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The AdcACB/AdcAII system is Essential for Zinc Homeostasis and an Important Contributor of Enterococcus faecalis Virulence

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Lam, Ling Ning, Brunson, Debra N., Molina, Jonathan J., Flores-Mireles, Ana L., Lemos, José A.·2021·Virulence
DOI 10.1101/2021.12.01.470831PMC PMC8966984

Preprint — published as 10.1080/21505594.2022.2056965

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Abstract

Bacterial pathogens require a variety of micronutrients for growth, including trace metals such as iron, manganese, and zinc (Zn). Despite their relative abundance in host environments, access to these metals is severely restricted during infection due to host-mediated defense mechanisms collectively known as nutritional immunity. Despite a growing appreciation of the importance of Zn in host-pathogen interactions, the mechanisms of Zn homeostasis and the significance of Zn to the pathophysiology of E. faecalis , a major pathogen of nosocomial and community-associated infections, have not been investigated. Here, we show that E. faecalis encoded an ABC-type transporter AdcACB and an orphan substrate-binding lipoprotein AdcAII that work cooperatively to maintain Zn homeostasis. Simultaneous inactivation of adcA and adcAII or the entire adcACB operon led to significant reduction in intracellular Zn under Zn-restricted conditions, heightened sensitivity to Zn-chelating agents including human calprotectin, aberrant cell morphology, and impaired fitness in serum ex vivo . Additionally, inactivation of adcACB and adcAII significantly reduced bacterial tolerance towards cell envelope-targeting antibiotics, which may be associated to altered fatty acid abundance and species. Lastly, we show that the AdcACB/AdcAII system contributes to E. faecalis virulence in an invertebrate ( Galleria mellonella ) infection model and in two catheter-associated mouse infection models that recapitulate many of the host conditions associated with enterococcal human infections. Collectively, this report reveals that high-affinity Zn import is essential for the pathogenesis of E. faecalis indicating that the surface-associated AdcA and AdcAII lipoproteins are potential therapeutic targets.

