Genes Important for Catalase Activity in Enterococcus faecalis

Heme is an essential cofactor and signaling molecule. Heme acquisition by proteins and heme signaling are ultimately reliant on the ability to mobilize labile heme (LH). However, the properties of LH pools, including concentration, oxidation state, distribution, speciation, and dynamics, are poorly understood. Herein, we elucidate the nature and dynamics of LH using genetically encoded ratiometric fluorescent heme sensors in the unicellular eukaryote Saccharomyces cerevisiae. We find that the subcellular distribution of LH is heterogeneous; the cytosol maintains LH at ∼20–40 nM, whereas the mitochondria and nucleus maintain it at concentrations below 2.5 nM. Further, we find that the signaling molecule nitric oxide can initiate the rapid mobilization of heme in the cytosol and nucleus from certain thiol-containing factors. We also find that the glycolytic enzyme glyceraldehyde phosphate dehydrogenase constitutes a major cellular heme buffer, and is responsible for maintaining the activity of the heme-dependent nuclear transcription factor heme activator protein (Hap1p). Altogether, we demonstrate that the heme sensors can be used to reveal fundamental aspects of heme trafficking and dynamics and can be used across multiple organisms, including Escherichia coli, yeast, and human cell lines.

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“…The tdh3Δ cells have normal Ctt1p activity as determined by a native in-gel colorimetric assay for catalase (34) (SI Appendix, Fig. S16C).…”

Section: Resultsmentioning

confidence: 99%

Heme dynamics and trafficking factors revealed by genetically encoded fluorescent heme sensors

Hanna

Harvey

Martinez-Guzman

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

166

405

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“…The steps of catalase folding and hemin (defined as protoporphyrin IX containing a ferric ion) insertion, as well as potential chaperone proteins involved in this process, remain unknown. A recent report investigating genes important for catalase biogenesis was carried out in Enterococcus faecalis (6). By screening for catalase activity using a transposon mutant library, seven genes important for catalase activity were identified which code for the catalase itself (katA), RNA turnover global regulators (rnjA and srmB), NADH peroxidase (npr), stress response regulator (etaR), and membrane transport proteins (oppBC) (6).…”

mentioning

confidence: 99%

“…A recent report investigating genes important for catalase biogenesis was carried out in Enterococcus faecalis (6). By screening for catalase activity using a transposon mutant library, seven genes important for catalase activity were identified which code for the catalase itself (katA), RNA turnover global regulators (rnjA and srmB), NADH peroxidase (npr), stress response regulator (etaR), and membrane transport proteins (oppBC) (6). Excluding katA, the precise function of these genes for catalase activity requires further investigation; however, Baureder and Hederstedt speculate that these genes have an indirect role in catalase activity within the cell (6).…”

mentioning

confidence: 99%

“…By screening for catalase activity using a transposon mutant library, seven genes important for catalase activity were identified which code for the catalase itself (katA), RNA turnover global regulators (rnjA and srmB), NADH peroxidase (npr), stress response regulator (etaR), and membrane transport proteins (oppBC) (6). Excluding katA, the precise function of these genes for catalase activity requires further investigation; however, Baureder and Hederstedt speculate that these genes have an indirect role in catalase activity within the cell (6). Furthermore, despite in-depth characterization of heme transport into the cytoplasmic space of bacteria, very little is known about the potential proteins involved in trafficking heme to heme proteins within the cytoplasmic space.…”

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confidence: 99%

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Cj1386, an Atypical Hemin-Binding Protein, Mediates Hemin Trafficking to KatA in Campylobacter jejuni

Flint

Stintzi

2015

J Bacteriol

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. While the structure and biochemical properties of catalase enzymes have been well characterized over many decades of research, it remained unclear how catalases acquire hemin. We have previously reported that Cj1386 is essential for ensuring proper hemin content in Campylobacter jejuni catalase (KatA) (A. Flint, Y. Q. Sun, and A. Stintzi, J Bacteriol 194:334 -345, 2012). In this report, an in-depth molecular characterization of Cj1386 was performed to elucidate the mechanistic details of this association. Coimmunoprecipitation assays revealed that KatA-Cj1386 transiently interact in vivo, and UV-visible spectroscopy demonstrated that purified Cj1386 protein binds hemin. Furthermore, hemin titration experiments determined that hemin binds to Cj1386 in a 1:1 ratio with hexacoordinate hemin binding. Mutagenesis of potential hemin-coordinating residues in Cj1386 showed that tyrosine 57 was essential for hemin coordination when Cj1386 was overexpressed in Escherichia coli. The importance of tyrosine 57 in hemin trafficking in vivo was confirmed by introducing the cj1386 Y57A allele into a C. jejuni ⌬cj1386 mutant background. The cj1386 Y57A mutation resulted in increased sensitivity toward H 2 O 2 relative to the wild type, suggesting that KatA was not functional in this strain. In support of this finding, KatA immunoprecipitated from the ⌬cj1386؉cj1386 Y57A mutant had significantly reduced hemin content compared to that of the cj1386 WT background. Overall, these findings indicate that Cj1386 is involved in directly trafficking hemin to KatA and that tyrosine 57 plays a key role in this function.

