A eukaryotic-type Ser/Thr kinase in
            <i>Enterococcus faecalis</i>
            mediates antimicrobial resistance and intestinal persistence

Kristich, Christopher J.; Wells, Carol L.; Dunny, Gary M.

doi:10.1073/pnas.0608742104

Cited by 142 publications

(180 citation statements)

References 35 publications

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“…Briefly, the codY coding sequence was amplified using the primers listed on Table S1 and was cloned into pCJK96 to yield plasmid pcodY-comp. The plasmid was electroporated into the ΔcodY strains using standard protocols (49).…”

Section: Methodsmentioning

confidence: 99%

Association of Metal Homeostasis and (p)ppGpp Regulation in the Pathophysiology of Enterococcus faecalis

2017

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In Enterococcus faecalis, the regulatory nucleotides pppGpp and ppGpp, collectively, (p)ppGpp, are required for growth in blood, survival within macrophages, and virulence. However, a clear understanding of how (p)ppGpp promotes virulence in E. faecalis and other bacterial pathogens is still lacking. In the host, the essential transition metals iron (Fe) and manganese (Mn) are not readily available to invading pathogens because of a host-driven process called nutritional immunity. Considering its central role in adaptation to nutritional stresses, we hypothesized that (p)ppGpp mediates E. faecalis virulence through regulation of metal homeostasis. Indeed, supplementation of serum with either Fe or Mn restored growth and survival of the Δrel ΔrelQ [(p)ppGpp 0 ] strain to wild-type levels. Using a chemically defined medium, we found that (p)ppGpp accumulates in response to either Fe depletion or Mn depletion and that the (p)ppGpp 0 strain has a strong growth requirement for Mn that is alleviated by Fe supplementation. Although inactivation of the nutrient-sensing regulator codY restored some phenotypes of the (p)ppGpp 0 strain, transcriptional analysis showed that the (p)ppGpp/CodY network does not promote transcription of known metal transporters. Interestingly, physiologic and enzymatic investigations suggest that the (p)ppGpp 0 strain requires higher levels of Mn in order to cope with high levels of endogenously produced reactive oxygen species (ROS). Because (p)ppGpp mediates antibiotic persistence and virulence in several bacteria, our findings have broad implications and provide new leads for the development of novel therapeutic and preventive strategies against E. faecalis and beyond. KEYWORDS (p)ppGpp, Enterococcus, manganese, metal homeostasis, nutritional immunity, oxidative stress E nterococcus faecalis is a common member of the human gut microbiota but also a leading cause of a number of hospital-acquired infections such as endocarditis and surgical wound and urinary tract infections (1). The association of this opportunistic pathogen with disease relies on its exceptional resilience, which allows it to prevail in the unfavorable hospital setting while providing a competitive advantage over other bacteria during both infection and treatment.Among the most prominent regulators involved in bacterial adaptation to stress are the nucleotide second messengers guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), collectively, (p)ppGpp, best known as the effectors of the stringent response (SR) (2). While initially discovered to accumulate in response to amino acid starvation, (p)ppGpp has also been shown to accumulate in response to restrictions in carbon, fatty acids, and iron, as well as in response to other nonnutritional stresses (3). During the SR, (p)ppGpp accumulation triggers transcriptional alterations that lead to general repression of rapid growth and simultaneous activation of stress survival, nutrient uptake, and biosynthesis pathways. In addition to its involvement in...

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Section: Methodsmentioning

confidence: 99%

Association of Metal Homeostasis and (p)ppGpp Regulation in the Pathophysiology of Enterococcus faecalis

2017

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“…This is the case in the genome of E. faecalis V583 that encodes 17 two-component systems and 158 one-component systems (9). One such system is that of PrkC, which contains a cytoplasmic kinase output domain fused to an extracellular input domain via a transmembrane region.…”

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confidence: 99%

“…One such system is that of PrkC, which contains a cytoplasmic kinase output domain fused to an extracellular input domain via a transmembrane region. This protein has been shown to modulate antimicrobial resistance and intestinal persistence, presumably in response to the perturbation of the cell wall by bile salts as detected by the extracellular domain (9). The majority of one-component systems are thought to be cytoplasmic, because 97% of those containing a helix-turn-helix output domain lack transmembrane regions.…”

mentioning

confidence: 99%

Molecular Analysis of BcrR, a Membrane-bound Bacitracin Sensor and DNA-binding Protein from Enterococcus faecalis

