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Genome-wide screen of Mycobacterium tuberculosis-infected macrophages revealed GID/CTLH complex-mediated modulation of bacterial growth

ABSTRACT: The eukaryotic GID/CTLH complex is a highly conserved E3 ubiquitin ligase involved in a broad range of biological processes. However, a role of this complex in host antimicrobial defenses has not been described. We exploited Mycobacterium tuberculosis (Mtb) induced cytotoxicity in macrophages in a FACS based CRISPR genetic screen to identify host determinants of intracellular Mtb growth restriction. Our screen identified 5 (GID8, YPEL5, WDR26, UBE2H, MAEA) of the 10 predicted members of the GID/CTLH complex as determinants of intracellular growth of both Mtb and Salmonella serovar Typhimurium. We show that the antimicrobial properties of the GID/CTLH complex knockdown macrophages are mediated by enhanced GABAergic signaling, activated AMPK, increased autophagic flux and resistance to cell death. Meanwhile, Mtb isolated from GID/CTLH knockdown macrophages are nutritionally starved and oxidatively stressed. Our study identifies the GID/CTLH complex activity as broadly suppressive of host antimicrobial responses against intracellular bacterial infections.

ORGANISM(S): Mus musculus Mycobacterium tuberculosis

PROVIDER: GSE267063 | GEO | 2024/05/08

REPOSITORIES: GEO

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Project description:The highly infectious bacterium Francisella tularensis is a facultative intracellular pathogen, whose virulence requires proliferation inside host cells, including macrophages. Although some Francisella determinants of intracellular growth have been identified, much remains to be understood about the pathogenesis of this organism. In particular, how Francisella responds to its intracellular environment could provide clues about its intracellular biology and reveal pathogenic determinants based on their intracellular expression profiles. Here we have performed a global transcriptional profiling of the highly virulent F. tularensis subsp. tularensis Schu S4 strain during its intracellular cycle within primary murine macrophages. Phagocytosed bacteria rapidly responded to their intracellular environment and progressively altered their transcriptional profile over time. Differential gene expression profiles were revealed that correlated with specific intracellular locations of the bacteria. Upregulation of general and oxidative stress response genes was a hallmark of the early phagosomal and late endosomal stages, while induction of a subset of transport and metabolic genes characterized the cytosolic replication stages. Expression of the Francisella Pathogenicity Island (FPI) genes, the functions of which are associated with intracellular proliferation, increased during the intracellular cycle. Similarly, 27 chromosomal loci putatively encoding hypothetical, secreted, outer membrane proteins or transcriptional regulators were identified as upregulated during the intracellular cycle. In-frame deletion of FTT0383, the Schu S4 ortholog of fevR, of FTT0369c or FTT1676 abolished the ability of Schu S4 to either survive or proliferate intracellularly, demonstrating that bacterial factors of intracellular pathogenesis can be identified based on their intracellular expression profile. In conclusion, establishing the intracellular transcriptome of Francisella has revealed important aspects of its intracellular biology and identified novel virulence determinants of this pathogen. Keywords: Time series Intracellular cycle within primary murine macrophages using the time series of zero, one, two, four, eight, twelve, sixteen and twenty-four hours

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Genome-wide screen of Mycobacterium tuberculosis-infected macrophages revealed GID/CTLH complex-mediated modulation of bacterial growth

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