De Novo Guanine Biosynthesis but Not the Riboswitch-Regulated Purine Salvage Pathway Is Required for Staphylococcus aureus Infection In Vivo

Abstract

De novo guanine biosynthesis is an evolutionarily conserved pathway that creates sufficient nucleotides to support DNA replication, transcription, and translation. Bacteria can also salvage nutrients from the environment to supplement the de novo pathway, but the relative importance of either pathway during Staphylococcus aureus infection is not known. In S. aureus, genes important for both de novo and salvage pathways are regulated by a guanine riboswitch. Bacterial riboswitches have attracted attention as a novel class of antibacterial drug targets because they have high affinity for small molecules, are absent in humans, and regulate the expression of multiple genes, including those essential for cell viability. Genetic and biophysical methods confirm the existence of a bona fide guanine riboswitch upstream of an operon encoding xanthine phosphoribosyltransferase (xpt), xanthine permease (pbuX), inosine-5=-monophosphate dehydrogenase (guaB), and GMP synthetase (guaA) that represses the expression of these genes in response to guanine. We found that S. aureus guaB and guaA are also transcribed independently of riboswitch control by alternative promoter elements. Deletion of xpt-pbuX-guaB-guaA genes resulted in guanine auxotrophy, failure to grow in human serum, profound abnormalities in cell morphology, and avirulence in mouse infection models, whereas deletion of the purine salvage genes xpt-pbuX had none of these effects. Disruption of guaB or guaA recapitulates the xpt-pbuXguaB-guaA deletion in vivo. In total, the data demonstrate that targeting the guanine riboswitch alone is insufficient to treat S. aureus infections but that inhibition of guaA or guaB could have therapeutic utility. IMPORTANCEDe novo guanine biosynthesis and purine salvage genes were reported to be regulated by a guanine riboswitch in Staphylococcus aureus. We demonstrate here that this is not true, because alternative promoter elements that uncouple the de novo pathway from riboswitch regulation were identified. We found that in animal models of infection, the purine salvage pathway is insufficient for S. aureus survival in the absence of de novo guanine biosynthesis. These data suggest targeting the de novo guanine biosynthesis pathway may have therapeutic utility in the treatment of S. aureus infections.

Host-derived fatty acids activate type VII secretion inStaphylococcus aureus

“…To briefly summarize, "USA300 WT strain" refers to NRS384 ΔhsdRΔmcr (35), and all mutants were constructed in this background. In-frame gene deletions were made by homologous recombination using pIMAY vector (36). Transposon mutants were obtained from the Nebraska Transposon Library and were transduced into the NRS384 ΔhsdRΔmcr strain by bacteriophage 85 (ATCC 27708-B1).…”

Influence of IS 256 on Genome Variability and Formation of Small-Colony Variants in Staphylococcus aureus

“…Indeed, an rsh mutant with an SCV phenotype was also isolated from a patient with a recurrent infection (42). In addition, knockout of guaA leads to thickened cell walls (43). However, guaA deletion mutants are not viable in human serum (43) and therefore represent an in vitro phenotype only.…”

“…In addition, knockout of guaA leads to thickened cell walls (43). However, guaA deletion mutants are not viable in human serum (43) and therefore represent an in vitro phenotype only. In contrast, inactivation of hemY led to a heme-auxotrophic phenotype, and recently, HemY has been shown to be an essential enzyme in the heme biosynthesis pathway in S. aureus (44).…”

Revealing 29 sets of independently modulated genes inStaphylococcus aureus, their regulators and role in key physiological responses

“…Concurrent activation of the CodY, PurR, and GR i-modulons in R10LB indicates that this media presents a guanine-limited environment, as activity of all three transcription factors decrease in response to falling cellular concentrations of various forms of guanine derivatives [13][14][15][16] . Consistent with this hypothesis, we also saw decreased activity of the Translation i-modulon in R10LB.…”