Dataset Information

Genome-wide screen for Mycobacterium tuberculosis genes that regulate host immunity.

ABSTRACT: In spite of its highly immunogenic properties, Mycobacterium tuberculosis (Mtb) establishes persistent infection in otherwise healthy individuals, making it one of the most widespread and deadly human pathogens. Mtb's prolonged survival may reflect production of microbial factors that prevent even more vigorous immunity (quantitative effect) or that divert the immune response to a non-sterilizing mode (qualitative effect). Disruption of Mtb genes has produced a list of several dozen candidate immunomodulatory factors. Here we used robotic fluorescence microscopy to screen 10,100 loss-of-function transposon mutants of Mtb for their impact on the expression of promoter-reporter constructs for 12 host immune response genes in a mouse macrophage cell line. The screen identified 364 candidate immunoregulatory genes. To illustrate the utility of the candidate list, we confirmed the impact of 35 Mtb mutant strains on expression of endogenous immune response genes in primary macrophages. Detailed analysis focused on a strain of Mtb in which a transposon disrupts Rv0431, a gene encoding a conserved protein of unknown function. This mutant elicited much more macrophage TNF?, IL-12p40 and IL-6 in vitro than wild type Mtb, and was attenuated in the mouse. The mutant list provides a platform for exploring the immunobiology of tuberculosis, for example, by combining immunoregulatory mutations in a candidate vaccine strain.

SUBMITTER: Beaulieu AM

PROVIDER: S-EPMC3000826 | biostudies-literature | 2010 Dec

REPOSITORIES: biostudies-literature

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Genome-wide screen for Mycobacterium tuberculosis genes that regulate host immunity.

Beaulieu Aimee M AM Rath Poonam P Imhof Marianne M Siddall Mark E ME Roberts Julia J Schnappinger Dirk D Nathan Carl F CF

PloS one 20101210 12

In spite of its highly immunogenic properties, Mycobacterium tuberculosis (Mtb) establishes persistent infection in otherwise healthy individuals, making it one of the most widespread and deadly human pathogens. Mtb's prolonged survival may reflect production of microbial factors that prevent even more vigorous immunity (quantitative effect) or that divert the immune response to a non-sterilizing mode (qualitative effect). Disruption of Mtb genes has produced a list of several dozen candidate im ...[more]

PMID: 21170273

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Project description:The interactions between a host cell and a pathogen can dictate disease outcomes and are important targets for host-directed therapies. Mycobacterium abscessus (Mab) is a highly antibiotic resistant, rapidly growing nontuberculous mycobacterium that infects patients with chronic lung diseases. Mab can infect host immune cells, such as macrophages, which contribute to its pathogenesis. However, our understanding of initial host-Mab interactions remains unclear. Here, we developed a functional genetic approach to define these host-Mab interactions by coupling a Mab fluorescent reporter with a genome-wide knockout library in murine macrophages. We used this approach to conduct a forward genetic screen to define host genes that contribute to the uptake of Mab by macrophages. We identified known regulators of phagocytosis, such as the integrin ITGB2, and uncovered a key requirement for glycosaminoglycan (sGAG) synthesis for macrophages to efficiently take up Mab. CRISPR-Cas9 targeting of three key sGAG biosynthesis regulators, Ugdh, B3gat3, and B4galt7 resulted in reduced uptake of both smooth and rough Mab variants by macrophages. Mechanistic studies suggest that sGAGs function upstream of pathogen engulfment and are required for the uptake of Mab, but not Escherichia coli or latex beads. Further investigation found that the loss of sGAGs reduced the surface expression, but not the mRNA expression, of key integrins, suggesting an important role for sGAGs in modulating surface receptor availability. Together, these studies globally define and characterize important regulators of macrophage-Mab interactions and are a first step to understanding host genes that contribute to Mab pathogenesis and disease. IMPORTANCE Pathogen interactions with immune cells like macrophages contribute to pathogenesis, yet the mechanisms underlying these interactions remain largely undefined. For emerging respiratory pathogens, like Mycobacterium abscessus, understanding these host-pathogen interactions is important to fully understand disease progression. Given that M. abscessus is broadly recalcitrant to antibiotic treatments, new therapeutic approaches are needed. Here, we leveraged a genome-wide knockout library in murine macrophages to globally define host genes required for M. abscessus uptake. We identified new macrophage uptake regulators during M. abscessus infection, including a subset of integrins and the glycosaminoglycan synthesis (sGAG) pathway. While ionic characteristics of sGAGs are known to drive pathogen-cell interactions, we discovered a previously unrecognized requirement for sGAGs to maintain robust surface expression of key uptake receptors. Thus, we developed a flexible forward-genetic pipeline to define important interactions during M. abscessus infection and more broadly identified a new mechanism by which sGAGs control pathogen uptake.

Dataset Information

Genome-wide screen for Mycobacterium tuberculosis genes that regulate host immunity.

Publications

Genome-wide screen for Mycobacterium tuberculosis genes that regulate host immunity.

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