EfaR Is a Major Regulator of Enterococcus faecalis Manganese Transporters and Influences Processes Involved in Host Colonization and Infection

ABSTRACTMetal ions, in particular manganese, are important modulators of bacterial pathogenicity. However, little is known about the role of manganese-dependent proteins in the nosocomial pathogenEnterococcus faecalis, a major cause of bacterial endocarditis. The present study demonstrates that the DtxR/MntR family metalloregulator EfaR ofE. faecaliscontrols the expression of several of its regulon members in a manganese-dependent way. We also show thatefaRinactivation impairs the ability ofE. faecalisto form biofilms, to survive inside macrophages, and to tolerate oxidative stress. Our results reveal that EfaR is an important modulator ofE. faecalisvirulence and link manganese homeostasis to enterococcal pathogenicity.

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Fitness Restoration of a Genetically Tractable Enterococcus faecalis V583 Derivative To Study Decoration-Related Phenotypes of the Enterococcal Polysaccharide Antigen

mSphere ◽

10.1128/msphere.00310-19 ◽

2019 ◽

Vol 4 (4) ◽

Author(s):

Sylviane Furlan ◽

Renata C. Matos ◽

Sean P. Kennedy ◽

Benoît Doublet ◽

Pascale Serror ◽

...

Keyword(s):

Enterococcus Faecalis ◽

Reference Strain ◽

Variable Region ◽

Genetic System ◽

Host Adaptation ◽

Dna Rearrangements ◽

Content Type ◽

Significant Growth ◽

Derivative Strain

ABSTRACT Commensal and generally harmless in healthy individuals, Enterococcus faecalis causes opportunistic infections in immunocompromised patients. Plasmid-cured E. faecalis strain VE14089, derived from sequenced reference strain V583, is widely used for functional studies due to its improved genetic amenability. Although strain VE14089 has no major DNA rearrangements, with the exception of an ∼20-kb integrated region of pTEF1 plasmid, the strain presented significant growth differences from the V583 reference strain of our collection (renamed VE14002). In the present study, genome sequencing of strain VE14089 identified additional point mutations. Excision of the integrated pTEF1 plasmid region and sequential restoration of wild-type alleles showing nonsilent mutations were performed to obtain the VE18379 reference-derivative strain. Recovery of the growth ability of the restored VE18379 strain at a level similar to that seen with the reference strain points to GreA and Spx as bacterial fitness determinants. Virulence potential in Galleria mellonella and intestinal colonization in mouse demonstrated host adaptation of the VE18379 strain equivalent to VE14002 host adaptation. We further demonstrated that deletion of the 16.8-kb variable region of the epa locus recapitulates the key role of Epa decoration in host adaptation, providing a genetic system to study the role of specific epa-variable regions in host adaptation independently of other genetic variations. IMPORTANCE E. faecalis strain VE14089 was derived from V583 cured of its plasmids. Although VE14089 had no major DNA rearrangements, it presented significant growth and host adaptation differences from the reference strain V583 of our collection. To construct a strain with better fitness, we sequenced the genome of VE14089, identified single nucleotide polymorphisms (SNPs), and repaired the genes that could account for these changes. Using this reference-derivative strain, we provide a novel genetic system to understand the role of the variable region of epa in the enterococcal lifestyle.

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The Catabolite Repressor/Activator Cra Is a Bridge Connecting Carbon Metabolism and Host Colonization in the Plant Drought Resistance-Promoting Bacterium Pantoea alhagi LTYR-11Z

Applied and Environmental Microbiology ◽

10.1128/aem.00054-18 ◽

2018 ◽

Vol 84 (13) ◽

Cited By ~ 3

Author(s):

Lei Zhang ◽

Muhang Li ◽

Qiqi Li ◽

Chaoqiong Chen ◽

Meng Qu ◽

...

