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 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 ◽  
Vol 20 (1) ◽  
Author(s):  
Jinxin Zheng ◽  
Yang Wu ◽  
Zhiwei Lin ◽  
Guangfu Wang ◽  
Sibo Jiang ◽  
...  
Keyword(s):  
Stress Tolerance ◽  
Enterococcus Faecalis ◽  
Biofilm Formation

scholarly journals RgpF Is Required for Maintenance of Stress Tolerance and Virulence in Streptococcus mutans

2017 ◽  
Vol 199 (24) ◽  
Author(s):  
C. J. Kovacs ◽  
R. C. Faustoferri ◽  
R. G. Quivey
Keyword(s):  
Cell Wall ◽  
Stress Tolerance ◽  
Streptococcus Mutans ◽  
Mutant Strain ◽  
Galleria Mellonella ◽  
Infection Model ◽  
Critical Function ◽  
Content Type ◽  
The Impact

ABSTRACT Bacterial cell wall dynamics have been implicated as important determinants of cellular physiology, stress tolerance, and virulence. In Streptococcus mutans, the cell wall is composed primarily of a rhamnose-glucose polysaccharide (RGP) linked to the peptidoglycan. Despite extensive studies describing its formation and composition, the potential roles for RGP in S. mutans biology have not been well investigated. The present study characterizes the impact of RGP disruption as a result of the deletion of rgpF, the gene encoding a rhamnosyltransferase involved in the construction of the core polyrhamnose backbone of RGP. The ΔrgpF mutant strain displayed an overall reduced fitness compared to the wild type, with heightened sensitivities to various stress-inducing culture conditions and an inability to tolerate acid challenge. The loss of rgpF caused a perturbation of membrane-associated functions known to be critical for aciduricity, a hallmark of S. mutans acid tolerance. The proton gradient across the membrane was disrupted, and the ΔrgpF mutant strain was unable to induce activity of the F1Fo ATPase in cultures grown under low-pH conditions. Further, the virulence potential of S. mutans was also drastically reduced following the deletion of rgpF. The ΔrgpF mutant strain produced significantly less robust biofilms, indicating an impairment in its ability to adhere to hydroxyapatite surfaces. Additionally, the ΔrgpF mutant lost competitive fitness against oral peroxigenic streptococci, and it displayed significantly attenuated virulence in an in vivo Galleria mellonella infection model. Collectively, these results highlight a critical function of the RGP in the maintenance of overall stress tolerance and virulence traits in S. mutans. IMPORTANCE The cell wall of Streptococcus mutans, the bacterium most commonly associated with tooth decay, is abundant in rhamnose-glucose polysaccharides (RGP). While these structures are antigenically distinct to S. mutans, the process by which they are formed and the enzymes leading to their construction are well conserved among streptococci. The present study describes the consequences of the loss of RgpF, a rhamnosyltransferase involved in RGP construction. The deletion of rgpF resulted in severe ablation of the organism's overall fitness, culminating in significantly attenuated virulence. Our data demonstrate an important link between the RGP and cell wall physiology of S. mutans, affecting critical features used by the organism to cause disease and providing a potential novel target for inhibiting the pathogenesis of S. mutans.

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 c-di-AMP is essential for the virulence of Enterococcus faecalis

2021 ◽  
Author(s):  
Shivani Kundra ◽  
Ling Ning Lam ◽  
Jessica K. Kajfasz ◽  
Leila Casella ◽  
Marissa J Andersen ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Biofilm Formation ◽  
Galleria Mellonella ◽  
Ex Vivo ◽  
Biological Significance ◽  
Opportunistic Pathogen ◽  
Bacterial Fitness ◽  
Key Aspects

Second messenger nucleotides are produced by bacteria in response to environmental stimuli and play a major role in the regulation of processes associated with bacterial fitness, including but not limited to osmoregulation, envelope homeostasis, central metabolism, and biofilm formation. In this study, we uncovered the biological significance of c-di-AMP in the opportunistic pathogen Enterococcus faecalis by isolating and characterizing strains lacking genes responsible for c-di-AMP synthesis (cdaA) and degradation (dhhP and gdpP). Using complementary approaches, we demonstrated that either complete loss of c-di-AMP (ΔcdaA strain) or c-di-AMP accumulation (ΔdhhP, ΔgdpP and ΔdhhPΔgdpP strains) drastically impaired general cell fitness and virulence of E. faecalis. In particular, the ΔcdaA strain was highly sensitive to envelope-targeting antibiotics, was unable to multiply and quickly lost viability in human serum or urine ex vivo, and was avirulent in an invertebrate (Galleria mellonella) and in two catheter-associated mouse infection models that recapitulate key aspects of enterococcal infections in humans. In addition to evidence linking these phenotypes to altered activity of metabolite and peptide transporters and inability to maintain osmobalance, we found that the attenuated virulence of ΔcdaA could be also attributed to a defect in Ebp pilus production and activity that severely impaired biofilm formation under both in vitro and in vivo conditions. Collectively, these results reveal that c-di-AMP signaling is essential for E. faecalis pathogenesis and a desirable target for drug development.

scholarly journals c-di-AMP is essential for the virulence of Enterococcus faecalis

2021 ◽  
Author(s):  
Shivani Kundra ◽  
Ling Ning Lam ◽  
Jessica K. Kajfasz ◽  
Leila G. Casella ◽  
Marissa J. Andersen ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Biofilm Formation ◽  
Galleria Mellonella ◽  
Ex Vivo ◽  
Biological Significance ◽  
Opportunistic Pathogen ◽  
Bacterial Fitness ◽  
Key Aspects

Second messenger nucleotides are produced by bacteria in response to environmental stimuli and play a major role in the regulation of processes associated with bacterial fitness, including but not limited to osmoregulation, envelope homeostasis, central metabolism, and biofilm formation. In this study, we uncovered the biological significance of c-di-AMP in the opportunistic pathogen Enterococcus faecalis by isolating and characterizing strains lacking genes responsible for c-di-AMP synthesis ( cdaA ) and degradation ( dhhP and gdpP ). Using complementary approaches, we demonstrated that either complete loss of c-di-AMP (Δ cdaA strain) or c-di-AMP accumulation (Δ dhhP , Δ gdpP and Δ dhhP Δ gdpP strains) drastically impaired general cell fitness and virulence of E. faecalis . In particular, the Δ cdaA strain was highly sensitive to envelope-targeting antibiotics, was unable to multiply and quickly lost viability in human serum or urine ex vivo , and was virtually avirulent in an invertebrate ( Galleria mellonella ) and in two catheter-associated mouse infection models that recapitulate key aspects of enterococcal infections in humans. In addition to evidence linking these phenotypes to altered activity of metabolite and peptide transporters and inability to maintain osmobalance, we found that the attenuated virulence of Δ cdaA could be also attributed to a defect in Ebp pilus production and activity that severely impaired biofilm formation under both in vitro and in vivo conditions. Collectively, these results demonstrate that c-di-AMP signaling is essential for E. faecalis pathogenesis and a desirable target for drug development.

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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