Abstract

Group A streptococcus (GAS) is a Gram-positive human pathogen that causes invasive infections with mild to life-threatening severity, like toxic shock syndrome, rheumatic heart disease, and necrotizing fasciitis (NF). NF is characterized by a clinical presentation of widespread tissue destruction due to the rapid spread of GAS infection into fascial planes. Despite quick medical interventions, mortality from NF is high. The early onset of the disease is difficult to diagnose because of non-specific clinical symptoms. Moreover, the unavailability of an effective vaccine against GAS warrants a genuine need for alternative treatments against GAS NF. One endoplasmic reticulum stress signaling pathway (PERK pathway) gets triggered in the host upon GAS infection. Bacteria utilize asparagine release as an output of this pathway for its pathogenesis. We reported that the combination of sub-cutaneous (SC) and intraperitoneal (IP) administration of PERK pathway inhibitors (GSK2656157 and ISRIB) cures local as well as systemic GAS infection in a NF murine model, by reducing asparagine release at the infection site. This protocol’s methodology is detailed below.

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.

Abdos Product Used

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

Author

Aparna Anand, Abhinay Sharma, Miriam Ravins, Atul Kumar Johri,  Boaz Tirosh, Emanuel Hanski

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