scholarly journals Physiological significance of ClpP in Enterococcus faecalis and its regulating proteins were identified by Tandem Mass Tag Mass Spectrometry

2019 ◽  
Author(s):  
Jinxin Zheng ◽  
Yang Wu ◽  
Zhiwei Lin ◽  
Guangfu Wang ◽  
Sibo Jiang ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Ribosomal Protein ◽  
Enterococcus Faecalis ◽  
Mutant Strain ◽  
Biofilm Formation ◽  
Protein Quality ◽  
Regulatory Protein ◽  
Physiological Significance ◽  
Tandem Mass ◽  
Acetyl Esterase

Abstract Background ClpP is important for bacterial growth and plays an indispensable role in cellular protein quality control systems by refolding or degrading damaged proteins, but the physiological significance of ClpP in Enterococcus faecalis is still obscure. Thus a clpP deletion mutant (△clpP) was constructed in E. faecalis OG1RF strain to elucidate a more comprehensive picture of the effect of ClpP on E. faecalis. The global abundance of proteins was determined by a mass spectrometer with Tandem Mass Tags labeling. Results The ΔclpP mutant strain showed impaired growth at 20°C or 45°C, at 5% NaCl or 2 mM H2O2. The surviving bacteria of the ΔclpP mutant strain reduced after exposure to the high concentration (50 x MIC) of linezolid or minocycline for 96 h. The ΔclpP mutant strain also demonstrated decreased biofilm formation but increased virulence in a Galleria mellonella model. The mass spectrometry proteomics data indicated that the abundances of 135 proteins changed (111 proteins increased, 24 proteins decreased) in the ΔclpP mutant strain. Among those differential abundance proteins, the abundances of stress response or virulence relating proteins: FsrA response regulator, gelatinase GelE, regulatory protein Spx (spxA), heat-inducible transcription repressor HrcA, transcriptional regulator CtsR, ATPase/chaperone ClpC, acetyl esterase/lipase, and chaperonin GroEL increased in the ΔclpP mutant strain; however, the abundances of ribosomal protein L4/L1 family protein (rplD), ribosomal protein L7/L12 (rplL2), 50S ribosomal protein L13 (rplM), L18 (rplR), L20 (rplT), 30S ribosomal protein S14 (rpsN2) and S18 (rpsR) all reduced. The abundances of biofilm formation related adapter protein MecA increased, while the abundances of dihydroorotase (pyrC), orotate phosphoribosyltransferase (pyrE) and orotidine-5'-phosphate decarboxylase (pyrF) all decreased in the ΔclpP mutant strain. Conclusion The present study demonstrates that ClpP participates in stress tolerance, biofilm formation, antimicrobials tolerance, and virulence of E. faecalis.

scholarly journals ClpP participates in stress tolerance, biofilm formation, antimicrobial tolerance, and virulence of Enterococcus faecalis

2020 ◽  
Author(s):  
Jinxin Zheng ◽  
Yang Wu ◽  
Zhiwei Lin ◽  
Guangfu Wang ◽  
Sibo Jiang ◽  
...  
Keyword(s):  
Ribosomal Protein ◽  
Stress Tolerance ◽  
Enterococcus Faecalis ◽  
Mutant Strain ◽  
Biofilm Formation ◽  
Protein Quality ◽  
Galleria Mellonella ◽  
Regulatory Protein ◽  
Proteomics Data ◽  
Acetyl Esterase

Abstract Background ClpP is important for bacterial growth and plays an indispensable role in cellular protein quality control systems by refolding or degrading damaged proteins, but the physiological significance of ClpP in Enterococcus faecalis remains obscure. A clpP deletion mutant (△ clpP ) was constructed using the E. faecalis OG1RF strain to clarify the effect of ClpP on E. faecalis. The global abundance of proteins was determined by a mass spectrometer with tandem mass tag labeling. Results The Δ clpP mutant strain showed impaired growth at 20°C or 45°C at 5% NaCl or 2 mM H 2 O 2 . The number of surviving Δ clpP mutants decreased after exposure to the high concentration (50× minimal inhibitory concentration) of linezolid or minocycline for 96 h. The Δ clpP mutant strain also demonstrated decreased biofilm formation but increased virulence in a Galleria mellonella model. The mass spectrometry proteomics data indicated that the abundances of 135 proteins changed (111 increased, 24 decreased) in the Δ clpP mutant strain. Among those, the abundances of stress response or virulence relating proteins: FsrA response regulator, gelatinase GelE, regulatory protein Spx ( spxA ), heat-inducible transcription repressor HrcA, transcriptional regulator CtsR, ATPase/chaperone ClpC, acetyl esterase/lipase, and chaperonin GroEL increased in the Δ clpP mutant strain; however, the abundances of ribosomal protein L4/L1 family protein ( rplD ), ribosomal protein L7/L12 ( rplL2 ), 50S ribosomal protein L13 ( rplM ), L18 ( rplR ), L20 ( rplT ), 30S ribosomal protein S14 ( rpsN2 ) and S18 ( rpsR ) all decreased. The abundances of biofilm formation-related adapter protein MecA increased, while the abundances of dihydroorotase ( pyrC ), orotate phosphoribosyltransferase ( pyrE ), and orotidine-5'-phosphate decarboxylase ( pyrF ) all decreased in the Δ clpP mutant strain. Conclusion The present study demonstrates that ClpP participates in stress tolerance, biofilm formation, antimicrobial tolerance, and virulence of E. faecalis.

