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Cited by 116Antimicrobial Resistance in StaphylococcusBacterial biofilms and quorum sensingDermatology and Skin Diseases

The DOI matches its Crossref record; 43 of the 43 references it cites were found in a registry or an index; none of the 36 sources its claims cite is checked for support yet.

Lack of nutritional immunity in diabetic skin infections promotes Staphylococcus aureus virulence

Open access
Thurlow, Lance R., Stephens, Amelia C., Hurley, Kelly E., Richardson, Anthony R.·2020·Science Advances
DOI 10.1126/sciadv.abc5569PMC PMC7673755
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Abstract

Nutritional immunity defects explain the severity of S. aureus skin infections in patients with diabetes.

Overview

CoChat Summary

TL;DR

In a streptozotocin-induced hyperglycemic mouse model, diabetic skin infections with *S. aureus* are more severe because infiltrating phagocytes fail to consume glucose and mount an oxidative burst, leaving excess tissue glucose. The excess glucose drives Agr-dependent *S. aureus* virulence factor production via glycolysis/ATP, and the glucose transporters GlcA/GlcC are required for full toxin production and invasive disease.

Key findings

  • Hyperglycemic STZ-treated mice infected with 1 × 10⁷ CFU *S. aureus* LAC had larger lesions, greater weight loss, higher abscess burden, and more dissemination than untreated mice; abscess glucose was >4-fold higher (later described as sixfold) despite only about a fourfold increase in blood glucose.
  • Phagocytes in STZ-treated infections lacked GLUT-1/GLUT-3 and nitrotyrosine, indicating no oxidative burst; glucose deprivation impaired RAW 264.7 macrophage killing of *S. aureus*, and GLUT-1 KO mice had increased abscess burden and dissemination.
  • Glucose activated Agr/RNAIII and increased *hla*, *psmα*, *aur*, and Hla; glycolytic mutants ΔpfkA, Δpyk, and ΔackA had lower intracellular ATP and Hla, with ATP-Hla correlation R² = 0.7, P = 0.019. An *agr* mutant caused no lesions despite increased burden in STZ-treated mice.
  • Phlorizin lowered blood glucose below 200 mg/dl in 7 of 10 treated mice and reduced virulence transcripts, lesion size, and dissemination with only about a half-log reduction in organism burden; ΔG4 and ΔG2 glucose transporter mutants had reduced ATP, Hla, and virulence, with ΔG4 still ~2 logs higher burden in STZ-treated mice but no observable lesions.

Methods

  • STZ-induced hyperglycemia in female C57BL/6J mice 8–12 weeks old; only mice with blood glucose >300 mg/dl were used as hyperglycemic; subcutaneous infection with 1 × 10⁷ CFU USA300 MRSA strain LAC; assessed day 7 for lesions, weight loss, CFU, and dissemination; phlorizin was given at 400 mg/kg twice daily.
  • In vitro: *S. aureus* grown in chemically defined media with glucose (PNG), casamino acids (PNCAA), or both; RNAIII-YFP reporter, qRT-PCR, Hla Western blot, intracellular ATP assays, immunohistochemistry, and macrophage infection/oxidative burst assays.
  • Statistics: two-tailed Student’s *t* test, significance at P < 0.05, SEM shown; sample sizes are indicated in figure legends.

Limitations

  • Stated: investigators were not blinded, studies were not randomized, and no statistical method was used to predetermine sample size; the mechanism by which insulin signaling is required for phagocyte GLUT-1/-3 expression remains unknown.
  • Evident: findings rely on an STZ-induced insulin-deficient hyperglycemia mouse model using only female mice 8–12 weeks old, so they may not capture all diabetes phenotypes or translate directly to humans.
  • Evident: conclusions about virulence versus bacterial burden rely on mutant strains and transcript/protein readouts; no human patient data are presented.

