Abstract

Fatty acid biosynthesis (FASII) enzymes are considered valid targets for antimicrobial drug development against the human pathogen Staphylococcus aureus However, incorporation of host fatty acids confers FASII antibiotic adaptation that compromises prospective treatments. S. aureus adapts to FASII inhibitors by first entering a nonreplicative latency period, followed by outgrowth. Here, we used transcriptional fusions and direct metabolite measurements to investigate the factors that dictate the duration of latency prior to outgrowth. We show that stringent response induction leads to repression of FASII and phospholipid synthesis genes. (p)ppGpp induction inhibits synthesis of malonyl-CoA, a molecule that derepresses FapR, a key regulator of FASII and phospholipid synthesis. Anti-FASII treatment also triggers transient expression of (p)ppGpp-regulated genes during the anti-FASII latency phase, with concomitant repression of FapR regulon expression. These effects are reversed upon outgrowth. GTP depletion, a known consequence of the stringent response, also occurs during FASII latency, and is proposed as the common signal linking these responses. We next showed that anti-FASII treatment shifts malonyl-CoA distribution between its interactants FapR and FabD, toward FapR, increasing expression of the phospholipid synthesis genes plsX and plsC during outgrowth. We conclude that components of the stringent response dictate malonyl-CoA availability in S. aureus FASII regulation, and contribute to latency prior to anti-FASII-adapted outgrowth. A combinatory approach, coupling a (p)ppGpp inducer and an anti-FASII, blocks S. aureus outgrowth, opening perspectives for bi-therapy treatment.IMPORTANCEStaphylococcus aureus is a major human bacterial pathogen for which new inhibitors are urgently needed. Antibiotic development has centered on the fatty acid synthesis (FASII) pathway, which provides the building blocks for bacterial membrane phospholipids. However, S. aureus overcomes FASII inhibition and adapts to anti-FASII by using exogenous fatty acids that are abundant in host environments. This adaptation mechanism comprises a transient latency period followed by bacterial outgrowth. Here, we use metabolite sensors and promoter reporters to show that responses to stringent conditions and to FASII inhibition intersect, in that both involve GTP and malonyl-CoA. These two signaling molecules contribute to modulating the duration of latency prior to S. aureus adaptation outgrowth. We exploit these novel findings to propose a bi-therapy treatment against staphylococcal infections.

Highlights

  • Bacterial infections that fail to respond to antibiotic treatments are on the rise, especially in the immunocompromised or weakened host, underlining the need for novel antimicrobial strategies [1]

  • Responded to mupirocin as expected in the parental strain, whereas no such responses were observed in the ppGpp0 background. These results indicate that stringent response controls these sensors in S. aureus

  • The PaccBC-lacZ reporter was inhibited by mupirocin in HG1-R, but not in the ppGpp0 strain (Fig. S2C). These results show that in S. aureus, stringent response induction leads to repression of malonyl-CoA synthesis [13]

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Summary

Introduction

Bacterial infections that fail to respond to antibiotic treatments are on the rise, especially in the immunocompromised or weakened host, underlining the need for novel antimicrobial strategies [1]. The second mode of adaptation occurs without FASII mutations and predominates in serum-supplemented medium In this case, full adaptation and eFA incorporation in actively growing cells is achieved after a latency phase, whose duration (6-12 h) depends on the strain and pre-growth in serum-containing medium. The. S. aureus FapR repressor reportedly regulates most FASII genes (except acc, encoding acetyl-CoA carboxylase, and FabZ, -hydroxyacyl-ACP dehydratase), and phospholipid synthesis genes plsX and plsC [13, 14]. We first show that stringent response induction exerts control over fatty acid and phospholipid synthesis in S. aureus by modulating FapR repressor activity. The chain of events revealed here indicate that (p)ppGpp/GTP and malonyl-CoA contribute to adjusting the timing of FASII-antibiotic-induced latency transition to outgrowth. We suggest a bi-therapy approach that combines FASII inhibitors and a (p)ppGpp inducer to prevent S. aureus adaptation

Results
Discussion
Materials and Methods
Findings
Declaration of Interests
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