scholarly journals ClpP participates in stress tolerance, biofilm formation, antimicrobial tolerance, and virulence of Enterococcus faecalis

2020 ◽  
Author(s):  
Jinxin Zheng ◽  
Yang Wu ◽  
Zhiwei Lin ◽  
Guangfu Wang ◽  
Sibo Jiang ◽  
...  
Keyword(s):  
Ribosomal Protein ◽  
Stress Tolerance ◽  
Enterococcus Faecalis ◽  
Mutant Strain ◽  
Biofilm Formation ◽  
Protein Quality ◽  
Galleria Mellonella ◽  
Regulatory Protein ◽  
Proteomics Data ◽  
Acetyl Esterase

Abstract Background ClpP is important for bacterial growth and plays an indispensable role in cellular protein quality control systems by refolding or degrading damaged proteins, but the physiological significance of ClpP in Enterococcus faecalis remains obscure. A clpP deletion mutant (△ clpP ) was constructed using the E. faecalis OG1RF strain to clarify the effect of ClpP on E. faecalis. The global abundance of proteins was determined by a mass spectrometer with tandem mass tag labeling.Results The Δ clpP mutant strain showed impaired growth at 20°C or 45°C at 5% NaCl or 2 mM H 2 O 2 . The number of surviving Δ clpP mutants decreased after exposure to the high concentration (50× minimal inhibitory concentration) of linezolid or minocycline for 96 h. The Δ clpP mutant strain also demonstrated decreased biofilm formation but increased virulence in a Galleria mellonella model. The mass spectrometry proteomics data indicated that the abundances of 135 proteins changed (111 increased, 24 decreased) in the Δ clpP mutant strain. Among those, the abundances of stress response or virulence relating proteins: FsrA response regulator, gelatinase GelE, regulatory protein Spx ( spxA ), heat-inducible transcription repressor HrcA, transcriptional regulator CtsR, ATPase/chaperone ClpC, acetyl esterase/lipase, and chaperonin GroEL increased in the Δ clpP mutant strain; however, the abundances of ribosomal protein L4/L1 family protein ( rplD ), ribosomal protein L7/L12 ( rplL2 ), 50S ribosomal protein L13 ( rplM ), L18 ( rplR ), L20 ( rplT ), 30S ribosomal protein S14 ( rpsN2 ) and S18 ( rpsR ) all decreased. The abundances of biofilm formation-related adapter protein MecA increased, while the abundances of dihydroorotase ( pyrC ), orotate phosphoribosyltransferase ( pyrE ), and orotidine-5'-phosphate decarboxylase ( pyrF ) all decreased in the Δ clpP mutant strain.Conclusion The present study demonstrates that ClpP participates in stress tolerance, biofilm formation, antimicrobial tolerance, and virulence of E. faecalis.

scholarly journals Functional Genomics of Enterococcus faecalis: Multiple Novel Genetic Determinants for Biofilm Formation in the Core Genome

2009 ◽  
Vol 191 (8) ◽  
pp. 2806-2814 ◽  
Author(s):  
Katie S. Ballering ◽  
Christopher J. Kristich ◽  
Suzanne M. Grindle ◽  
Ana Oromendia ◽  
David T. Beattie ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Biofilm Formation ◽  
Core Genome ◽  
Genetic Locus ◽  
Regulatory Protein ◽  
Genetic Determinants ◽  
Genetic Loci ◽  
Putative Promoter Region ◽  
Biofilm Growth ◽  
The Core

ABSTRACT The ability of Enterococcus faecalis to form robust biofilms on host tissues and on abiotic surfaces such as catheters likely plays a major role in the pathogenesis of opportunistic antibiotic-resistant E. faecalis infections and in the transfer of antibiotic resistance genes. We have carried out a comprehensive analysis of genetic determinants of biofilm formation in the core genome of E. faecalis. Here we describe 68 genetic loci predicted to be involved in biofilm formation that were identified by recombinase in vivo expression technology (RIVET); most of these genes have not been studied previously. Differential expression of a number of these determinants during biofilm growth was confirmed by quantitative reverse transcription-PCR, and genetic complementation studies verified a role in biofilm formation for several candidate genes. Of particular interest was genetic locus EF1809, predicted to encode a regulatory protein of the GntR family. We isolated 14 independent nonsibling clones containing the putative promoter region for this gene in the RIVET screen; EF1809 also showed the largest increase in expression during biofilm growth of any of the genes tested. Since an in-frame deletion of EF1809 resulted in a severe biofilm defect that could be complemented by the cloned wild-type gene, we have designated EF1809 ebrA (enterococcal biofilm regulator). Most of the novel genetic loci identified in our studies are highly conserved in gram-positive bacterial pathogens and may thus constitute a pool of uncharacterized genes involved in biofilm formation that may be useful targets for drug discovery.

