NAD LIMITATION AND ITS IMPACT ON C. GLABRATA SURVIVAL AND VIRULENCE
Embargo until
2020-12-01
Date
2016-10-05
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Johns Hopkins University
Abstract
This work describes what happens in cells that are starved for NAD+. Candida glabrata is an opportunistic yeast pathogen that is an NAD+ auxotroph and limitation of NAD+ in media leads to transcriptional activation of key adhesin genes encoded in C. glabrata. Additionally, starvation for NAD+ leads to a dramatic increase in the virulence of C. glabrata in a mouse model of infection. Interestingly, NAD+ depletion does not result in cell death. Although intracellular NAD+ levels diminish dramatically, the cell somehow is able to maintain its viability over the course of weeks. The work in this thesis sheds light on both cellular survival in response to NAD+ limitation and the effect of NAD+ on virulence.
In the first part of this thesis, I have identified two major pathways which regulate the TORC1 kinase and PKA kinase altering the ability to survive NAD+ depletion. Both the Sea1/Npr2/Npr3 complex and Ira1 function as GTPase activating proteins (GAPs). The Sea1/Npr2/Npr3 complex is the GAP for the small G protein GTR1, which regulates TORC1 activation, and Ira1 is the GAP for RAS, the G protein responsible for PKA activation. Loss of SEA1, NPR2, NPR3, or IRA1 results in marked defects in the cells ability to survive NAD+ starvation, illustrating the importance of these particular genes. Based on these data, we suggest a model where the cell responds to NAD+ depletion by inactivation both of the major pro-growth pathways by altering the activation state of TORC1 and PKA. Failure to appropriately sense this stress and respond with inactivation of the major growth pathways results in a loss in viability.
In the second part of the thesis, I followed up on a previous observation made in the lab which showed transcriptional up-regulation of de novo purine biosynthesis in response to NAD+ limitation, independent of the previously known regulators of the cellular NAD+ response. Naturally, this led to the question: why would NAD+ limitation impact purine biosynthesis? Through a series of metabolomics experiments my work demonstrated that in response to NAD+ limitation there is a massive accumulation of inosine monophosphate (IMP) and its breakdown products inosine and hypoxanthine. Importantly, metabolic flux labeling shows clearly that the de novo purine biosynthesis pathway is functional in starved cells, leading to new synthesis of IMP and downstream products. Through the generation of multiple mutants in the de novo pathway, I was able to demonstrate that de novo purine biosynthesis is absolutely required for virulence, whereas the salvage purine biosynthesis pathway was not required for virulence. Together these data minimally suggest that the upregulation of purine biosynthesis genes in response NAD+ starvation has an effect on virulence. An additional piece of information received from the metabolomics experiments was that in response to NAD+ limitation there is a definitive purine nucleotide imbalance between AMP and GMP. This raised the possibility that when cells are NAD+ starved the signal they may actually be sensing is a purine nucleotide imbalance. Treatment of C. glabrata with the drugs MPA or 6AU, which function to specifically deplete cellular guanine pools, showed a transcriptional response which echoed that of NAD+ depletion. Moreover, treatment of C. glabrata with MPA or 6AU prior to mouse infection phenocopied the hyper virulence phenotype we observe with NAD+ depletion. This work suggests a model which connects NAD+ status and purine metabolism and potentially hints at a novel pathway relevant to virulence regulation by NAD+ depletion.
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Keywords
NAD+, survival, virulence, purine metabolism