Luciferase Reporter Gene System to Detect Cell Wall Stress Stimulon Induction in Staphylococcus aureus

Dengler, Vanina; McCallum, Nadine

doi:10.1007/978-1-4939-3676-2_11

“…We hypothesized that increased c-di-AMP levels can lead to cell wall thickness increase and beta-lactam resistance through activation of the CWSS and tested this hypothesis using the ΔgdpP mutant. CWSS basal expression was assessed in the absence of antibiotic-induced stress by quantifying the activity of the sas016 gene promoter, widely used as a proxy for CWSS activation (11,24,25). Confirming our hypothesis, the CWSS activity was increased in the ∆gdpP mutant (up to 4.5 times) as compared to the WT (Figure 5A).…”

Section: High C-di-amp Levels Increase the Activation Of The Cell Wal...supporting

confidence: 74%

“…The manipulated regions were checked by Sanger sequencing. The sas016 promoter luciferase fusion plasmid psas016 p -luc (24)was transduced into LAC* WT, ΔgdpP and ΔgdpP/ΔvraR mutant background using phage 80α.…”

Section: Plasmid Transduction and Genetic Manipulationsmentioning

confidence: 99%

C-di-AMP levels modulateStaphylococcus aureuscell wall thickness as well as virulence and contribute to antibiotic resistance and tolerance

Haunreiter

¹

,

Tarnutzer

²

,

Bär

³

et al. 2023

Preprint

0

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Beta-lactam antibiotics are widely used to treat infections caused by the important human pathogen Staphylococcus aureus. Resistance to beta-lactams, as found in methicillin-resistant S. aureus (MRSA), renders effective treatment difficult. The second messenger cyclic di-3′,5′-adenosine monophosphate (c-di-AMP) promotes beta-lactam resistance in clinical S. aureus isolates. C-di-AMP plays a crucial role in the regulation of cellular processes such as virulence, cell wall homeostasis and resistance to beta-lactams in many bacterial species. In S. aureus, c-di-AMP synthesis is mediated by the diadenylate cyclase DacA, while its degradation is carried out by the phosphodiesterases GdpP and Pde2. In this work, we assessed the effect of altered c-di-AMP levels due to mutations in cacA, gdpP or gdpP/pde2 on virulence determinants. We report that a previously described growth defect in bacteria producing high c-di-AMP levels is mainly attributable to smaller cell size. High c-di-AMP levels also led to decreased survival upon oxidative stress, reduced production of the antioxidant staphyloxanthin, increased oxacillin and fosfomycin resistance and increased cell wall thickness. While resistance to ceftaroline was not affected, high c-di-AMP levels promoted tolerance to this antibiotic. In response to cell wall stress induced by antibiotics, the three-component regulatory system VraTSR mediates an increase in cell wall synthesis via the cell wall stress stimulon (CWSS). Increased c-di-AMP levels led to an activation of the CWSS. Upon deletion of vraR, resistance to oxacillin and fosfomycin as well as cell wall thickness diminished in the ΔgdpP mutant, indicating a contribution of the VraTSR system to the cell wall related phenotypes.

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“…We hypothesized that increased c-di-AMP levels can lead to cell wall thickness increase and beta-lactam resistance through the activation of the CWSS and tested this hypothesis using the Δ gdpP mutant as an example for a strain with increased c-di-AMP levels. CWSS basal expression was assessed in the absence of antibiotic-induced stress by quantifying the activity of the sas016 gene promoter, widely used as a proxy for CWSS activation ( 17 , 29 , 30 ). Confirming our hypothesis, the CWSS activity was significantly increased in the ∆ gdpP mutant compared to the WT ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The manipulated regions were checked by Sanger sequencing. The sas016 promoter luciferase fusion plasmid p sas016 p - luc ( 29 ) was transduced into LAC* WT, Δ gdpP , and Δ gdpP/ Δ vraR mutant background using phage 80α.…”

Section: Methodsmentioning

confidence: 99%

C-di-AMP levels modulate Staphylococcus aureus cell wall thickness, response to oxidative stress, and antibiotic resistance and tolerance