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Integrity signals

Registry

Crossref · 10.1126/sciadv.abc5569

Cites

43 of 43 references verified0 citation errors · 0 not checkable

Statistics

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Figures

Fig. 1 Diminished oxidative burst in hyperglycemic abscesses correlates with worse infection outcomes. Untreated (UT) (N = 9 and N = 11) and STZ-treated (N = 11 and N = 8) mice were infected with 1 × 107 CFU of the USA300 methicillin-resistant S. aureus (MRSA) strain LAC. WT-infected STZ-treated mice have a larger dermonecrotic lesion (A), increased abscess burden (B), and increased dissemination to the kidney (C) compared with similarly infected untreated mice. Pictures of dermonecrotic lesions from an untreated mouse (red arrows) (D) and a STZ-treated mouse (E) show enhanced dermonecrosis in hyperglycemic infection. Immunohistochemistry (IHC) was performed on tissues from untreated and STZ-treated infections using antibodies against iNOS, nitrotyrosine (Y-NO2), or GLUT-1 (red) and counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). (F) Tissues from both untreated and STZ-treated mice show iNOS staining. (G) However, while untreated mice exhibit robust Y-NO2 staining, this adduct is largely absent in tissue from STZ-treated animals. (H) Similarly, tissues from untreated mice show robust GLUT-1 staining that is mostly absent in tissue from STZ-treated animals. Photo credit: Lance Thurlow, University of Pittsburgh.
Fig. 1 Diminished oxidative burst in hyperglycemic abscesses correlates with worse infection outcomes. Untreated (UT) (N = 9 and N = 11) and STZ-treated (N = 11 and N = 8) mice were infected with 1 × 107 CFU of the USA300 methicillin-resistant S. aureus (MRSA) strain LAC. WT-infected STZ-treated mice have a larger dermonecrotic lesion (A), increased abscess burden (B), and increased dissemination to the kidney (C) compared with similarly infected untreated mice. Pictures of dermonecrotic lesions from an untreated mouse (red arrows) (D) and a STZ-treated mouse (E) show enhanced dermonecrosis in hyperglycemic infection. Immunohistochemistry (IHC) was performed on tissues from untreated and STZ-treated infections using antibodies against iNOS, nitrotyrosine (Y-NO2), or GLUT-1 (red) and counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). (F) Tissues from both untreated and STZ-treated mice show iNOS staining. (G) However, while untreated mice exhibit robust Y-NO2 staining, this adduct is largely absent in tissue from STZ-treated animals. (H) Similarly, tissues from untreated mice show robust GLUT-1 staining that is mostly absent in tissue from STZ-treated animals. Photo credit: Lance Thurlow, University of Pittsburgh.CC BY-NC · PubMed Central
Fig. 2 Agr is required for invasive infections in hyperglycemic mice and requires glucose for activation. Glucose levels were measured in abscesses from untreated and STZ-treated animals and normalized to protein levels. (A) Abscesses from STZ-treated animals had significantly higher glucose levels than those from untreated animals. (B) S. aureus strain LAC harboring a plasmid with YPF linked to the RNAIII promoter (filled symbols) were grown in PNG (red symbols) or PNCAA (blue symbols). Fluorescence is only observed in strains grown in PNG (left axis) (B). (C) Relative RNAIII:YFP fluorescence normalized to OD660 at 10 hours shows greater induction of RNAIII in strains LAC and COL grown in PNG (n = 3). Normal (N = 9 and N = 11) and diabetic (N = 11 and N = 8) mice were infected with 1 × 107 CFU of the USA300 MRSA strain LAC or LAC agr::Tn (n = 10 for untreated and N = 8 for STZ-treated). (D) Neither treated nor STZ-treated mice infected with agr::Tn formed necrotic lesions. (E) The agr::Tn abscesses had lower bacterial burdens than WT-infected mice in both untreated and STZ-treated mice; however, the reduced burden in STZ-treated mice was not as notable. (F) STZ-treated mice infected with agr::Tn had significantly less dissemination to the kidneys compared with STZ-treated mice infected with WT LAC.
Fig. 2 Agr is required for invasive infections in hyperglycemic mice and requires glucose for activation. Glucose levels were measured in abscesses from untreated and STZ-treated animals and normalized to protein levels. (A) Abscesses from STZ-treated animals had significantly higher glucose levels than those from untreated animals. (B) S. aureus strain LAC harboring a plasmid with YPF linked to the RNAIII promoter (filled symbols) were grown in PNG (red symbols) or PNCAA (blue symbols). Fluorescence is only observed in strains grown in PNG (left axis) (B). (C) Relative RNAIII:YFP fluorescence normalized to OD660 at 10 hours shows greater induction of RNAIII in strains LAC and COL grown in PNG (n = 3). Normal (N = 9 and N = 11) and diabetic (N = 11 and N = 8) mice were infected with 1 × 107 CFU of the USA300 MRSA strain LAC or LAC agr::Tn (n = 10 for untreated and N = 8 for STZ-treated). (D) Neither treated nor STZ-treated mice infected with agr::Tn formed necrotic lesions. (E) The agr::Tn abscesses had lower bacterial burdens than WT-infected mice in both untreated and STZ-treated mice; however, the reduced burden in STZ-treated mice was not as notable. (F) STZ-treated mice infected with agr::Tn had significantly less dissemination to the kidneys compared with STZ-treated mice infected with WT LAC.CC BY-NC · PubMed Central