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Figure 1. Growth characteristics of E. faecalis and its Zn-deficient mutants under Zn-restricted conditions. (a) Schematic of the gene locus of the Zn transport system in E. faecalis OG1RF core genome. Growth curves of E. faecalis wild type (WT) and its isogenic mutants in BHI (b), BHI supplemented with 100 µmZnSo4 (c), 10 µm TPEN (d), combination of TPEN and ZnSo4 (e), TPEN and MnSo4 (f) or TPEN and Fe SO4(g). In (b) and (d-f), data points represent the average of nine biological replicates. Finally, the growth curve of E. faecalis wild type (WT) and genetically complemented ∆adc mutants in BHI supplemented with 10 µm TPEN (h). In (c) and (g), data points represent the average of six biological replicates. Error bar represents the standard error of margin (SEM). Statistical analysis was performed using simple linear regression ofthe exponential growth phase, and slope of each mutant’s growth kinetics was compared with that of the parent strain.
Figure 1. Growth characteristics of E. faecalis and its Zn-deficient mutants under Zn-restricted conditions. (a) Schematic of the gene locus of the Zn transport system in E. faecalis OG1RF core genome. Growth curves of E. faecalis wild type (WT) and its isogenic mutants in BHI (b), BHI supplemented with 100 µmZnSo4 (c), 10 µm TPEN (d), combination of TPEN and ZnSo4 (e), TPEN and MnSo4 (f) or TPEN and Fe SO4(g). In (b) and (d-f), data points represent the average of nine biological replicates. Finally, the growth curve of E. faecalis wild type (WT) and genetically complemented ∆adc mutants in BHI supplemented with 10 µm TPEN (h). In (c) and (g), data points represent the average of six biological replicates. Error bar represents the standard error of margin (SEM). Statistical analysis was performed using simple linear regression ofthe exponential growth phase, and slope of each mutant’s growth kinetics was compared with that of the parent strain.CC BY · PubMed Central
Figure 2. Growth characteristics of E. faecalis and its Zn-deficient mutants in the presence of calprotectin. Growth curves of E. faecalis OG1RF WT and its isogenic mutants in BHI supplemented with CP buffer media (a) and 150 µg ml −1 of WT hCP (b) or hCp∆mn-tail (c). Data points represent the average, and error bar represents the standard error of margin (SEM) of at least six biological replicates. Statistical analysis was performed using simple linear regression of exponential growth phase, and the slope of each mutant’s growth kinetics was compared with that of the parent strain.
Figure 2. Growth characteristics of E. faecalis and its Zn-deficient mutants in the presence of calprotectin. Growth curves of E. faecalis OG1RF WT and its isogenic mutants in BHI supplemented with CP buffer media (a) and 150 µg ml −1 of WT hCP (b) or hCp∆mn-tail (c). Data points represent the average, and error bar represents the standard error of margin (SEM) of at least six biological replicates. Statistical analysis was performed using simple linear regression of exponential growth phase, and the slope of each mutant’s growth kinetics was compared with that of the parent strain.CC BY · PubMed Central
Figure 3. Intracellular Zn quantification and transcriptional profiles of E. faecalis and its Zn-deficient mutants. (a) ICP-OES quantifications of intracellular Zn of mid-log grown E. faecalis OG1RF WT and derivatives grown in BHI and BHI supplemented with 7.5 µm TPEN. Data points represent five biological replicates. Statistical analysis was performed using two-way ANOVA with Dunnett’s multiple comparison test. ** p ≤0.01, *** p ≤0.001, and **** p ≤0.0001. (b) Comparison of reversed transcribed cDNA copy of adcA and adcAII in E. faecalis OG1RF and the ∆zur mutant grown for 1 hour in BHI, BHI with 30 µm TPEN, and BHI with 4 mM ZnSo4. Data points represent the average of six biological replicates. Statistical analysis was performed using the unpaired t-test with Welch’s correction. * p ≤.05. Error bars represent the standard error of margin (SEM).
Figure 3. Intracellular Zn quantification and transcriptional profiles of E. faecalis and its Zn-deficient mutants. (a) ICP-OES quantifications of intracellular Zn of mid-log grown E. faecalis OG1RF WT and derivatives grown in BHI and BHI supplemented with 7.5 µm TPEN. Data points represent five biological replicates. Statistical analysis was performed using two-way ANOVA with Dunnett’s multiple comparison test. ** p ≤0.01, *** p ≤0.001, and **** p ≤0.0001. (b) Comparison of reversed transcribed cDNA copy of adcA and adcAII in E. faecalis OG1RF and the ∆zur mutant grown for 1 hour in BHI, BHI with 30 µm TPEN, and BHI with 4 mM ZnSo4. Data points represent the average of six biological replicates. Statistical analysis was performed using the unpaired t-test with Welch’s correction. * p ≤.05. Error bars represent the standard error of margin (SEM).CC BY · PubMed Central
Figure 4. Growth and survival of E. faecalis in serum and urine. Colony-forming units (CFU) of E. faecalis OG1RF WT and its mutants incubated in pooled (a) human serum or (b) serum supplemented with 500 µm ZnSo4 and pooled human urine (d). (c) CFU counts of E. faecalis OG1RF WT, its mutants, and genetic complemented mutants after 24 hours of incubation in pooled human serum. In (a-d), data points represent the average and error bar represents the standard error of margin (SEM) of nine biological replicates. Statistical analysis was performed using one-way ANOVA with Welch’s correction. *** p ≤0.001 and **** p ≤0.0001.
Figure 4. Growth and survival of E. faecalis in serum and urine. Colony-forming units (CFU) of E. faecalis OG1RF WT and its mutants incubated in pooled (a) human serum or (b) serum supplemented with 500 µm ZnSo4 and pooled human urine (d). (c) CFU counts of E. faecalis OG1RF WT, its mutants, and genetic complemented mutants after 24 hours of incubation in pooled human serum. In (a-d), data points represent the average and error bar represents the standard error of margin (SEM) of nine biological replicates. Statistical analysis was performed using one-way ANOVA with Welch’s correction. *** p ≤0.001 and **** p ≤0.0001.CC BY · PubMed Central
Figure 5. Bright-field microscopic images of E. faecalis and its indicated mutants. Images shown are representative of 10 images that are acquired from one biological sample from each strain grown in BHI, respectively, and imaged at 100x magnification. Black bars represent five microns in length.
Figure 5. Bright-field microscopic images of E. faecalis and its indicated mutants. Images shown are representative of 10 images that are acquired from one biological sample from each strain grown in BHI, respectively, and imaged at 100x magnification. Black bars represent five microns in length.CC BY · PubMed Central
Figure 6. Characterization of E. faecalis virulence traits at the cell surface interface. (a) Biofilm biomass quantification of E. faecalis OG1RF WT and its mutants grown in BHI for 24 hours. Statistical analysis was performed using One-way ANOVA with Welch’s correction. (b-e) Final growth yields of E. faecalis OG1RF WT and its mutants after 24 hours of incubation in BHI supplemented with 2-fold increasing concentrations of (b) ampicillin, (c) daptomycin, (d) bacitracin, and (e) vancomycin. Data points represent the average of nine biological replicates. Statistical analysis was performed using One-way ANOVA with Welch’s correction. * p ≤0.05, *** p ≤0.001, and **** p ≤0.0001. Error bars represent the standard error of margin (SEM).
Figure 6. Characterization of E. faecalis virulence traits at the cell surface interface. (a) Biofilm biomass quantification of E. faecalis OG1RF WT and its mutants grown in BHI for 24 hours. Statistical analysis was performed using One-way ANOVA with Welch’s correction. (b-e) Final growth yields of E. faecalis OG1RF WT and its mutants after 24 hours of incubation in BHI supplemented with 2-fold increasing concentrations of (b) ampicillin, (c) daptomycin, (d) bacitracin, and (e) vancomycin. Data points represent the average of nine biological replicates. Statistical analysis was performed using One-way ANOVA with Welch’s correction. * p ≤0.05, *** p ≤0.001, and **** p ≤0.0001. Error bars represent the standard error of margin (SEM).CC BY · PubMed Central
Figure 7. Virulence of E. faecalis in different animal models. (a) Percentage survival of G. mellonella larvae 96 hours post-infection with E. faecalis WT or indicated mutants. Each curve represents a group of 15 larvae injected with ~1 x 105 CFU of selected E. faecalis strain. Data points represent the average of 6 biological replicates. Statistical analysis was performed using the log-rank (Mantel-Cox) test. (b) Total CFU recovered after 48 hours from spleen, peritoneal wash, and catheter of mice infected with 2 × 108 CFU of bacteria. (c) Total CFU recovered after 24 hours from bladder, kidney, spleen, and catheter of mice infected with 1 × 107 CFU of WT or indicated mutants. In (b and c), ten mice were infected with two biological replicates and data points shown were a result of using the ROUT outlier test. The black line represents the median. Statistical analysis was performed using the Mann-Whitney test. * p ≤0.05, ** p ≤0.01, *** p ≤0.001, and **** p ≤0.0001. The dashed line represents the limit of detection (LOD = 50 CFUs).
Figure 7. Virulence of E. faecalis in different animal models. (a) Percentage survival of G. mellonella larvae 96 hours post-infection with E. faecalis WT or indicated mutants. Each curve represents a group of 15 larvae injected with ~1 x 105 CFU of selected E. faecalis strain. Data points represent the average of 6 biological replicates. Statistical analysis was performed using the log-rank (Mantel-Cox) test. (b) Total CFU recovered after 48 hours from spleen, peritoneal wash, and catheter of mice infected with 2 × 108 CFU of bacteria. (c) Total CFU recovered after 24 hours from bladder, kidney, spleen, and catheter of mice infected with 1 × 107 CFU of WT or indicated mutants. In (b and c), ten mice were infected with two biological replicates and data points shown were a result of using the ROUT outlier test. The black line represents the median. Statistical analysis was performed using the Mann-Whitney test. * p ≤0.05, ** p ≤0.01, *** p ≤0.001, and **** p ≤0.0001. The dashed line represents the limit of detection (LOD = 50 CFUs).CC BY · PubMed Central

