Ongoing evolution of theMycobacterium tuberculosislactate dehydrogenase reveals the pleiotropic effects of bacterial adaption to host pressure

Abstract

AbstractThe bacterial determinants that facilitateMycobacterium tuberculosis(Mtb) adaptation to the human host environment are poorly characterized. We have sought to decipher the pressures facing the bacteriumin vivoby assessing Mtb genes that are under positive selection in clinical isolates. One of the strongest targets of selection in the Mtb genome islldD2, which encodes a quinone-dependent L-lactate dehydrogenase (LldD2) that catalyzes the oxidation of lactate to pyruvate. Lactate accumulation is a salient feature of the intracellular environment during infection andlldD2is essential for Mtb growth in macrophages. We determined the extent oflldD2variation across a set of global clinical isolates and defined how prevalent mutations modulates Mtb fitness. We show the stepwise nature oflldD2evolution that occurs as a result of ongoinglldD2selection in the background of ancestral lineage defining mutations and demonstrate that the genetic evolution oflldD2additively augments Mtb growth in lactate. Using quinone-dependent antibiotic susceptibility as a functional reporter, we also find that the evolvedlldD2mutations functionally increase the quinone-dependent activity of LldD2. Using13C-lactate metabolic flux tracing, we find thatlldD2is necessary for robust incorporation of lactate into central carbon metabolism. In the absence oflldD2, label preferentially accumulates in methylglyoxal precursors dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P) and is associated with a discernible growth defect, providing experimental evidence for accumulated lactate toxicity via a methylglyoxal pathway that has been proposed previously. The evolvedlldD2variants increase lactate incorporation to pyruvate but also alter flux in the methylglyoxal pathway, suggesting both an anaplerotic and detoxification benefit tolldD2evolution. We further show that the mycobacterial cell is transcriptionally sensitive to the changes associated with alteredlldD2activity which affect the expression of genes involved in cell wall lipid metabolism and the ESX-1 virulence system. Together, these data illustrate a multifunctional role of LldD2 that provide context for the selective advantage oflldD2mutations in adapting to host stress.