L-arginine supplementation abrogates hypoxia-induced virulence of Staphylococcus aureus in a murine diabetic pressure wound model

Abstract

Diabetic foot ulcers (DFUs) are the most common complications of diabetes resulting from hyperglycemia leading to ischemic hypoxic tissue and nerve damage. Staphylococcus aureus is the most frequently isolated bacteria from DFUs and causes severe necrotic infections leading to amputations with a poor 5-year survival rate. However, very little is known about the mechanisms by which S. aureus dominantly colonizes and causes severe disease in DFUs. Herein, we utilized a pressure wound model in diabetic TALLYHO/JngJ mice to reproduce ischemic hypoxic tissue damage seen in DFUs and demonstrated that anaerobic fermentative growth of S. aureus significantly increased the virulence and the severity of disease by activating two-component regulatory systems leading to expression of virulence factors. Our in vitro studies showed that supplementation of nitrate as a terminal electron acceptor promotes anaerobic respiration and suppresses the expression of S. aureus virulence factors through inactivation of two-component regulatory systems, suggesting potential therapeutic benefits by promoting anaerobic nitrate respiration. Our in vivo studies revealed that dietary supplementation of L-arginine (L-Arg) significantly attenuated the severity of disease caused by S. aureus in the pressure wound model by providing nitrate. Collectively, these findings highlight the importance of anaerobic fermentative growth in S. aureus pathogenesis and the potential of dietary L-Arg supplementation as a therapeutic to prevent severe S. aureus infection in DFUs.IMPORTANCES. aureus is the most common cause of infection in DFUs, often resulting in lower-extremity amputation with a distressingly poor 5-year survival rate. Treatment for S. aureus infections has largely remained unchanged for decades and involves tissue debridement with antibiotic therapy. With high levels of conservative treatment failure, recurrence of ulcers, and antibiotic resistance, a new approach is necessary to prevent lower-extremity amputations. Nutritional aspects of DFU treatment have largely been overlooked as there has been contradictory clinical trial evidence, but very few in vitro and in vivo modelings of nutritional treatment studies have been performed. Here we demonstrate that dietary supplementation of L-Arg in a diabetic mouse model significantly reduced duration and severity of disease caused by S. aureus. These findings suggest that L-Arg supplementation could be useful as a potential preventive measure against severe S. aureus infections in DFUs.

Keywords: L-arginine; Staphylococcus aureus; diabetic foot ulcers; hypoxia; virulence.

Fig 1

Hypoxic condition induced by a pressure wound model significantly increases the virulence of S. aureus in diabetic TALLYHO/JngJ mice. (A) Diabetic TALLYHO/JngJ mice (n = 8 mice/group) or non-diabetic SWR/J control mice (n = 8 mice/group) were subcutaneously infected with 1 × 10⁷ of S. aureus LAC strain in the ischemic pressure wound. As comparisons, diabetic TALLYHO/JngJ mice were subcutaneously infected without ischemic pressure wound or were not infected with ischemic pressure wound. Progress of wound healing was monitored daily for 21 days. (B) Analysis of areas of wound measured using the SilhouetteStar camera. Representative images from three independent experiments are shown. (C) Analysis of bacterial burden of infected site at day 21 post-infection. (D and E) Quantitative real-time PCR analysis of (D) virulence factors and (E) TCRS at the infection site with the pressure wound compared to that without pressure wound at day 7 post-infection. Data are presented as the mean ± standard deviation of three independent experiments. Statistical significance was evaluated by Student’s t-test. *P < 0.01. LOD, limit of detection.

Fig 2

In vitro S. aureus growth under hypoxic and elevated sugar conditions increases expression of S. aureus virulence factors. (A) Aerobic or anaerobic growth of S. aureus LAC strain in BHI-G6P broth was determined by measuring the OD₆₀₀ for 24 hours. (B) S. aureus LAC strain chromosomally integrated with the gfp gene aerobically or anaerobically was cultured in BHI-G6P broth for 24 hours, and biofilm formation was analyzed using a confocal microscopy by measuring the mean fluorescence intensity (n = 8). Representative images from three independent experiments are shown. (C) qRT- PCR analysis of the genes related to biofilm formation in anaerobic conditions compared to aerobic conditions. (D) Growth analysis of S. aureus LAC strain aerobically or anaerobically cultured in BHI-G6P broth supplemented with the indicated amount of vancomycin and cefoxitin was determined by measuring the OD₆₀₀ after 24 hours. (E) qRT- PCR analysis of the genes related to cell wall synthesis in anaerobic conditions compared to aerobic conditions. (F) Human RBC was incubated with supernatants from S. aureus LAC strain aerobically or anaerobically cultured in BHI-G6P broth at 37°C for 1 hour, positive control (LukF and LukS, 1 µg of each) or negative control (medium). The OD₅₄₀ value relative to the positive control was calculated to assess hemolytic activity. Data are presented as the mean ± standard deviation of three independent experiments. Statistical significance was evaluated by Student’s t-test. *P < 0.01.