Keyword(s):

Carbon Source ◽

Carbon Metabolism ◽

Carbon Sources ◽

Endophytic Bacterium ◽

Bacterial Attachment ◽

Gene Encoding ◽

Wheat Roots ◽

Host Colonization ◽

Content Type

ABSTRACT Efficient root colonization is a prerequisite for application of plant growth-promoting (PGP) bacteria in improving health and yield of agricultural crops. We have recently identified an endophytic bacterium, Pantoea alhagi LTYR-11Z, with multiple PGP properties that effectively colonizes the root system of wheat and improves its growth and drought tolerance. To identify novel regulatory genes required for wheat colonization, we screened an LTYR-11Z transposon (Tn) insertion library and found cra to be a colonization-related gene. By using transcriptome (RNA-seq) analysis, we found that transcriptional levels of an eps operon, the ydiV gene encoding an anti-FlhD 4 C 2 factor, and the yedQ gene encoding an enzyme for synthesis of cyclic dimeric GMP (c-di-GMP) were significantly downregulated in the Δ cra mutant. Further studies demonstrated that Cra directly binds to the promoters of the eps operon, ydiV , and yedQ and activates their expression, thus inhibiting motility and promoting exopolysaccharide (EPS) production and biofilm formation. Consistent with previous findings that Cra plays a role in transcriptional regulation in response to carbon source availability, the activating effects of Cra were much more pronounced when LTYR-11Z was grown within a gluconeogenic environment than when it was grown within a glycolytic environment. We further demonstrate that the ability of LTYR-11Z to colonize wheat roots is modulated by the availability of carbon sources. Altogether, these results uncover a novel strategy utilized by LTYR-11Z to achieve host colonization in response to carbon nutrition in the environment, in which Cra bridges a connection between carbon metabolism and colonization capacity of LTYR-11Z. IMPORTANCE Rapid and appropriate response to environmental signals is crucial for bacteria to adapt to competitive environments and to establish interactions with their hosts. Efficient colonization and persistence within the host are controlled by various regulatory factors that respond to specific environmental cues. The most common is nutrient availability. In this work, we unraveled the pivotal role of Cra in regulation of colonization ability of Pantoea alhagi LTYR-11Z in response to carbon source availability. Moreover, we identified three novel members of the Cra regulon involved in EPS synthesis, regulation of flagellar biosynthesis, and synthesis of c-di-GMP and propose a working model to explain the Cra-mediated regulatory mechanism that links carbon metabolism to host colonization. This study elucidates the regulatory role of Cra in bacterial attachment and colonization of plants, which raises the possibility of extending our studies to other bacteria associated with plant and human health.

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Effects of Oxygen Availability on Acetic Acid Tolerance and Intracellular pH in Dekkera bruxellensis

Applied and Environmental Microbiology ◽

10.1128/aem.00515-16 ◽

2016 ◽

Vol 82 (15) ◽

pp. 4673-4681 ◽

Cited By ~ 6

Author(s):

Claudia Capusoni ◽

Stefania Arioli ◽

Paolo Zambelli ◽

M. Moktaduzzaman ◽

Diego Mora ◽

...

Keyword(s):

Oxidative Stress ◽

Acetic Acid ◽

Intracellular Ph ◽

Acid Tolerance ◽

Oxygen Availability ◽

Industrial Processes ◽

Dekkera Bruxellensis ◽

Acetic Acid Tolerance ◽

Content Type

ABSTRACTThe yeastDekkera bruxellensis, associated with wine and beer production, has recently received attention, because its high ethanol and acid tolerance enables it to compete withSaccharomyces cerevisiaein distilleries that produce fuel ethanol. We investigated how different cultivation conditions affect the acetic acid tolerance ofD. bruxellensis. We analyzed the ability of two strains (CBS 98 and CBS 4482) exhibiting different degrees of tolerance to grow in the presence of acetic acid under aerobic and oxygen-limited conditions. We found that the concomitant presence of acetic acid and oxygen had a negative effect onD. bruxellensisgrowth. In contrast, incubation under oxygen-limited conditions resulted in reproducible growth kinetics that exhibited a shorter adaptive phase and higher growth rates than those with cultivation under aerobic conditions. This positive effect was more pronounced in CBS 98, the more-sensitive strain. Cultivation of CBS 98 cells under oxygen-limited conditions improved their ability to restore their intracellular pH upon acetic acid exposure and to reduce the oxidative damage to intracellular macromolecules caused by the presence of acetic acid. This study reveals an important role of oxidative stress in acetic acid tolerance inD. bruxellensis, indicating that reduced oxygen availability can protect against the damage caused by the presence of acetic acid. This aspect is important for optimizing industrial processes performed in the presence of acetic acid.IMPORTANCEThis study reveals an important role of oxidative stress in acetic acid tolerance inD. bruxellensis, indicating that reduced oxygen availability can have a protective role against the damage caused by the presence of acetic acid. This aspect is important for the optimization of industrial processes performed in the presence of acetic acid.