Dengler Haunreiter,

Tarnutzer,

Bär

et al. 2023

Microbiol Spectr

3

0

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Beta-lactam antibiotics are widely used to treat infections caused by the important human pathogen Staphylococcus aureus . Resistance to beta-lactams, as found in methicillin-resistant S. aureus , renders effective treatment difficult. The second messenger cyclic di-3′,5′-adenosine monophosphate (c-di-AMP) promotes beta-lactam resistance in clinical S. aureus isolates. C-di-AMP plays a crucial role in the regulation of cellular processes such as virulence, cell wall homeostasis, and resistance to beta-lactams in many bacterial species. In S. aureus, c-di-AMP synthesis is mediated by the diadenylate cyclase DacA, while its degradation is carried out by the phosphodiesterases GdpP and Pde2. In this work, we assessed the effect of altered c-di-AMP levels due to mutations in dacA , gdpP, or gdpP/pde2 on virulence determinants. We report that a previously described growth defect in bacteria producing high c-di-AMP levels is mainly attributable to smaller cell size. High c-di-AMP levels also led to decreased survival upon oxidative stress, reduced production of the antioxidant staphyloxanthin, increased oxacillin and fosfomycin resistance, and increased cell wall thickness. While resistance to ceftaroline was not affected, high c-di-AMP levels promoted tolerance to this antibiotic. In response to cell wall stress induced by antibiotics, the three-component regulatory system VraTSR mediates an increase in cell wall synthesis via the cell wall stress stimulon (CWSS). Increased c-di-AMP levels led to an activation of the CWSS. Upon deletion of vraR , resistance to oxacillin and fosfomycin as well as cell wall thickness diminished in the Δ gdpP mutant, indicating a contribution of the VraTSR system to the cell wall related phenotypes. IMPORTANCE Antibiotic resistance and tolerance are substantial healthcare-related problems, hampering effective treatment of bacterial infections. Mutations in the phosphodiesterase GdpP, which degrades cyclic di-3′, 5′-adenosine monophosphate (c-di-AMP), have recently been associated with resistance to beta-lactam antibiotics in clinical Staphylococcus aureus isolates. In this study, we show that high c-di-AMP levels decreased the cell size and increased the cell wall thickness in S. aureus mutant strains. As a consequence, an increase in resistance to cell wall targeting antibiotics, such as oxacillin and fosfomycin as well as in tolerance to ceftaroline, a cephalosporine used to treat methicillin-resistant S. aureus infections, was observed. These findings underline the importance of investigating the role of c-di-AMP in the development of tolerance and resistance to antibiotics in order to optimize treatment in the clinical setting.

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“…Thus, reporter gene approaches for antibiotic mode of action analysis are most common in the standard Gram-positive and Gram-negative model organisms Bacillus subtilis and E. coli (Table 2) (Bianchi, 1999;Hutter et al, 2004a;Urban et al, 2007;Wenzel et al, 2014). However, reporter gene studies in general are common tools in many organisms, including pathogens like S. aureus, and also strains that were not developed as antibiotic mode of action analysis tool can prove useful as such (Mesak et al, 2008;Chanda et al, 2009;Mondal et al, 2010;Dengler and McCallum, 2016;Bojer et al, 2017).…”

Section: Reporter Gene Fusionsmentioning

confidence: 99%

A How-To Guide for Mode of Action Analysis of Antimicrobial Peptides

Schäfer

¹

,

Wenzel

²

2020

Front. Cell. Infect. Microbiol.

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Antimicrobial peptides (AMPs) are a promising alternative to classical antibiotics in the fight against multi-resistant bacteria. They are produced by organisms from all domains of life and constitute a nearly universal defense mechanism against infectious agents. No drug can be approved without information about its mechanism of action. In order to use them in a clinical setting, it is pivotal to understand how AMPs work. While many pore-forming AMPs are well-characterized in model membrane systems, non-pore-forming peptides are often poorly understood. Moreover, there is evidence that pore formation may not happen or not play a role in vivo. It is therefore imperative to study how AMPs interact with their targets in vivo and consequently kill microorganisms. This has been difficult in the past, since established methods did not provide much mechanistic detail. Especially, methods to study membrane-active compounds have been scarce. Recent advances, in particular in microscopy technology and cell biological labeling techniques, now allow studying mechanisms of AMPs in unprecedented detail. This review gives an overview of available in vivo methods to investigate the antibacterial mechanisms of AMPs. In addition to classical mode of action classification assays, we discuss global profiling techniques, such as genomic and proteomic approaches, as well as bacterial cytological profiling and other cell biological assays. We cover approaches to determine the effects of AMPs on cell morphology, outer membrane, cell wall, and inner membrane properties, cellular macromolecules, and protein targets. We particularly expand on methods to examine cytoplasmic membrane parameters, such as composition, thickness, organization, fluidity, potential, and the functionality of membrane-associated processes. This review aims to provide a guide for researchers, who seek a broad overview of the available methodology to study the mechanisms of AMPs in living bacteria.

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Luciferase Reporter Gene System to Detect Cell Wall Stress Stimulon Induction in Staphylococcus aureus

Cited by 6 publications

References 28 publications

C-di-AMP levels modulateStaphylococcus aureuscell wall thickness as well as virulence and contribute to antibiotic resistance and tolerance

C-di-AMP levels modulateStaphylococcus aureuscell wall thickness as well as virulence and contribute to antibiotic resistance and tolerance

C-di-AMP levels modulate Staphylococcus aureus cell wall thickness, response to oxidative stress, and antibiotic resistance and tolerance

A How-To Guide for Mode of Action Analysis of Antimicrobial Peptides

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