Gauntlett

Gebhard

Keis

et al. 2008

Journal of Biological Chemistry

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BcrR has been identified as a novel regulatory protein of high level bacitracin resistance encoded by the bcrABD operon in Enterococcus faecalis. The N-terminal domain of BcrR has similarity to the helix-turn-helix motif of DNAbinding proteins, and topological modeling predicts that the C-terminal domain contains four transmembrane ␣-helices. These data have led to the hypothesis that BcrR functions as both a membrane-bound sensor and transducer of bacitracin availability to regulate bcrABD expression. To characterize the bcrABD promoter and identify the promoter elements to which BcrR binds, a series of bcrA-lacZ fusions were constructed. A 69-bp region was identified that was essential for bacitracin-dependent bcrA-lacZ expression. Mutations that targeted this region were used to identify two inverted repeat sequences, each with the sequence 5-GACA(N) 7 TGTC-3, on the bcrABD promoter that were required for bcrA-lacZ expression. To study BcrR binding to this region, we overproduced BcrR with a C-terminal hexa-histidine tag in Escherichia coli membranes, extracted the protein with n-dodecyl-␤-D-maltoside, and subsequently purified it via Ni 2؉ -nitrilotriacetic acid and gel filtration chromatography to apparent homogeneity. Purified BcrR was reconstituted into liposomes, and BcrR binding to bcrABD promoter DNA was analyzed using electrophoretic mobility shift assays. Both inverted repeat sequences were required for BcrR binding, both in the presence and absence of bacitracin. These data demonstrate that membrane-bound BcrR binds specifically to the bcrABD promoter, irrespective of bacitracin concentration. We therefore propose that bacitracin-dependent induction of bcrABD expression by BcrR occurs after DNA binding.Bacitracin is a polypeptide antibiotic that functions by binding to and sequestering undecaprenyl pyrophosphate (UPP) 4 (1). Because UPP acts as a carrier of peptidoglycan monomeric units across the cell membrane (2), the antibacterial nature of bacitracin results from its ability to block cell wall synthesis. High level bacitracin resistance in Enterococcus faecalis is encoded by the bcrABD operon that is under the control of a putative membrane-bound DNA-binding protein, BcrR (3). In the presence of bacitracin, transcription of the bcrABD operon leads to the production of BcrA and BcrB, which are proposed to act as an ABC exporter of bacitracin, thus conferring high level bacitracin resistance (minimum inhibitory concentration Ն 256 g/ml) to the cell (3). BcrD is proposed to function as an undecaprenyl pyrophosphate phosphatase that functions to increase the amount of undecaprenyl available to the cell (3). It has been demonstrated that bcrR, which is transcribed constitutively, is essential for high level bacitracin resistance in E. faecalis and that transcription of bcrABD is abolished in the absence of bcrR (3). The expression of bcrABD was found to be inducible with increasing concentrations of bacitracin (3). The N-terminal domain (amino acid residues 5-61) of BcrR has homology to the Xre famil...

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“…In order to tackle this disadvantage, the NICE system was optimised to be used in Enterococcus as a single-plasmid expression vector [34]. Other inducible expression vectors for E. faecalis are available: the rhamnose-inducible system [35] and the recently described one that is under control of the pheromone cCF10 [54]. The present work describes an alternative system for controlled gene expression in E. faecalis using a single-plasmid expression system.…”

Section: Discussionmentioning

confidence: 99%

“…Beside this, the widely used Lactococcus lactis NICE system is suitable for use in other Gram-positive species including Enterococcus, either by using two simultaneous compatible plasmids -one harbouring the expression cassette and the other carrying the nisRK genes- [33], or as a single-plasmid expression vector including both cassettes [34]. In addition, other E. faecalis expression systems based on rhamnose as inducer [35] or under control of the pheromone cCF10 [36] have been described. Yet, the molecular genetic tools available for E. faecalis are more limited than for some other microorganisms.…”

Section: Introductionmentioning

confidence: 99%

An agmatine-inducible system for the expression of recombinant proteins in Enterococcus faecalis

et al. 2014

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Background: Scientific interest in Enterococcus faecalis has increased greatly over recent decades. Some strains are involved in food fermentation and offer health benefits, whereas others are vancomycin-resistant and cause infections that are difficult to treat. The limited availability of vectors able to express cloned genes efficiently in E. faecalis has hindered biotechnological studies on the bacterium's regulatory and pathogenicity-related genes. The agmatine deiminase (AGDI) pathway of E. faecalis, involved in the conversion of agmatine into putrescine, is driven by a response inducer gene aguR.Results: This study describes that the exposure to the induction factor (agmatine) results in the transcription of genes under the control of the aguB promoter, including the aguBDAC operon. A novel E. faecalis expression vector, named pAGEnt, combining the aguR inducer gene and the aguB promoter followed by a cloning site and a stop codon was constructed. pAGEnt was designed for the overexpression and purification of a protein fused to a 10-amino-acid His-tag at the C-terminus. The use of GFP as a reporter of gene expression in E. faecalis revealed that under induction with 60 mM agmatine, fluorescence reached 40 arbitrary units compared to 0 in uninduced cells. Conclusion: pAGEnt vector can be used for the overexpression of recombinant proteins under the induction of agmatine in E. faecalis, with a close correlation between agmatine concentration and fluorescence when GFP was used as reporter.

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A eukaryotic-type Ser/Thr kinase in Enterococcus faecalis mediates antimicrobial resistance and intestinal persistence

Cited by 142 publications

References 35 publications

Association of Metal Homeostasis and (p)ppGpp Regulation in the Pathophysiology of Enterococcus faecalis

Association of Metal Homeostasis and (p)ppGpp Regulation in the Pathophysiology of Enterococcus faecalis

Molecular Analysis of BcrR, a Membrane-bound Bacitracin Sensor and DNA-binding Protein from Enterococcus faecalis

An agmatine-inducible system for the expression of recombinant proteins in Enterococcus faecalis

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