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Observations on the Role of TcdE Isoforms in Clostridium difficile Toxin Secretion

Journal of Bacteriology ◽

10.1128/jb.00224-15 ◽

2015 ◽

Vol 197 (15) ◽

pp. 2600-2609 ◽

Cited By ~ 20

Author(s):

Revathi Govind ◽

Leah Fitzwater ◽

Rebekah Nichols

Keyword(s):

Amino Acids ◽

Clostridium Difficile ◽

Cell Viability ◽

Virulence Factors ◽

Start Codon ◽

Nosocomial Pathogen ◽

Content Type ◽

Antibiotic Associated Diarrhea ◽

Toxin Secretion

ABSTRACTClostridium difficileis a major nosocomial pathogen and the principal causative agent of antibiotic-associated diarrhea. The toxigenicC. difficilestrains that cause disease secrete virulence factors, toxin A and toxin B, that cause colonic injury and inflammation.C. difficiletoxins have no export signature and are secreted by an unusual mechanism that involves TcdE, a holin-like protein. We isolated a TcdE mutant of the epidemic R20291 strain with impaired toxin secretion, which was restored by complementation with functional TcdE. In the TcdE open reading frame (ORF), we identified three possible translation start sites; each translated isoform may play a specific role in TcdE-controlled toxin release. We created plasmid constructs that express only one of the three TcdE isoforms and complemented the TcdE mutant with these isoforms. Western blot analysis of the complemented strains demonstrated that TcdE is translated efficiently from the start codon at the 25th and 27th positions in the predicted ORF, producing proteins with 142 amino acids (TcdE142) and 140 amino acids (TcdE140), respectively. TcdE166was not detected when expressed from its own ribosomal binding site (RBS). The effects of all three TcdE isoforms onC. difficilecell viability and toxin release were determined. Among the three isoforms, overexpression of TcdE166and TcdE142had a profound effect on cell viability compared to the TcdE140isoform. Similarly, TcdE166and TcdE142facilitated toxin release more efficiently than did TcdE140. The importance of these variations among TcdE isoforms and their role in toxin release are discussed.IMPORTANCEC. difficileis a nosocomial pathogen that has become the most prevalent cause of antibiotic-associated diarrhea in North America and in several countries in Europe. Most strains ofC. difficileproduce two high-molecular-weight toxins that are regarded as the primary virulence factors. The mechanism by which these large toxins are secreted from bacterial cells is not yet clear but involves TcdE, a holin-like protein. In this work, we show that TcdE could be translated from three different start codons, resulting in the production of three TcdE isoforms. Furthermore, we investigated the role of these isoforms in toxin release and cell lysis inC. difficile. An understanding of TcdE-dependent toxin secretion may be helpful for the development of strategies for preventing and treatingC. difficileinfections.

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SlyA Is a Transcriptional Regulator Involved in the Virulence of Enterococcus faecalis

Infection and Immunity ◽

10.1128/iai.01132-10 ◽

2011 ◽

Vol 79 (7) ◽

pp. 2638-2645 ◽

Cited By ~ 39

Author(s):

Charlotte Michaux ◽

Maurizio Sanguinetti ◽

Fany Reffuveille ◽

Yanick Auffray ◽

Brunella Posteraro ◽

...

Keyword(s):

Enterococcus Faecalis ◽

Galleria Mellonella ◽

Peritoneal Macrophages ◽

Bacterial Virulence ◽

Transcriptional Analysis ◽

Infection Model ◽

Wild Type ◽

Content Type ◽

Wild Type Parent

ABSTRACTPhylogenetic analysis of the crystal structure of theEnterococcus faecalisSlyA (EF_3002) transcriptional factor places it between the SlyA and MarR regulator subfamilies. Proteins of these families are often involved in the regulation of genes important for bacterial virulence and stress response. To gather evidence for the role of this putative regulator inE. faecalisbiology, we dissected the genetic organization of theslyA-EF_3001 locus and constructed aslyAdeletion mutant as well as complemented strains. Interestingly, compared to the wild-type parent, the ΔslyAmutant is more virulent in an insect infection model (Galleria mellonella), exhibits increased persistence in mouse kidneys and liver, and survives better inside peritoneal macrophages. In order to identify a possible SlyA regulon, global microarray transcriptional analysis was performed. This study revealed that theslyA-EF_3001 locus appears to be autoregulated and that 117 genes were differentially regulated in the ΔslyAmutant. In the mutant strain, 111 were underexpressed and 6 overexpressed, indicating that SlyA functions mainly as an activator of transcription.

