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 TNFα, 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.

Genetic regulation of vesiculogenesis and immunomodulation in Mycobacterium tuberculosis

“…Under protocols approved by the Weill Cornell Medical College or Rockefeller University Institutional Animal Care and Use Committees, mice were infected by aerosol with ∼100 bacilli. Tissues were collected at time points and processed to enumerate cfu as described (17).…”

“…Earlier work demonstrated that the transposon disrupting virR had inserted between codons 121 and 122 (17). However, it remained to be established whether the VirR protein was absent, truncated, or modified with a neo-C terminus, whether the mutant Significance Bacteria stimulate host cells in part via secreted products, some of which are packaged in membrane vesicles (MV).…”

“…Recently, a forward genetic screen using a library of more than 10,000 loss-of-function transposon mutants of Mtb identified rv0431 as an endogenous regulator of Mtb's immunostimulatory potential (17). Infection with Mtb rv0431:Tn led to markedly increased release of TNF-α, IL-12 p40, and IL-6 from primary mouse macrophages.…”

Mycobacterial Shuttle Vectors Designed for High-Level Protein Expression in Infected Macrophages

“…However, relatively few gene products have been identified that directly attenuate innate immune cell functions (6,19,24,27,28,33,34,39,42,45). Several distinct approaches have been used to identify putative virulence factors, including transposon mutagenesis of M. tuberculosis, isolation and characterization of regions of difference (RD), and studies of eukaryotic-like proteins encoded by the M. tuberculosis genome.…”

“…This stable cell line provides a 40-fold range of GFP expression in every cell infected. Another report described the use of immunoreporter macrophage cell lines (6). In that case, the macrophages only provided transient reporter expression that had a wide fluorescence distribution.…”