Fig. 3 Toxin production by S. aureus requires ATP generation from glycolysis. (A) Representative Western blot showing Hla in spent media from LAC grown in PNG (left) or PNCAA (right). (B) Quantification of Hla normalized to OD660 in LAC grown in PNG or PNCAA (n = 3). AU, arbirtrary unit. (C)Quantification of intracellular ATP normalized to OD660 relative to LAC grown in PNG versus PNCAA (n = 3). (D) Representative Hla Western blots from culture supernatants from LAC (left), LAC ΔpfkA (center), and LAC Δpyk (right) grown in PNMix. (E) Quantification of Western blots from LAC, LAC ΔpfkA, and LAC Δpyk (n = 3). (F) Quantification of intracellular ATP normalized to OD660 relative to LAC. (G) Representative Hla Western blots of culture supernatant from LAC (left) and LAC ΔackA (right) grown in PNG. (H) Quantification of α-toxin Western blots from LAC and LAC ΔackA (n = 3). (I) Quantification of intracellular ATP normalized to OD660 relative to LAC (n = 3). (J) Regression analysis revealed a significant correlation between normalized intracellular ATP and the amount of normalized excreted Hla (R2 = 0.7, nonzero slope, P = 0.019). Untreated and STZ-treated mice were infected with 107 CFU of WT LAC or LAC ΔackA (WT UT, n = 10; WT STZ, n = 7; ΔackA UT, n = 10; and ΔackA STZ, n = 11). (K) Untreated mice infected with WT or ΔackA have similar lesion sizes. STZ-treated mice infected with ΔakcA have significantly larger lesion sizes than similarly infected untreated mice, but smaller lesion sizes than STZ-treated mice infected with WT (K). (L) STZ-treated mice infected with WT have significantly increased bacterial burden in the subcutaneous abscess compared with similarly infected untreated mice. STZ-treated mice infected with ΔackA have significantly increased bacterial burden in the subcutaneous abscess compared with similarly infected untreated mice (L). There is no significant difference in abscess burden in STZ-treated mice infected with WT or ΔackA (L). N.S., not significant.
Fig. 3 Toxin production by S. aureus requires ATP generation from glycolysis. (A) Representative Western blot showing Hla in spent media from LAC grown in PNG (left) or PNCAA (right). (B) Quantification of Hla normalized to OD660 in LAC grown in PNG or PNCAA (n = 3). AU, arbirtrary unit. (C)Quantification of intracellular ATP normalized to OD660 relative to LAC grown in PNG versus PNCAA (n = 3). (D) Representative Hla Western blots from culture supernatants from LAC (left), LAC ΔpfkA (center), and LAC Δpyk (right) grown in PNMix. (E) Quantification of Western blots from LAC, LAC ΔpfkA, and LAC Δpyk (n = 3). (F) Quantification of intracellular ATP normalized to OD660 relative to LAC. (G) Representative Hla Western blots of culture supernatant from LAC (left) and LAC ΔackA (right) grown in PNG. (H) Quantification of α-toxin Western blots from LAC and LAC ΔackA (n = 3). (I) Quantification of intracellular ATP normalized to OD660 relative to LAC (n = 3). (J) Regression analysis revealed a significant correlation between normalized intracellular ATP and the amount of normalized excreted Hla (R2 = 0.7, nonzero slope, P = 0.019). Untreated and STZ-treated mice were infected with 107 CFU of WT LAC or LAC ΔackA (WT UT, n = 10; WT STZ, n = 7; ΔackA UT, n = 10; and ΔackA STZ, n = 11). (K) Untreated mice infected with WT or ΔackA have similar lesion sizes. STZ-treated mice infected with ΔakcA have significantly larger lesion sizes than similarly infected untreated mice, but smaller lesion sizes than STZ-treated mice infected with WT (K). (L) STZ-treated mice infected with WT have significantly increased bacterial burden in the subcutaneous abscess compared with similarly infected untreated mice. STZ-treated mice infected with ΔackA have significantly increased bacterial burden in the subcutaneous abscess compared with similarly infected untreated mice (L). There is no significant difference in abscess burden in STZ-treated mice infected with WT or ΔackA (L). N.S., not significant.CC BY-NC · PubMed Central
Fig. 4 Reducing blood glucose in hyperglycemic mice with phlorizin reverses infection severity. Mice were treated with phlorizin (Phlo) 1 day before infection and throughout the course of infection to lower blood glucose levels without influencing insulin signaling. (A) STZ-treated mice treated with phlorizin had smaller lesions than mice treated with STZ alone, but larger lesions than untreated mice. (B) Infected STZ-treated mice display significant weight loss that is reversed with phlorizin. (C) STZ-treated mice that were further treated with phlorizin display lower abscess burdens than mice treated with STZ-alone, but higher burdens than untreated mice. Phlorizin treatment resulted in decreased dissemination to the peripheral organs in STZ-treated mice including the kidneys (D), liver (E), and heart (F). (G) Quantitative real-time PCR S. aureus isolated from untreated, STZ-treated, and dual STZ phlorizin abscesses showed significantly increased expression (P < 0.05) of hla, psmα, and aur in STZ-treated mice that is reversed by phlorizin.
