Functionalizing Lists of Unknown TB Entities (FLUTE)

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Program director: Eric J. Rubin, Harvard School of Public Health

Investigators: Sarah Fortune, Chris Sassetti, Kyu Y. Rhee, Sabine Ehrt, Dirk Schnappinger, Steven A. Carr, Jonathan Livny, James Sacchettini, Thomas R. Ioerger, Christoph Grundner

Funding source: NIH U19 AI107774

Project home page: http://orca2.tamu.edu/U19/

Project objectives: Mycobacterium tuberculosis is one of the most successful pathogens in the world, still responsible for millions of deaths each year. Nearly half of its protein coding genes have functions that are unknown. FLUTE a Functional Genomics Resource Center funded by NIAID, with the goal of defining functions for unknown ORFs, hypothetical genes, and non-coding RNAs in Mtb.


High-Resolution Phenotypic Profiling Defines Genes Essential for Mycobacterial Growth and Cholesterol Catabolism

Publication: PMC3182942

The pathways that comprise cellular metabolism are highly interconnected, and alterations in individual enzymes can have far-reaching effects. As a result, global profiling methods that measure gene expression are of limited value in predicting how the loss of an individual function will affect the cell. In this work, we employed a new method of global phenotypic profiling to directly define the genes required for the growth of Mycobacterium tuberculosis. A combination of high-density mutagenesis and deep-sequencing was used to characterize the composition of complex mutant libraries exposed to different conditions. This allowed the unambiguous identification of the genes that are essential for Mtb to grow in vitro, and proved to be a significant improvement over previous approaches. To further explore functions that are required for persistence in the host, we defined the pathways necessary for the utilization of cholesterol, a critical carbon source during infection. Few of the genes we identified had previously been implicated in this adaptation by transcriptional profiling, and only a fraction were encoded in the chromosomal region known to encode sterol catabolic functions. These genes comprise an unexpectedly large percentage of those previously shown to be required for bacterial growth in mouse tissue. Thus, this single nutritional change accounts for a significant fraction of the adaption to the host. This work provides the most comprehensive genetic characterization of a sterol catabolic pathway to date, suggests putative roles for uncharacterized virulence genes, and precisely maps genes encoding potential drug targets.

Data sets and additional information can be found here.


Peptidoglycan synthesis in Mycobacterium tuberculosis is organized into networks with varying drug susceptibility

Publication: PMCID: PMC4620856

Peptidoglycan (PG), a complex polymer composed of saccharide chains cross-linked by short peptides, is a critical component of the bacterial cell wall. PG synthesis has been extensively studied in model organisms but remains poorly understood in mycobacteria, a genus that includes the important human pathogen Mycobacterium tuberculosis (Mtb). The principle PG synthetic enzymes have similar and, at times, overlapping functions. To determine how these are functionally organized,we carried out whole genome transposon mutagenesis screens in Mtb strains deleted for ponA1, ponA2, and ldtB, major PG synthetic enzymes. We identified distinct factors required to sustain bacterial growth in the absence of each of these enzymes. We find that even the homologues PonA1 and PonA2 have unique sets of genetic interactions, suggesting there are distinct PG synthesis pathways in Mtb. Either PonA1 or PonA2 is required for growth of Mtb, but both genetically interact with LdtB, which has its own distinct genetic network. We further provide evidence that each interaction network is differentially susceptible to antibiotics. Thus, Mtb uses alternative pathways to produce PG, each with its own biochemical characteristics and vulnerabilities.

Data sets and additional information can be found here.