scholarly journals Biosynthesis of arsenolipids by the cyanobacterium Synechocystis sp. PCC 6803

2014 ◽  
Vol 11 (5) ◽  
pp. 506 ◽  
Author(s):  
Xi-Mei Xue ◽  
Georg Raber ◽  
Simon Foster ◽  
Song-Can Chen ◽  
Kevin A. Francesconi ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
Mutant Strain ◽  
High Performance ◽  
Tandem Mass ◽  
Wild Type ◽  
Arsenic Compounds ◽  
Synechocystis Sp ◽  
Pcc 6803

Environmental context Arsenic biotransformation processes play a key role in the cycling of arsenic in aquatic systems. We show that a freshwater cyanobacterium can convert inorganic arsenic into arsenolipids, and the conversion efficiency depends on the arsenic concentration. The role of these novel arsenic compounds remains to be elucidated. Abstract Although methylated arsenic and arsenosugars have been verified in various freshwater organisms, lipid-soluble arsenic compounds have not been identified. Here, we report investigations with the model organism cyanobacterium Synechocystis sp. PCC 6803 wild type and ΔarsM (arsenic(III) S-adenosylmethionine methyltransferase) mutant strain, which lacks the enzymes for arsenic methylation cultured in various concentrations of arsenate (AsV). Although Synechocystis accumulated higher arsenic concentrations at the higher exposure levels, the bioaccumulation factor decreased with increasing AsV. The accumulated arsenic in the cells was partitioned into water-soluble and lipid-soluble fractions; lipid-soluble arsenic was found in Synechocystis wild type cells (3–35% of the total depending on the level of arsenic exposure), but was not detected in Synechocystis ΔarsM mutant strain showing that ArsM was required for arsenolipid biosynthesis. The arsenolipids present in Synechocystis sp. PCC 6803 were analysed by high performance liquid chromatography–inductively coupled plasma–mass spectrometry, high performance liquid chromatography–electrospray mass spectrometry, and high resolution tandem mass spectrometry. The two major arsenolipids were characterised as arsenosugar phospholipids based on their assigned molecular formulas C47H88O14AsP and C47H90O14AsP, and tandem mass spectrometric data demonstrated the presence of the phosphate arsenosugar and acylated glycerol groups.

scholarly journals An AraC-Type Transcriptional Regulator Encoded on the Enterococcus faecalis Pathogenicity Island Contributes to Pathogenesis and Intracellular Macrophage Survival

2008 ◽  
Vol 76 (12) ◽  
pp. 5668-5676 ◽  
Author(s):  
Phillip S. Coburn ◽  
Arto S. Baghdayan ◽  
GT Dolan ◽  
Nathan Shankar
Keyword(s):  
Enterococcus Faecalis ◽  
Mutant Strain ◽  
Biofilm Formation ◽  
Transcriptional Regulator ◽  
Pathogenicity Island ◽  
Nucleotide Sequence Analysis ◽  
Infection Model ◽  
Wild Type ◽  
Altered Expression

ABSTRACT A gene encoding a putative AraC-type transcriptional regulator was identified on the 153-kb pathogenicity island (PAI) found among virulent Enterococcus faecalis strains. In an effort to understand the function of this regulator, designated PerA (for pathogenicity island-encoded regulator), we first examined the expression of the perA gene in the original PAI strain MMH594 and in an unrelated clinical isolate E99 by reverse transcription-PCR. Interestingly, expression analysis revealed no detectable perA transcript in MMH594, whereas a transcript was observed in strain E99. Nucleotide sequence analysis revealed that this altered expression between the two strains was attributable to the differential location of an IS1191 element within the putative promoter region upstream of the perA gene. In order to determine the role of this putative regulator in E. faecalis pathogenesis, a perA-deficient mutant was created in strain E99, and the wild-type and mutant pair were compared for phenotypic differences. In in vitro biofilm assays, the mutant strain showed a significantly higher level of growth medium-specific biofilm formation compared to the wild type. However, in a murine intraperitoneal infection model, the mutant strain was significantly less pathogenic. The mutant was also attenuated for survival within macrophages in vitro. These findings highlight the importance of PerA as a regulator of biofilm formation and survival within macrophages and is likely a regulator controlling determinants important to pathogenesis.

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