Enterococcus faecalis Manganese Exporter MntE Alleviates Manganese Toxicity and Is Required for Mouse Gastrointestinal Colonization

ABSTRACT Bacterial pathogens encounter a variety of nutritional environments in the human host, including nutrient metal restriction and overload. Uptake of manganese (Mn) is essential for Enterococcus faecalis growth and virulence; however, it is not known how this organism prevents Mn toxicity. In this study, we examine the role of the highly conserved MntE transporter in E. faecalis Mn homeostasis and virulence. We show that inactivation of mntE results in growth restriction in the presence of excess Mn, but not other metals, demonstrating its specific role in Mn detoxification. Upon growth in the presence of excess Mn, an mntE mutant accumulates intracellular Mn, iron (Fe), and magnesium (Mg), supporting a role for MntE in Mn and Fe export and a role for Mg in offsetting Mn toxicity. Growth of the mntE mutant in excess Fe also results in increased levels of intracellular Fe, but not Mn or Mg, providing further support for MntE in Fe efflux. Inactivation of mntE in the presence of excess iron also results in the upregulation of glycerol catabolic genes and enhanced biofilm growth, and addition of glycerol is sufficient to augment biofilm growth for both the mntE mutant and its wild-type parental strain, demonstrating that glycerol availability significantly enhances biofilm formation. Finally, we show that mntE contributes to colonization of the antibiotic-treated mouse gastrointestinal (GI) tract, suggesting that E. faecalis encounters excess Mn in this niche. Collectively, these findings demonstrate that the manganese exporter MntE plays a crucial role in E. faecalis metal homeostasis and virulence.

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Enterococcus faecalis manganese exporter MntE alleviates manganese toxicity and is required for mouse gastrointestinal colonization

10.1101/2020.02.06.936633 ◽

2020 ◽

Author(s):

Ling Ning Lam ◽

Jun Jie Wong ◽

Kelvin Kian Long Chong ◽

Kimberly A. Kline

Keyword(s):

Enterococcus Faecalis ◽

Wild Type ◽

Gi Tract ◽

Biofilm Growth ◽

Mn Toxicity ◽

Excess Iron ◽

Catabolic Genes ◽

Gastrointestinal Colonization ◽

Excess Fe

AbstractBacterial pathogens encounter a variety of nutritional environments in the human host, including nutrient metal restriction and overload. Uptake of manganese (Mn) is essential for Enterococcus faecalis growth and virulence; however, it is not known how this organism prevents Mn toxicity. In this study, we examine the role of the highly conserved MntE transporter in E. faecalis Mn homeostasis and virulence. We show that inactivation of mntE results in growth restriction in the presence of excess Mn, but not other metals, demonstrating its specific role in Mn detoxification. Upon growth in the presence of excess Mn, an mntE mutant accumulates intracellular Mn, iron (Fe), and magnesium (Mg), supporting a role for MntE in Mn and Fe export, and a role for Mg in offsetting Mn toxicity. Growth of the mntE mutant in excess Fe also results in increased levels of intracellular Fe, but not Mn or Mg, providing further support for MntE in Fe efflux. Inactivation of mntE in the presence of excess iron also results in the upregulation of glycerol catabolic genes and enhanced biofilm growth, and addition of glycerol is sufficient to augment biofilm growth for both the mntE mutant and its wild type parental strain, demonstrating that glycerol availability significantly enhances biofilm formation. Finally, we show that mntE contributes to infection of the antibiotic-treated mouse gastrointestinal (GI) tract, suggesting that E. faecalis encounters excess Mn in this niche. Collectively, these findings demonstrate that the manganese exporter MntE plays a crucial role in E. faecalis metal homeostasis and virulence.

