The YjbH Adaptor Protein Enhances Proteolysis of the Transcriptional Regulator Spx in Staphylococcus aureus

Engman, Jakob; Rogstam, Annika; Frees, Dorte; Ingmer, Hanne; Wachenfeldt, Claes von

doi:10.1128/jb.06414-11

Cited by 59 publications

(89 citation statements)

References 41 publications

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“…ClpX in association with ClpP degrades the global transcriptional regulator Spx that controls a general stress regulon important for survival during severe heat stress (26,27,39,40). We therefore hypothesized that Spx accumulation contributed to the improved stress tolerance of the SADR-3 and SADR-4 isolates.…”

Section: Resultsmentioning

confidence: 99%

Stepwise Decrease in Daptomycin Susceptibility in Clinical Staphylococcus aureus Isolates Associated with an Initial Mutation in rpoB and a Compensatory Inactivation of the clpX Gene

Bæk

Thøgersen

Mogenssen

et al. 2015

Antimicrob Agents Chemother

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dDaptomycin is a lipopeptide antibiotic used clinically for the treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections. The emergence of daptomycin-nonsusceptible S. aureus isolates during therapy is often associated with multiple genetic changes; however, the relative contributions of these changes to resistance and other phenotypic changes usually remain unclear. The present study was undertaken to investigate this issue using a genetically characterized series of four isogenic clinical MRSA strains derived from a patient with bacteremia before and during daptomycin treatment. The first strain obtained after daptomycin therapy carried a single-nucleotide polymorphism (SNP) in rpoB (RpoB A 477 D) that decreased susceptibility not only to daptomycin but also to vancomycin, ␤-lactams, and rifampin. Furthermore, the rpoB mutant exhibited pleiotropic phenotypes, including increased cell wall thickness, reduced expression of virulence traits, induced expression of the stress-associated transcriptional regulator Spx, and slow growth. A subsequently acquired loss-of-function mutation in clpX partly alleviated the growth defect conferred by the rpoB mutation without changing antibiotic susceptibility. The final isolate acquired three additional mutations, including an SNP in mprF (MprF S 295 L) known to confer daptomycin nonsusceptibility, and accordingly, this isolate was the only daptomycin-nonsusceptible strain of this series. Interestingly, in this isolate, the cell wall had regained the same thickness as that of the parental strain, while the level of transcription of the vraSR (cell wall stress regulator) was increased. In conclusion, this study illustrates how serial genetic changes selected in vivo contribute to daptomycin nonsusceptibility, growth fitness, and virulence traits. Staphylococcus aureus is a commensal bacterium that can cause a variety of both localized and more invasive infections. Due to its profound ability to acquire resistance to clinically relevant antibiotics, S. aureus remains a major clinical challenge worldwide (1). The most challenging antimicrobial resistance issue in S. aureus has been the evolution and dissemination of methicillinresistant S. aureus (MRSA) strains first in hospitals and later in the general community (1). MRSA infections are typically treated with last-line antibiotics, such as vancomycin and, more recently, daptomycin (DAP), a cyclic lipopeptide derived from Streptomyces roseosporus with bactericidal activity against a broad range of Gram-positive bacteria (2, 3). The mechanism of action of daptomycin has not been fully elucidated, but it is generally accepted that daptomycin inserts into the cytoplasmic membrane of Grampositive bacteria via a calcium-dependent pathway, leading to potassium efflux, membrane depolarization, and, eventually, cell death (2).The development of daptomycin nonsusceptibility (DAP-NS) in S. aureus seems to be a relatively rare phenomenon not involving horizontal gene transfer but, instead, taking place by a stepwise acqu...

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Section: Resultsmentioning

confidence: 99%

Stepwise Decrease in Daptomycin Susceptibility in Clinical Staphylococcus aureus Isolates Associated with an Initial Mutation in rpoB and a Compensatory Inactivation of the clpX Gene

Bæk

Thøgersen

Mogenssen

et al. 2015

Antimicrob Agents Chemother

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“…However, following the addition of 5 mM diamide, spx transcription was greatly reduced after 30 min (OD 650 ϭ 1.1 with diamide) and recovered after 60 min as the diamide effect tapered (OD 650 ϭ 1.4 with diamide). Interestingly, protein levels of Spx actually increase following diamide treatment in both B. subtilis and S. aureus (31,47). This increase in Spx levels alongside a corresponding decrease in spx transcript levels following diamide treatment (Fig.…”

Section: Figmentioning

confidence: 92%

“…Details of the protein targets of these S. aureus adaptors are limited, and to date, only two target proteins have been described to be regulated by S. aureus adaptors. McsB targets CtsR for ClpCP degradation (30), and the levels of the redox-sensing transcriptional regulator Spx have been shown to be controlled by the adaptor YjbH (31).…”

