocytic cells infected with Mycobacterium bovis BCG (Bacillus Calmette-Guérin), as a proxy for Mycobacterium tuberculosis (Mtb). Many of these perturbations constrained the growth of intracellular mycobacteria. We identified over 100 genes associated with diverse biological pathways as potential HDT targets. We validated key components of the type I interferon and aryl hydrocarbon receptor signaling pathways that respond to the small-molecule inhibitors cerdulatinib and CH223191, respectively; these inhibitors enhanced human macrophage survival and limited the intracellular growth of Mtb. Thus, high-throughput functional genomic screens, by elucidating highly complex host-pathogen interactions, can serve to identify HDTs to potentially improve TB treatment.
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•High-throughput functional genomic screens elucidate host-pathogen interactions•Screens find over 100 host targets inhibiting intracellular bacterial pathogenesis•AHR/ARNT pathway inhibition blocks bacterial pathogenesis via oxylipin metabolism•Cerdulatinib and CH223191 are potential host-directed therapies for TB
Lai et al. approach tuberculosis (TB) intervention from the point of view of the host by establishing large-scale CRISPR screen platforms in human innate immune cells. By either knocking out or reducing the expression of individual genes, the authors determine how human cells respond, on the genetic level, to intracellular mycobacteria, which cause TB. Specifically, they find which genes promote or suppress bacterial survival. On this basis, the authors identify potential targets for host-directed therapies.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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