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Effects of Biofilm Growth on Plasmid Copy Number and Expression of Antibiotic Resistance Genes in Enterococcus faecalis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02010-12 ◽

2013 ◽

Vol 57 (4) ◽

pp. 1850-1856 ◽

Cited By ~ 17

Author(s):

L. C. Cook ◽

G. M. Dunny

Keyword(s):

Antibiotic Resistance ◽

Enterococcus Faecalis ◽

Resistance Genes ◽

Copy Number ◽

Antibiotic Resistance Genes ◽

Plasmid Copy Number ◽

Plasmid Copy ◽

Rapid Reduction ◽

Biofilm Growth ◽

Content Type

ABSTRACTBiofilm growth causes increased average plasmid copy number as well as increased copy number heterogeneity inEnterococcus faecaliscells carrying plasmid pCF10. In this study, we examined whether biofilm growth affected the copy number and expression of antibiotic resistance determinants for several plasmids with diverse replication systems. Four differentE. faecalisplasmids, unrelated to pCF10, demonstrated increased copy number in biofilm cells. In biofilm cells, we also observed increased transcription of antibiotic resistance genes present on these plasmids. The increase in plasmid copy number correlated with increased plating efficiency on high concentrations of antibiotics. Single-cell analysis of strains carrying two different plasmids suggested that the increase in plasmid copy number associated with biofilm growth was restricted to a subpopulation of biofilm cells. Regrowth of harvested biofilm cells in liquid culture resulted in a rapid reduction of plasmid copy number to that observed in the planktonic state. These results suggest a possible mechanism by which biofilm growth could reduce susceptibility to antibiotics in clinical settings.

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Loss of Ethanolamine Utilization in Enterococcus faecalis Increases Gastrointestinal Tract Colonization

mBio ◽

10.1128/mbio.00790-18 ◽

2018 ◽

Vol 9 (3) ◽

Cited By ~ 8

Author(s):

Karan Gautam Kaval ◽

Kavindra V. Singh ◽

Melissa R. Cruz ◽

Sruti DebRoy ◽

Wade C. Winkler ◽

...

Keyword(s):

Enterococcus Faecalis ◽

Opposite Effect ◽

Wild Type Strain ◽

Food Poisoning ◽

Wild Type ◽

Gi Tract ◽

Human Gut ◽

Content Type ◽

Serovar Typhimurium ◽

High Concentrations

ABSTRACT Enterococcus faecalis is paradoxically a dangerous nosocomial pathogen and a normal constituent of the human gut microbiome, an environment rich in ethanolamine. E. faecalis carries the eut (ethanolamine utilization) genes, which enable the catabolism of ethanolamine (EA) as a valuable source of carbon and/or nitrogen. EA catabolism was previously shown to contribute to the colonization and growth of enteric pathogens, such as Salmonella enterica serovar Typhimurium and enterohemorrhagic Escherichia coli (EHEC), in the gut environment. We tested the ability of eut mutants of E. faecalis to colonize the gut using a murine model of gastrointestinal (GI) tract competition and report the surprising observation that these mutants outcompete the wild-type strain. IMPORTANCE Some bacteria that are normal, harmless colonizers of the human body can cause disease in immunocompromised patients, particularly those that have been heavily treated with antibiotics. Therefore, it is important to understand the factors that promote or negate these organisms’ ability to colonize. Previously, ethanolamine, found in high concentrations in the GI tract, was shown to promote the colonization and growth of bacteria associated with food poisoning. Here, we report the surprising, opposite effect of ethanolamine utilization on the commensal colonizer E. faecalis , namely, that loss of this metabolic capacity made it a better colonizer.

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Ethanolamine Utilization and Bacterial Microcompartment Formation Are Subject to Carbon Catabolite Repression

Journal of Bacteriology ◽

10.1128/jb.00703-18 ◽

2019 ◽

Vol 201 (10) ◽

Cited By ~ 2

Author(s):

Karan Gautam Kaval ◽

Margo Gebbie ◽

Jonathan R. Goodson ◽

Melissa R. Cruz ◽

Wade C. Winkler ◽

...