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confidence: 99%

Role of Adaptor TrfA and ClpPC in Controlling Levels of SsrA-Tagged Proteins and Antitoxins in Staphylococcus aureus

2014

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bStaphylococcus aureus responds to changing extracellular environments in part by adjusting its proteome through alterations of transcriptional priorities and selective degradation of the preexisting pool of proteins. In Bacillus subtilis, the proteolytic adaptor protein MecA has been shown to play a role in assisting with the proteolytic degradation of proteins involved in competence and the oxidative stress response. However, the targets of TrfA, the MecA homolog in S. aureus, have not been well characterized. In this work, we investigated how TrfA assists chaperones and proteases to regulate the proteolysis of several classes of proteins in S. aureus. By fusing the last 3 amino acids of the SsrA degradation tag to Venus, a rapidly folding yellow fluorescent protein, we obtained both fluorescence-based and Western blot assay-based evidence that TrfA and ClpCP are the adaptor and protease, respectively, responsible for the degradation of the SsrA-tagged protein in S. aureus. Notably, the impact of TrfA on degradation was most prominent during late log phase and early stationary phase, due in part to a combination of transcriptional regulation and proteolytic degradation of TrfA by ClpCP. We also characterized the temporal transcriptional regulation governing TrfA activity, wherein Spx, a redox-sensitive transcriptional regulator degraded by ClpXP, activates trfA transcription while repressing its own promoter. Finally, the scope of TrfA-mediated proteolysis was expanded by identifying TrfA as the adaptor that works with ClpCP to degrade antitoxins in S. aureus. Together, these results indicate that the adaptor TrfA adds temporal nuance to protein degradation by ClpCP in S. aureus.

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“…However, the putative Zn-binding N-terminal region is not conserved among all YjbH orthologues. Recent studies of S. aureus YjbH have provided evidence that the Cys residues associated with the N-terminal region of YjbH are not necessary for activity (Engman et al, 2012). Alternative explanations for regulation of YjbH-mediated proteolysis of Spx may have to be considered.…”

Section: Introductionmentioning

confidence: 99%

Geobacillus thermodenitrificans YjbH recognizes the C-terminal end of Bacillus subtilis Spx to accelerate Spx proteolysis by ClpXP

et al. 2012

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Proteolytic control can govern the levels of specific regulatory factors, such as Spx, a transcriptional regulator of the oxidative stress response in Gram-positive bacteria. Under oxidative stress, Spx concentration is elevated and upregulates transcription of genes that function in the stress response. When stress is alleviated, proteolysis of Spx catalysed by ClpXP reduces Spx concentration. Proteolysis is enhanced by the substrate recognition factor YjbH, which possesses a His-Cys-rich region at its N terminus. However, mutations that generate H12A, C13A, H14A, H16A and C31/34A residue substitutions in the N terminus of Bacillus subtilis YjbH (BsYjbH) do not affect functionality in Spx proteolytic control in vivo and in vitro. Because of difficulties in obtaining soluble BsYjbH, the Geobacillus thermodenitrificans yjbH gene was cloned, which yielded soluble GtYjbH protein. Despite its lack of a His-Cys-rich region, GtYjbH complements a B. subtilis yjbH null mutant, and shows high activity in vitro when combined with ClpXP and Spx in an approximately 30 : 1 (ClpXP/Spx : GtYjbH) molar ratio. In vitro interaction experiments showed that Spx and the protease-resistant Spx DD (in which the last two residues of Spx are replaced with two Asp residues) bind to GtYjbH, but deletion of 12 residues from the Spx C terminus (SpxDC) significantly diminished interaction and proteolytic degradation, indicating that the C terminus of Spx is important for YjbH recognition. These experiments also showed that Spx, but not GtYjbH, interacts with ClpX. Kinetic measurements for Spx proteolysis by ClpXP in the presence and absence of GtYjbH suggest that YjbH overcomes non-productive Spx-ClpX interaction, resulting in rapid degradation.

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The YjbH Adaptor Protein Enhances Proteolysis of the Transcriptional Regulator Spx in Staphylococcus aureus

Cited by 59 publications

References 41 publications

Stepwise Decrease in Daptomycin Susceptibility in Clinical Staphylococcus aureus Isolates Associated with an Initial Mutation in rpoB and a Compensatory Inactivation of the clpX Gene

Stepwise Decrease in Daptomycin Susceptibility in Clinical Staphylococcus aureus Isolates Associated with an Initial Mutation in rpoB and a Compensatory Inactivation of the clpX Gene

Role of Adaptor TrfA and ClpPC in Controlling Levels of SsrA-Tagged Proteins and Antitoxins in Staphylococcus aureus

Geobacillus thermodenitrificans YjbH recognizes the C-terminal end of Bacillus subtilis Spx to accelerate Spx proteolysis by ClpXP

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