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“…However, an ace mutant was not attenuated in the same peritonitis model (6), indicating that the GrvRS system regulates other virulence determinants in addition to ace. Indeed, a kat gene encoding catalase was reported to be regulated by the GrvRS system in OG1RF (37). Catalase plays an importance role in protection against cellular oxidative stress by degrading hydrogen peroxide (38).…”

Section: Discussionmentioning

confidence: 99%

The Two-Component System GrvRS (EtaRS) Regulates ace Expression in Enterococcus faecalis OG1RF

et al. 2015

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e Expression of ace (adhesin to collagen of Enterococcus faecalis), encoding a virulence factor in endocarditis and urinary tract infection models, has been shown to increase under certain conditions, such as in the presence of serum, bile salts, urine, and collagen and at 46°C. However, the mechanism of ace/Ace regulation under different conditions is still unknown. In this study, we identified a two-component regulatory system GrvRS as the main regulator of ace expression under these stress conditions. Using Northern hybridization and ␤-galactosidase assays of an ace promoter-lacZ fusion, we found transcription of ace to be virtually absent in a grvR deletion mutant under the conditions that increase ace expression in wild-type OG1RF and in the complemented strain. Moreover, a grvR mutant revealed decreased collagen binding and biofilm formation as well as attenuation in a murine urinary tract infection model. Here we show that GrvR plays a major role in control of ace expression and E. faecalis virulence. Enterococcus faecalis is a Gram-positive commensal bacterium of the gastrointestinal tract and also a common cause of hospital-acquired infections and endocarditis (1). During experimental infections, the adhesin to collagen of E. faecalis (Ace) plays an important role which has been attributed to mediating binding of E. faecalis to human extracellular matrix (ECM) proteins such as collagen (types I and IV), laminin, and dentin (2-4). Different infection models have shown attenuation of ace deletion mutants (5-7) and specific anti-recombinant Ace (rAce) antibodies were shown to confer protection against E. faecalis infection in an experimental endocarditis model (6). Therefore, Ace is considered a virulence factor in E. faecalis infection.In order to better understand the role of Ace, it is important to study the regulation mechanisms of ace expression and surface display. Previous works have identified several environmental factors regulating ace expression; e.g., transcription of ace was increased when E. faecalis was grown at 46°C and grown in the presence of 40% horse serum, urine, and bile salts (5,8,9). In addition, levels of Ace on the cell surface are dependent on the E. faecalis strain and growth phase (10-12). With E. faecalis OG1RF, Ace is increased in the early exponential phase but reduced in the stationary phase; however, with E. faecalis JH2-2, it is maintained in later growth phases (10-12). The decrease in stationary-phase Ace in strain OG1RF was shown to be dependent on a functional fsr quorum-sensing system controlling the expression of gelatinase (GelE), which cleaves Ace from the OG1RF cell surface in latephase cultures (11). Strains such as JH2-2 (lacking a complete fsr system operon) as well as fsr and gelE mutants of OG1RF do not cleave Ace from the surface (11), since they do not produce gelatinase. In other words, the amount of Ace on OG1RF strains is regulated in part at the posttranslational level. At the transcriptional level, the enterococcal regulator of survival (Ers) was prev...

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Genes Important for Catalase Activity in Enterococcus faecalis

Cited by 38 publications

References 31 publications

Heme dynamics and trafficking factors revealed by genetically encoded fluorescent heme sensors

Heme dynamics and trafficking factors revealed by genetically encoded fluorescent heme sensors

Cj1386, an Atypical Hemin-Binding Protein, Mediates Hemin Trafficking to KatA in Campylobacter jejuni

The Two-Component System GrvRS (EtaRS) Regulates ace Expression in Enterococcus faecalis OG1RF

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