Keyword(s):

Gene Expression ◽

Enterococcus Faecalis ◽

Small Rna ◽

Catabolite Repression ◽

Carbon Catabolite Repression ◽

Bioinformatics Analysis ◽

Maximum Efficiency ◽

Gi Tract ◽

Content Type ◽

Regulatory Factors

ABSTRACT Ethanolamine (EA) is a compound prevalent in the gastrointestinal (GI) tract that can be used as a carbon, nitrogen, and/or energy source. Enterococcus faecalis, a GI commensal and opportunistic pathogen, contains approximately 20 ethanolamine utilization (eut) genes encoding the necessary regulatory, enzymatic, and structural proteins for this process. Here, using a chemically defined medium, two regulatory factors that affect EA utilization were examined. First, the functional consequences of loss of the small RNA (sRNA) EutX on the efficacy of EA utilization were investigated. One effect observed, as loss of this negative regulator causes an increase in eut gene expression, was a concomitant increase in the number of catabolic bacterial microcompartments (BMCs) formed. However, despite this increase, the growth of the strain was repressed, suggesting that the overall efficacy of EA utilization was negatively affected. Second, utilizing a deletion mutant and a complement, carbon catabolite control protein A (CcpA) was shown to be responsible for the repression of EA utilization in the presence of glucose. A predicted cre site in one of the three EA-inducible promoters, PeutS, was identified as the target of CcpA. However, CcpA was shown to affect the activation of all the promoters indirectly through the two-component system EutV and EutW, whose genes are under the control of the PeutS promoter. Moreover, a bioinformatics analysis of bacteria predicted to contain CcpA and cre sites revealed that a preponderance of BMC-containing operons are likely regulated by carbon catabolite repression (CCR). IMPORTANCE Ethanolamine (EA) is a compound commonly found in the gastrointestinal (GI) tract that can affect the behavior of human pathogens that can sense and utilize it, such as Enterococcus faecalis and Salmonella. Therefore, it is important to understand how the genes that govern EA utilization are regulated. In this work, we investigated two regulatory factors that control this process. One factor, a small RNA (sRNA), is shown to be important for generating the right levels of gene expression for maximum efficiency. The second factor, a transcriptional repressor, is important for preventing expression when other preferred sources of energy are available. Furthermore, a global bioinformatics analysis revealed that this second mechanism of transcriptional regulation likely operates on similar genes in related bacteria.

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

Infection and Immunity ◽

10.1128/iai.06377-11 ◽

2013 ◽

Vol 81 (3) ◽

pp. 935-944 ◽

Cited By ~ 15

Author(s):

M. C. Abrantes ◽

J. Kok ◽

M. de F. Lopes

Keyword(s):

Oxidative Stress ◽

Metal Ions ◽

Enterococcus Faecalis ◽

Bacterial Endocarditis ◽

Nosocomial Pathogen ◽

Host Colonization ◽

Content Type ◽

Bacterial Pathogenicity

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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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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Candida albicans and Bacterial Microbiota Interactions in the Cecum during Recolonization following Broad-Spectrum Antibiotic Therapy

Infection and Immunity ◽

10.1128/iai.00449-12 ◽

2012 ◽

Vol 80 (10) ◽

pp. 3371-3380 ◽

Cited By ~ 126

Author(s):

Katie L. Mason ◽

John R. Erb Downward ◽

Kelly D. Mason ◽

Nicole R. Falkowski ◽

Kathryn A. Eaton ◽

...

Keyword(s):

Candida Albicans ◽

Bacterial Diversity ◽

Vital Role ◽

Gi Tract ◽

Content Type ◽

Healthy Humans ◽

Life Threatening Illness ◽

Bacterial Microbiota

ABSTRACTCandida albicansis a normal member of the gastrointestinal (GI) tract microbiota of healthy humans, but during host immunosuppression or alterations in the bacterial microbiota,C. albicanscan disseminate and cause life-threatening illness. The bacterial microbiome of the GI tract, including lactic acid bacteria (LAB), plays a vital role in preventing fungal invasion. However, little is known about the role ofC. albicansin shaping the bacterial microbiota during antibiotic recovery. We investigated the fungal burdens in the GI tracts of germfree mice and mice with a disturbed microbiome to demonstrate the role of the microbiota in preventingC. albicanscolonization. Histological analysis demonstrated that colonization withC. albicansduring antibiotic treatment does not trigger overt inflammation in the murine cecum. Bacterial diversity is reduced long term following cefoperazone treatment, but the presence ofC. albicansduring antibiotic recovery promoted the recovery of bacterial diversity. Cefoperazone diminishesBacteroidetespopulations long term in the ceca of mice, but the presence ofC. albicansduring cefoperazone recovery promotedBacteroidetespopulation recovery. However, the presence ofC. albicansresulted in a long-term reduction inLactobacillusspp. and promotedEnterococcus faecalispopulations. Previous studies have focused on the ability of bacteria to alterC. albicans; this study addresses the ability ofC. albicansto alter the bacterial microbiota during nonpathogenic colonization.