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Sigma S-Dependent Antioxidant Defense Protects Stationary-Phase Escherichia coli against the Bactericidal Antibiotic Gentamicin

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.03683-14 ◽

2014 ◽

Vol 58 (10) ◽

pp. 5964-5975 ◽

Cited By ~ 20

Author(s):

Jing-Hung Wang ◽

Rachna Singh ◽

Michael Benoit ◽

Mimi Keyhan ◽

Matthew Sylvester ◽

...

Keyword(s):

Oxidative Stress ◽

Escherichia Coli ◽

Stationary Phase ◽

Antioxidant Defense ◽

Physiological State ◽

Pentose Phosphate ◽

Phase Cell ◽

Content Type

ABSTRACTStationary-phase bacteria are important in disease. The σs-regulated general stress response helps them become resistant to disinfectants, but the role of σsin bacterial antibiotic resistance has not been elucidated. Loss of σsrendered stationary-phaseEscherichia colimore sensitive to the bactericidal antibiotic gentamicin (Gm), and proteomic analysis suggested involvement of a weakened antioxidant defense. Use of the psfiAgenetic reporter, 3′-(p-hydroxyphenyl) fluorescein (HPF) dye, and Amplex Red showed that Gm generated more reactive oxygen species (ROS) in the mutant. HPF measurements can be distorted by cell elongation, but Gm did not affect stationary-phase cell dimensions. Coadministration of the antioxidantN-acetyl cysteine (NAC) decreased drug lethality particularly in the mutant, as did Gm treatment under anaerobic conditions that prevent ROS formation. Greater oxidative stress, due to insufficient quenching of endogenous ROS and/or respiration-linked electron leakage, therefore contributed to the greater sensitivity of the mutant; infection by a uropathogenic strain in mice showed this to be the case alsoin vivo. Disruption of antioxidant defense by eliminating the quencher proteins, SodA/SodB and KatE/SodA, or the pentose phosphate pathway proteins, Zwf/Gnd and TalA, which provide NADPH for ROS decomposition, also generated greater oxidative stress and killing by Gm. Thus, besides its established mode of action, Gm also kills stationary-phase bacteria by generating oxidative stress, and targeting the antioxidant defense ofE. colican enhance its efficacy. Relevant aspects of the current controversy on the role of ROS in killing by bactericidal drugs of exponential-phase bacteria, which represent a different physiological state, are discussed.

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Role of the Filifactor alocis Hypothetical Protein FA519 in Oxidative Stress Resistance

Microbiology Spectrum ◽

10.1128/spectrum.01212-21 ◽

2021 ◽

Author(s):

Ezinne Aja ◽

Arunima Mishra ◽

Yuetan Dou ◽

Hansel M. Fletcher

Keyword(s):

Oxidative Stress ◽

Periodontal Disease ◽

Stress Resistance ◽

Hypothetical Protein ◽

Content Type ◽

Genetic Tools ◽

Oxidative Stress Resistance ◽

Filifactor Alocis ◽

Diagnostic Indicator

Filifactor alocis is an emerging member of the periodontal community and is now proposed to be a diagnostic indicator of periodontal disease. However, due to the lack of genetic tools available to study this organism, not much is known about its virulence attributes.

