Use of random barcode transposon site sequencing as a tool for identifying nutrient acquisition strategies utilized by Mycobacterium tuberculosis during infection of human macrophages - Mycobacterium tuberculosis (Mtb) is a highly successful pathogen that infects one-quarter of the world population and causes more deaths annually than any other single pathogen. Mtb can cause infection that lasts for a person’s lifetime. How Mtb can persist in vivo during infection is a question that is central to unlocking new therapeutics for treating this devastating disease. Intracellular pathogens like Mtb must acquire all their nutrients from host cells that have evolved mechanisms to starve pathogens of nutrients. What nutrients Mtb can obtain from host cells, and which are essential for its survival, is poorly understood. Genetic screens are critical for understanding the mechanistic basis of microbial fitness and pathogenesis. Transposon Sequencing screens have been a powerful tool for understanding the biology of Mtb yet are inherently limited in throughput. Thus, it is challenging to screen for genes required to assimilate the myriad of possible nutrients that Mtb could exploit for in vivo growth. We have developed a variant of TnSeq, random barcode transpson site sequencing (RB-TnSeq), in which each transposon inserted into a gene is randomly barcoded. Upon completion of a genetic screen, the presence of specific transposon mutants in a library can be detected by a simple PCR reaction, thus dramatically increasing the possible throughput of screening. To date we have performed a total of 45 RB-TnSeq screens, including on 18 individual carbon sources. From our screening data we propose a hypothesis that we will test in aim 1 of this proposal: that Mtb utilizes both D- and L-lactate as crucial carbon sources for infection of human macrophages. Both enantiomers of lactate are produced abundantly by activated macrophages. Further, we propose that lactate crosses the outer membrane of Mtb through a pore formed by the PPE3 protein. We hypothesize that the type VII alternative secretion system is required for PPE3 export to the cell surface. We show for the first time that Mtb utilizes D-lactate as a carbon source and have identified a possible D-lactate dehydrogenase that we will demonstrate is required for D- lactate assimilation into central metabolism. To broaden our understanding of metabolites used as nutrients by Mtb in human macrophages, in aim 2 we will use RB-TnSeq to identify genes required by Mtb for growth in human primary macrophages. By comparing the results of this screen from the results of our in vitro screens on numerous carbon sources, we will identify a set of candidate nutrients pathways required for Mtb to replicate and persist inside host cells.
