Abstract
ABSTRACTWe have engineered Saccharomyces cerevisiae to inducibly synthesize the prokaryotic signaling nucleotides cyclic di-GMP (cdiGMP), cdiAMP, and ppGpp in order to characterize the range of effects these nucleotides exert on eukaryotic cell function during bacterial pathogenesis. Synthetic genetic array (SGA) and transcriptome analyses indicated that, while these compounds elicit some common reactions in yeast, there are also complex and distinctive responses to each of the three nucleotides. All three are capable of inhibiting eukaryotic cell growth, with the guanine nucleotides exhibiting stronger effects than cdiAMP. Mutations compromising mitochondrial function and chromatin remodeling show negative epistatic interactions with all three nucleotides. In contrast, certain mutations that cause defects in chromatin modification and ribosomal protein function show positive epistasis, alleviating growth inhibition by at least two of the three nucleotides. Uniquely, cdiGMP is lethal both to cells growing by respiration on acetate and to obligately fermentative petite mutants. cdiGMP is also synthetically lethal with the ribonucleotide reductase (RNR) inhibitor hydroxyurea. Heterologous expression of the human ppGpp hydrolase Mesh1p prevented the accumulation of ppGpp in the engineered yeast and restored cell growth. Extensive in vivo interactions between bacterial signaling molecules and eukaryotic gene function occur, resulting in outcomes ranging from growth inhibition to death. cdiGMP functions through a mechanism that must be compensated by unhindered RNR activity or by functionally competent mitochondria. Mesh1p may be required for abrogating the damaging effects of ppGpp in human cells subjected to bacterial infection.
Highlights
We have engineered Saccharomyces cerevisiae to inducibly synthesize the prokaryotic signaling nucleotides cyclic di-GMP, cyclic di-AMP (cdiAMP), and ppGpp in order to characterize the range of effects these nucleotides exert on eukaryotic cell function during bacterial pathogenesis
To generate strains of S. cerevisiae where the intracellular synthesis of ppGpp, cyclic di-GMP (cdiGMP), and cdiAMP nucleotides could be controllably induced, we heterologously expressed genes encoding selected bacterial enzymes specifying their synthesis by using the dual tetracycline activator/repressor system of Belli et al [25]
A Caulobacter crescentus gene variant, dgcA0244, which encodes a synthetase that is insensitive to product feedback inhibition, was chosen to provide cdiGMP synthesis [26], while genes generating truncated enzymes which exhibit constitutively active synthetase activity were used for cdiAMP and ppGpp production
Summary
We have engineered Saccharomyces cerevisiae to inducibly synthesize the prokaryotic signaling nucleotides cyclic di-GMP (cdiGMP), cdiAMP, and ppGpp in order to characterize the range of effects these nucleotides exert on eukaryotic cell function during bacterial pathogenesis. CdiGMP and cdiAMP are significant participants in the metabolic cross talk between host and pathogen, as they are required both for the effective functioning of the invading bacterial cells and for their detection by the host’s defenses. Apart from their induction of host immune responses, other influences of these bacterial signaling molecules on host cell physiology and metabolism remain poorly defined. A study of the impact of these bacterial nucleotides on eukaryotic cell function has great relevance to the interaction of bacteria with their hosts, including both pathogenic and symbiotic relationships with plants and animals [21,22,23]. The impact of intracellular cdiAMP on yeast cells was less marked, and the expression levels of only 75 genes were significantly altered following intracellular accumulation of cdiAMP to levels Ͼ5-fold higher than those observed for ATP
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