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Inhibition of the Classical Pathway of Complement Activation Impairs Bacterial Clearance during Enterococcus faecalis Infection

Infection and Immunity ◽

10.1128/iai.00660-20 ◽

2021 ◽

Vol 89 (5) ◽

Author(s):

Eman M. Rabie Shehab El-Din ◽

Abdelaziz Elgaml ◽

Youssif M. Ali ◽

Ramadan Hassan

Keyword(s):

Enterococcus Faecalis ◽

Microbial Pathogens ◽

Health Concern ◽

Classical Pathway ◽

Bacterial Clearance ◽

Limited Information ◽

Content Type ◽

Fab Fragments

ABSTRACT Enterococcus faecalis infections are considered a major public health concern worldwide. The complement system has a crucial role in the protection against different microbial pathogens, including E. faecalis. Complement can be activated through three different pathways, including the classical, lectin, and alternative pathways. There is limited information on the role of the classical pathway (CP) in protection against infections caused by E. faecalis. In the present study, we generated Fab fragments that successfully block the CP in mouse via inhibition of a key enzyme, C1s-A. Our results showed that anti-C1s-A Fab fragments block CP-mediated C3b and C4b deposition in vitro. We further showed that administration of anti-C1s-A Fab fragments significantly impairs the CP functional activity in vivo. Moreover, treatment of mice infected with E. faecalis using anti-C1s-A Fab fragments significantly impairs bacterial clearance as determined from the viable bacterial counts recovered from blood, kidneys, spleens, livers, and lungs of infected mice. Overall, this study highlights the essential role of the CP in host defense against E. faecalis.

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Capsaicin, a Powerful •OH-Inactivating Ligand

Antioxidants ◽

10.3390/antiox9121247 ◽

2020 ◽

Vol 9 (12) ◽

pp. 1247

Author(s):

Adriana Pérez-González ◽

Mario Prejanò ◽

Nino Russo ◽

Tiziana Marino ◽

Annia Galano

Keyword(s):

Oxidative Stress ◽

Density Functional Theory ◽

Metal Ions ◽

Density Functional ◽

Functional Theory ◽

Diffusion Limited

Oxidative conditions are frequently enhanced by the presence of redox metal ions. In this study, the role of capsaicin (8-methyl-N-vanillyl-6-nonenamide, CAP) in copper-induced oxidative stress was investigated using density functional theory simulations. It was found that CAP has the capability to chelate Cu(II), leading to complexes that are harder to reduce than free Cu(II). CAP fully turns off the Cu(II) reduction by Asc−, and slows down the reduction in this cation by O2•−. Therefore, CAP is proposed as an •OH-inactivating ligand by impeding the reduction in metal ions (OIL-1), hindering the production of •OH via Fenton-like reactions, at physiological pH. CAP is also predicted to be an excellent antioxidant as a scavenger of •OH, yielded through Fenton-like reactions (OIL-2). The reactions between CAP-Cu(II) chelates and •OH were estimated to be diffusion-limited. Thus, these chelates are capable of deactivating this dangerous radical immediately after being formed by Fenton-like reactions.

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Ergothioneine Is a Secreted Antioxidant in Mycobacterium smegmatis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02572-12 ◽

2013 ◽

Vol 57 (7) ◽

pp. 3202-3207 ◽

Cited By ~ 47

Author(s):

Carine Sao Emani ◽

Monique J. Williams ◽

Ian J. Wiid ◽

Nicholas F. Hiten ◽

Albertus J. Viljoen ◽

...

Keyword(s):

Oxidative Stress ◽

Mycobacterium Smegmatis ◽

Double Mutant ◽

Culture Media ◽

Growth Cycle ◽

Low Molecular Weight ◽

Deficient Mutant ◽

Wild Type ◽

Content Type

ABSTRACTErgothioneine (ERG) and mycothiol (MSH) are two low-molecular-weight thiols synthesized by mycobacteria. The role of MSH has been extensively investigated in mycobacteria; however, little is known about the role of ERG in mycobacterial physiology. In this study, quantification of ERG at various points in the growth cycle ofMycobacterium smegmatisrevealed that a significant portion of ERG is found in the culture media, suggesting that it is actively secreted. A mutant ofM. smegmatislackingegtD(MSMEG_6247) was unable to synthesize ERG, confirming its role in ERG biosynthesis. Deletion ofegtDfrom wild-typeM. smegmatisand an MSH-deficient mutant did not affect their susceptibility to antibiotics tested in this study. The ERG- and MSH-deficient double mutant was significantly more sensitive to peroxide than either of the single mutants lacking either ERG or MSH, suggesting that both thiols play a role in protectingM. smegmatisagainst oxidative stress and that ERG is able to partly compensate for the loss of MSH.

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