Fig. 4 Reducing blood glucose in hyperglycemic mice with phlorizin reverses infection severity. Mice were treated with phlorizin (Phlo) 1 day before infection and throughout the course of infection to lower blood glucose levels without influencing insulin signaling. (A) STZ-treated mice treated with phlorizin had smaller lesions than mice treated with STZ alone, but larger lesions than untreated mice. (B) Infected STZ-treated mice display significant weight loss that is reversed with phlorizin. (C) STZ-treated mice that were further treated with phlorizin display lower abscess burdens than mice treated with STZ-alone, but higher burdens than untreated mice. Phlorizin treatment resulted in decreased dissemination to the peripheral organs in STZ-treated mice including the kidneys (D), liver (E), and heart (F). (G) Quantitative real-time PCR S. aureus isolated from untreated, STZ-treated, and dual STZ phlorizin abscesses showed significantly increased expression (P < 0.05) of hla, psmα, and aur in STZ-treated mice that is reversed by phlorizin.CC BY-NC · PubMed Central
Fig. 5 Glucose transporters are essential for ATP and toxin production. (A) Representative Hla Western blot from supernatants of WT LAC, LAC ΔG4 (lacking all glucose transporters), and LAC ΔG2 (lacking the recently acquired GlcC and GlcA transporters) grown in PNG. (B) Quantification of Hla Western blots (n = 3) showing significantly decreased Hla production by LAC ΔG2 and LAC ΔG4 compared to WT LAC. (C) The LAC ΔG4 (lacking all glucose transporters) and LAC ΔG2 have reduced intracellular ATP compared to WT LAC when grown in PNG. (D) Quantitative real-time PCR from WT LAC and LAC ΔG4 grown in PNG shows significant decreases in aur, psmα, and hla transcripts in LAC ΔG4 (n = 3). (E) Quantitative real-time PCR from WT LAC and LAC ΔG4 isolated from STZ-treated mice showed reduced transcript levels of hla, psmα, and aur in LAC ΔG4. (F) Day 7 lesion sizes measured from untreated mice infected with LAC ΔG4 were virtually undetectable, and abscesses in STZ-treated mice infected with LAC ΔG4 also rarely had small lesions. (G) LAC ΔG4 is attenuated in untreated and STZ-treated mice compared with WT LAC but has higher CFU in STZ-treated animals.
Fig. 5 Glucose transporters are essential for ATP and toxin production. (A) Representative Hla Western blot from supernatants of WT LAC, LAC ΔG4 (lacking all glucose transporters), and LAC ΔG2 (lacking the recently acquired GlcC and GlcA transporters) grown in PNG. (B) Quantification of Hla Western blots (n = 3) showing significantly decreased Hla production by LAC ΔG2 and LAC ΔG4 compared to WT LAC. (C) The LAC ΔG4 (lacking all glucose transporters) and LAC ΔG2 have reduced intracellular ATP compared to WT LAC when grown in PNG. (D) Quantitative real-time PCR from WT LAC and LAC ΔG4 grown in PNG shows significant decreases in aur, psmα, and hla transcripts in LAC ΔG4 (n = 3). (E) Quantitative real-time PCR from WT LAC and LAC ΔG4 isolated from STZ-treated mice showed reduced transcript levels of hla, psmα, and aur in LAC ΔG4. (F) Day 7 lesion sizes measured from untreated mice infected with LAC ΔG4 were virtually undetectable, and abscesses in STZ-treated mice infected with LAC ΔG4 also rarely had small lesions. (G) LAC ΔG4 is attenuated in untreated and STZ-treated mice compared with WT LAC but has higher CFU in STZ-treated animals.CC BY-NC · PubMed Central
Fig. 6 The evolutionary acquisition of glucose transporters by S. aureus coincides with acquisition of virulence factors. A phylogenetic tree based on 16S ribosomal DNA sequences showing the relation of S. aureus to other staphylococcal species. Red arrows show where S. aureus and other related species evolutionarily acquired lactate dehydrogenase 1 (ldh1), glucose transporter A (glcA), glucose transporter C (glcC), and virulence factors including α-, β-, and γ-hemolysins (A). Untreated and STZ-treated mice were infected with 1 × 107 CFU of WT and ∆G2 (WT UT, n = 10; WT STZ, n = 7 to 8; ∆G2 UT, n = 10; and ∆G2 STZ, n = 11). STZ-treated mice infected with WT had larger lesion sizes than infected untreated mice. STZ-treated mice infected with ΔG2 had smaller lesion sizes than WT infected STZ-treated mice (B). (C) STZ-treated mice infected with WT or ΔG2 had significantly higher abscess bacterial burdens than their untreated infected controls. (D) STZ-treated mice infected with WT displayed increased dissemination to the kidneys compared to similarly infected untreated mice. STZ-treated mice infected with ΔG2 had increased dissemination to the kidneys compared to similarly infected untreated mice (D).
Fig. 6 The evolutionary acquisition of glucose transporters by S. aureus coincides with acquisition of virulence factors. A phylogenetic tree based on 16S ribosomal DNA sequences showing the relation of S. aureus to other staphylococcal species. Red arrows show where S. aureus and other related species evolutionarily acquired lactate dehydrogenase 1 (ldh1), glucose transporter A (glcA), glucose transporter C (glcC), and virulence factors including α-, β-, and γ-hemolysins (A). Untreated and STZ-treated mice were infected with 1 × 107 CFU of WT and ∆G2 (WT UT, n = 10; WT STZ, n = 7 to 8; ∆G2 UT, n = 10; and ∆G2 STZ, n = 11). STZ-treated mice infected with WT had larger lesion sizes than infected untreated mice. STZ-treated mice infected with ΔG2 had smaller lesion sizes than WT infected STZ-treated mice (B). (C) STZ-treated mice infected with WT or ΔG2 had significantly higher abscess bacterial burdens than their untreated infected controls. (D) STZ-treated mice infected with WT displayed increased dissemination to the kidneys compared to similarly infected untreated mice. STZ-treated mice infected with ΔG2 had increased dissemination to the kidneys compared to similarly infected untreated mice (D).CC BY-NC · PubMed Central