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From Commensal to Pathogen: Translocation of Enterococcus faecalis from the Midgut to the Hemocoel of Manduca sexta

mBio ◽

10.1128/mbio.00065-11 ◽

2011 ◽

Vol 2 (3) ◽

Cited By ~ 68

Author(s):

Katie L. Mason ◽

Taylor A. Stepien ◽

Jessamina E. Blum ◽

Jonathan F. Holt ◽

Normand H. Labbe ◽

...

Keyword(s):

Immune Response ◽

Gastrointestinal Tract ◽

Bacillus Thuringiensis ◽

Mortality Rate ◽

Enterococcus Faecalis ◽

Manduca Sexta ◽

Insecticidal Toxin ◽

Content Type ◽

Robust Immune Response

ABSTRACTA dynamic homeostasis is maintained between the host and native bacteria of the gastrointestinal tract in animals, but migration of bacteria from the gut to other organs can lead to disease or death.Enterococcus faecalisis a commensal of the gastrointestinal tract; however,Enterococcusspp. are increasingly frequent causes of nosocomial infections with a high mortality rate. We investigated the commensal-to-pathogen switch undergone byE. faecalisOG1RF in the lepidopteran model hostManduca sextaassociated with its location in the host.E. faecalispersists in the harsh midgut environment ofM. sextalarvae without causing apparent illness, but injection ofE. faecalisdirectly into the larval hemocoel is followed by rapid death. Additionally, oral ingestion ofE. faecalisin the presence ofBacillus thuringiensisinsecticidal toxin, a pore-forming toxin that targets the midgut epithelium, induces an elevated mortality rate. We show that the loss of gut integrity due toB. thuringiensistoxin correlates with the translocation ofE. faecalisfrom the gastrointestinal tract into the hemolymph. Upon gaining access to the hemolymph,E. faecalisinduces an innate immune response, illustrated by hemocyte aggregation, in larvae prior to death. The degree of hemocyte aggregation is dependent upon the route ofE. faecalisentry. Our data demonstrate the efficacy of theM. sextalarval model system in investigatingE. faecalis-induced sepsis and clarifies controversies in the field regarding the events leading to larval death followingB. thuringiensistoxin exposure.IMPORTANCEThis study advances our knowledge ofEnterococcus faecalis-induced sepsis following translocation from the gut and provides a model for mammalian diseases in which the spatial distribution of bacteria determines disease outcomes. We demonstrate thatE. faecalisis a commensal in the gut ofManduca sextaand a pathogen in the hemocoel, resulting in a robust immune response and rapid death, a process we refer to as the “commensal-to-pathogen” switch. While controversy remains regardingBacillus thuringiensistoxin-induced killing, our laboratory previously found that under some conditions, the midgut microbiota is essential forB. thuringiensistoxin killing ofLymantria dispar(N. A. Broderick, K. F. Raffa, and J. Handelsman, Proc. Natl. Acad. Sci. U. S. A. 103:15196–15199, 2006; B. Raymond, et al., Environ. Microbiol. 11:2556–2563, 2009; P. R. Johnston, and N. Crickmore, Appl. Environ. Microbiol. 75:5094–5099, 2009). We and others have demonstrated that the role of the midgut microbiota inB. thuringiensistoxin killing is dependent upon the lepidopteran species and formulation ofB. thuringiensistoxin (N. A. Broderick, K. F. Raffa, and J. Handelsman, Proc. Natl. Acad. Sci. U. S. A. 103:15196–15199, 2006; N. A. Broderick, et al., BMC Biol. 7:11, 2009). This work reconciles much of the apparently contradictory previous data and reveals that theM. sexta-E. faecalissystem provides a model for mammalian sepsis.

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