Fig 3

Supplementation of sodium nitrate suppresses expression of S. aureus virulence factors under hypoxic conditions. (A) qRT-PCR analysis of the gene related to anaerobic nitrate respiration in S. aureus LAC strain anaerobically or aerobically cultured in BHI-G6P broth for 24 hours. (B) Growth of S. aureus LAC strain cultured in BHI-G6P broth supplemented with or without 2-mM NaNO₃ was determined by measuring the OD₆₀₀ at the indicated time points for 24 hours. (C) Confocal microscopy images and the mean fluorescence intensity of biofilm formation by S. aureus LAC strain chromosomally integrated with the gfp gene anaerobically cultured in BHI-G6P supplemented with or without 2 mM NaNO₃ for 24 hours. (D) qRT- PCR analysis of the genes related to biofilm formation in S. aureus LAC strain anaerobically cultured in BHI-G6P broth supplemented with or without 2 mM NaNO₃. (E) Growth analysis of S. aureus LAC strain anaerobically cultured in BHI-G6P broth supplemented with or without 2 mM NaNO₃ and the indicated amount of vancomycin and cefoxitin was determined by measuring the OD₆₀₀ after 24 hours. (F) qRT- PCR analysis of the genes related to cell wall synthesis in S. aureus LAC strain anaerobically cultured in BHI-G6P broth supplemented with or without 2 mM NaNO₃. (G) Human RBC was incubated with supernatants from S. aureus LAC strain anaerobically cultured in BHI-G6P broth supplemented with or without 2 mM NaNO₃ for 1 hour, positive control (LukF and LukS, 1 µg of each) or negative control (medium). The OD₅₄₀ value relative to the positive control was calculated to assess hemolytic activity. Data are presented as the mean ± standard deviation of three independent experiments. Statistical significance was evaluated by Student’s t-test. *P < 0.01.

Fig 4

Sodium nitrate induces transcriptional decrease of TCRS and dephosphorylation of response regulatory leading to decreased expression of S. aureus virulence factors. (A) qRT-PCR analysis of global regulatory TCRS in S. aureus LAC strain anaerobically cultured for 24 hours in BHI-G6P broth supplemented with or without 2 mM NaNO₃. (B) Phosphorylation of Agr, SaeR, or HptR fused with 3× FLAG in S. aureus LAC strain anaerobically cultured in BHI-G6P broth with or without supplementation of 2 mM NaNO₃ for 24 hours was analyzed using a Phos-tag PAGE, followed by Western blot using an anti-FLAG mAb. Phos-tag bound phosphorylated protein has a slower migration rate than unbound non-phosphorylated protein, resulting in detectable separation of phosphorylated protein from non-phosphorylated protein. Representative images from two independent experiments are shown. (C) qRT- PCR analysis of staphylococcal cytotoxin genes in S. aureus LAC strain anaerobically cultured for 24 hours in BHI-G6P broth supplemented with or without 2 mM NaNO₃ (n = 3).

Fig 5

Dietary L-Arg supplementation maintains body weight and serum glucose and increases ROS and nitrate production in diabetic TALLYHO/JngJ mice. (A) Body weight and (B) serum glucose level in diabetic TALLYHO/JngJ mice before and after gavage with PBS (n = 12) or L-Arg for 6 weeks (n = 35). (C) Serum nitrite/nitrate (NOx) level and (D) ROS response of bone marrow-derived neutrophils from diabetic TALLYHO/JngJ mice after gavage with PBS or L-Arg for 6 weeks. (E) Representative images of in vivo ROS/RNS response to pressure wound visualized by L-012 luminol. The alphabet indicates statistical significance determined by the Mann-Whitney test (P < 0.0001). The asterisk indicates statistical significance determined by the Student’s t-test. *P < 0.01.

Fig 6

Dietary L-Arg supplementation significantly decreases the severity of disease caused by S. aureus-infected pressure wound in diabetic TALLYHO/JngJ mice. (A) Representative images of diabetic TALLYHO/JngJ mice gavaged with L-Arg or PBS subcutaneously infected with 1 × 10⁷ of S. aureus LAC strain to the ischemic pressure wound (n = 8). Progress of wound healing was monitored for 21 days. (B) Analysis of areas of wound measured using the SilhouetteStar camera. (C) Analysis of bacterial burden of infected pressure wound at day 21 post-infection. (D and E) qRT-PCR analysis of (D) TCRS and (E) virulence factors of infected pressure wounds of L-Arg fed over PBS fed diabetic TALLYHO/JngJ mice at day 7 post-infection. Data are presented as the mean ± standard deviation of three independent experiments. The asterisk indicates statistical significance evaluated by Student’s t-test. *P < 0.01. LOD, limit of detection.