DESIGNER ARRAYS FOR DEFINED MUTANT ANALYSIS IN A MACAQUE MODEL OF TUBERCULOSIS

Tulane University Of Louisiana, New Orleans LA

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Abstract

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The basic mechanisms employed by M. tuberculosis (Mtb) to successfully infect and persist in mammalian lungs are poorly understood. DeADMAn (Designer Arrays for Defined Mutant Analysis) represents a high-throughput approach for subtractive identification of mutants attenuated for survival in animal tissues. This technology uses a catalog of well-defined transposon mutants in Mtb genes to infect animals in pools. Genomic DNA from the input pool (all members of a mutant pool) as well as the output pool (Mtb colonies that grow from bacilli harvested from the infected organ), is PCR amplified using transposon-junction specific primers and labeled with fluorescent dyes (Cy5 for input pool and Cy3 for output pool) and hybridized to a cognate Mtb microarray. Mutants in genes that are essential for growth in-vivo are not represented in the output pool, since these mutants are unable to survive in animal tissues. Monkeys infected with Mtb represent an excellent model of TB - they exhibit pathology remarkably similar to humans and develop human-like latent TB. We conducted experiments with the DeADMAn technology in monkeys. We identified over 75 genes, mutants in which were attenuated for in-vivo growth in macaque lungs, in this model of acute TB. Some of these genes have previously been shown to be implicated to have a role during growth in macrophage or mouse studies. We also identified numerous novel genes with no previous report of being important for in-vivo growth. These include the cadI, cobL, drrA, lpqP, fadD21, cycA, yrbE3B, lipU, lprO, lppV and mce1E genes. We propose to study these differences using the identical mutant screen, but in a model of latent disease, during co-infection with SIV, during dissemination and following BCG vaccination. These studies will shed light on the differences in virulence-mechanisms employed by tubercle bacilli for infecting various animal models.