Background

Group A Streptococcus (GAS), or Streptococcus pyogenes , is a human-specific Gram-positive bacterial pathogen. It remains among the top ten pathogens causing several diseases with mild to life-threatening clinical symptoms (Bisno and Stevens, 1996;Efstratiou and Lamagni, 2016). The clinical presentation of these diseases includes pharyngitis, impetigo, erysipelas, tonsilitis, scarlet fever, necrotizing fasciitis, rheumatic fever, glomerulonephritis, septicemia, and others. More than 220 different GAS serotypes are circulating in the human population worldwide. M1T1 is the dominant serotype spread globally and causing diseases in the population of economically well-equipped countries (Aziz and Kotb, 2008; Nelson et al., 2016; Lynskey et al., 2019). GAS classification is based on variation in the N-terminal amino acid sequence of the surface M-protein. Antibiotic regimens are the only definitive strategy to cure GAS infections, because of the unavailability of any licensed universal vaccine on the market. Recent reports on the development of antibiotic resistance in GAS are worrisome and have motivated us to develop a new alternative therapeutic approach to cure its infections.

Invasive GAS infections, like sepsis, bacteremic pneumonia, and necrotizing fasciitis (NF), spread in sterile sites (Metzgar and Zampolli, 2011). NF begins as a small lesion with the appearance of mild erythema, which rapidly progresses with inflammation in the subcutaneous tissue, and destructs skin and soft tissue over large body areas (Walker et al., 2014). Antibiotic administration, surgical debridement of infected tissues, and supportive care are the main line of treatment for invasive GAS diseases (Stevens and Bryant, 2017). Unfortunately, the mortality rate from NF ranges from 23 to 35% in resource-rich settings, despite quick medical interventions (Davies et al., 1996; Carapetis et al., 2005;Allen and Moore, 2010;Olsen and Musser, 2010; Cole et al., 2011;Ralph and Carapetis, 2013). Therefore, alternative therapeutic approaches are urgently needed to combat invasive soft-tissue GAS infections.

Our recent studies showed that GAS causes endoplasmic reticulum stress (ER stress) and triggers the unfolded protein response (UPR) in the host cells upon infection. This results in enhanced asparagine synthesis and release, which is utilized by GAS to increase virulence and rate of proliferation. Furthermore, the PERK-eIF2α-ATF4 branch of UPR was exclusively triggered during GAS infection (Anand et al., 2021). When we treated GAS-infected mice with PERK pathway inhibitors (GSK2656157 and ISRIB), the bacteria were cleared faster than in untreated infected mice. We discuss the method of treatment using PERK pathway inhibitors in a murine model of human soft tissue infection.

Materials and reagents

1. Preparation of bacteria for mice infection

   1. 15 mL conical tubes (Greiner Bio-One CELLSTAR, catalog number: 188621)

   2. 50 mL conical tubes (MiniPlast, catalog number: 835-050-21-111)

   3. Microcentrifuge tubes (Corning^® Axygen, catalog number: MCT-175-C)

   4. Spectrophotometric cuvettes (Polystyrene cuvette, 10 × 4 × 45 mm, Sarstedt AG & Co. KG, Germany, catalog number: 67.742)

   5. GAS strains (serotype M14 strain JS14, which was isolated from a patient with pneumonia, and serotype M1T1 strain 5448, a clinical isolate from a patient with necrotizing fasciitis and toxic shock syndrome)

   6. Sterile Dulbecco's Phosphate Buffered Saline (without calcium chloride and magnesium chloride, pH 7.1–7.5, Biological Industries, catalog number: 02-023-1A)

   7. THY media (see Recipes)

      Todd-Hewitt broth (Becton, Dickenson, and Company, catalog number: 249240)

      Yeast extract (Becton, Dickenson, and Company, catalog number: 212750)

   8. Blood Agar plates (see Recipes)

      Defibrinated sheep blood (DSB) (hylabs^®, catalog number: PD049)

      
      

2. Mice infection

   1. 1 mL syringes without needles (BD Plastipak^TM 1 mL Luer, catalog number: 303172)

   2. Needle (30G × 1/2") for syringe (BD Microlance^TM 3, catalog number: 2025-01)

   3. Bacterial culture prepared for injection (suspended in PBS)

   4. Female BALB/c OlaHsd mice, aged 3–4 weeks, weighing 10–12 g (ENVIGO RMS, Israel Ltd.)

   5. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: 472301)

   6. Polyethylene glycol (PEG-400) (Sigma-Aldrich, catalog number: 202398)

   7. 0.9% sodium chloride saline (Teva Medical, catalog number: AWN1324)

   8. Ketamine-xylazine mix (mixed in 1:6 ratio, respectively, Ketamine-Vetoquinol, Xylazine-20 mg/mL, Eurovet Animal Health B.V.)

   9. Isoflurane (Piramal Critical Care Inc, USA, catalog number: 66794-013-25)

   10. GSK2656157 (Sigma-Aldrich, catalog number: 504651) (see Recipes)

   11. Integrated stress response inhibitor (ISRIB) (Sigma-Aldrich, catalog number: SML0843) (see Recipes)

       
       

3. CFU quantification of GAS in skin biopsy samples

   1. Sterile 2 mL tubes (Abdos Labtech Private Limited, catalog number: P10203)

   2. Sterile Dulbecco's Phosphate Buffered Saline (without calcium chloride and magnesium chloride, pH 7.1–7.5, Biological Industries, catalog number: 02-023-1A)

   3. 70% Ethanol (Romaical, 19-19-009102-80)

   4. 8.0-mm Punch biopsy (Acu-Punch, Acuderm Inc., catalog number: P825)

   5. Dissection kit—surgical scissors and fine forceps

   6. Blood Agar plates (see Recipes)

      Defibrinated sheep blood (DSB) (hylabs^®, catalog number: PD049)

Equipment

1. Spectrophotometer (Amersham Biosciences, Ultraspec 2100 pro, 80-2112-21)

2. Centrifuge with swing rotor (Sigma, model: 3-16PK)

3. Vortex mixer (Heidolph reax top, Z655988)

4. -80 °C freezer (Thermo Scientific, 8926)

5. 37 °C incubator (BINDER GmbH, BD 56)

6. Digital vernier caliper (Bar Naor Ltd., BN30087-00)

7. Electronic homogenizer (POLYTRON^® 2100 Homogenizers, Kinematica, 05400261)

8. Electric shaver (Phillips, MG3730/15)

9. Desiccator jar (SP BEL-ART polycarbonate/polypropylene desiccator, F42032-0000)

Software

1. GraphPad Prism 5.03 (for statistical analysis and graphical presentation)

Procedure