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Metadata

Authors
Thurlow, L. R., Stephens, A. C., Hurley, K. E., & Richardson, A. R.
Affiliations
University of Pittsburgh (US)
Corresponding author
Richardson, Anthony R.
ORCID
Venue
Science Advances
Publisher
American Association for the Advancement of Science (AAAS)
Published
2020-11-13
Volume
6
Issue
46
ISSN
2375-2548 (ISSN-L)
DOI
10.1126/sciadv.abc5569
PMC
PMC7673755
OpenAlex
W3101718346
Semantic Scholar
8d286177e32bdfed8ec5286bb4823b3213f85fc8
Cited by
116
References
43
Citations by year
2026: 30 · 2025: 27 · 2024: 25 · 2023: 21 · 2022: 8 · 2021: 4 · 2020: 1
FWCI
1.24
Type
journal article
Language
en
Open access
Licence
Locations
  • Science Advances (publishedVersion, cc-by-nc)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Funders
NIH Office of the Director (R01AI093613 and R21AI111707)
Topics
  • Health Sciences › Medicine › Infectious Diseases › Antimicrobial Resistance in Staphylococcus
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Bacterial biofilms and quorum sensing
  • Health Sciences › Medicine › Dermatology › Dermatology and Skin Diseases
Keywords
nutritional immunity, diabetes mellitus, Staphylococcus aureus, skin infections, virulence factors, hyperglycemia, host-pathogen interaction, bacterial virulence
MeSH terms
Diabetes Mellitus, Glucose, Humans, Hyperglycemia, Staphylococcal Infections, Staphylococcus aureus, Virulence

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Thurlow, L. R., Stephens, A. C., Hurley, K. E., & Richardson, A. R. (2020). Lack of nutritional immunity in diabetic skin infections promotes Staphylococcus aureus virulence. Science Advances. https://doi.org/10.1126/sciadv.abc5569

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