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Metadata

Authors
Lam, L. N., Brunson, D. N., Molina, J. J., Flores-Mireles, A. L., & Lemos, J. A.
Affiliations
  • University of Florida Health (US)
  • University of Notre Dame (US)
ORCID
Venue
Virulence
Publisher
openRxiv
Published
2021-12-01
Volume
13
Issue
1
Pages
592-608
ISSN
2150-5594 (ISSN-L)
DOI
10.1101/2021.12.01.470831
PMC
PMC8966984
OpenAlex
W4220830726
Cited by
23
References
1
Citations by year
2026: 5 · 2025: 7 · 2024: 4 · 2023: 4 · 2022: 3
FWCI
2.71
Type
journal article
Language
en
Open access
Licence
cc-by
Locations
  • Virulence (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • University of Notre Dame
  • National Institutes of Health
  • National Institute of Allergy and Infectious Diseases (AI135158, R21AI135158)
  • National Institute of Diabetes and Digestive and Kidney Diseases (R01DK128805)
  • National Institute of Dental and Craniofacial Research (DE021990, T90DE021990)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
  • Life Sciences › Immunology and Microbiology › Microbiology › Escherichia coli research studies
  • Health Sciences › Nursing › Nutrition and Dietetics › Trace Elements in Health
Keywords
Enterococcus faecalis, zinc homeostasis, ABC transporters, nutritional immunity, bacterial virulence, catheter-related infection, Galleria mellonella infection model, antibiotic tolerance, calprotectin, zinc chelation, therapeutic target
MeSH terms
Animals, Bacterial Proteins, Homeostasis, Enterococcus faecalis, Virulence, Zinc, Mice

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Lam, L. N., Brunson, D. N., Molina, J. J., Flores-Mireles, A. L., & Lemos, J. A. (2021). The AdcACB/AdcAII system is Essential for Zinc Homeostasis and an Important Contributor of Enterococcus faecalis Virulence. Virulence. https://doi.org/10.1101/2021.12.01.470831