Murine Soft Tissue Infection Model to Study Group A Streptococcus (GAS) Pathogenesis in Necrotizing Fasciitis.

Group A streptococcus (GAS) necrotizing fasciitis (NF) causes high morbidity and mortality despite prompt intravenous administration of antibiotics, surgical soft-tissue debridement, and supportive treatment in the intensive care unit. Since there is no effective vaccine against GAS infections, a comprehensive understanding of NF pathogenesis is required to design more efficient treatments. To increase our understanding of NF pathogenesis, we need a reliable animal model that mirrors, at least in part, the infectious process in humans. This chapter describes a reliable murine model of human NF that mimics the histopathology observed in humans, namely the destruction of soft tissue, a paucity of infiltrating neutrophils, and the presence of many gram-positive cocci at the center of the infection.

PERK Pathway Inhibitors Cure Group A Streptococcal Necrotizing Fasciitis in a Murine Model.

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. This protocol was validated in: Sci Transl Med (2021), DOI: 10.1126/scitranslmed.abd7465.

Unfolded protein response inhibitors cure group A streptococcal necrotizing fasciitis by modulating host asparagine.

Group A streptococcus (GAS) is among the top 10 causes of mortality from an infectious disease, producing mild to invasive life-threatening manifestations. Necrotizing fasciitis (NF) is characterized by a rapid GAS spread into fascial planes followed by extensive tissue destruction. Despite prompt treatments of antibiotic administration and tissue debridement, mortality from NF is still high. Moreover, there is no effective vaccine against GAS, and early diagnosis of NF is problematic because its clinical presentations are not specific. Thus, there is a genuine need for effective treatments against GAS NF. Previously, we reported that GAS induces endoplasmic reticulum (ER) stress to gain asparagine from the host. Here, we demonstrate that GAS-mediated asparagine induction and release occur through the PERK-eIF2α-ATF4 branch of the unfolded protein response. Inhibitors of PERK or integrated stress response (ISR) blocked the formation and release of asparagine by infected mammalian cells, and exogenously added asparagine overcame this inhibition. Moreover, in a murine model of NF, we show that the inhibitors minimized mortality when mice were challenged with a lethal dose of GAS and reduced bacterial counts and lesion size when mice were challenged with a sublethal dose. Immunohistopathology studies demonstrated that PERK/ISR inhibitors protected mice by enabling neutrophil infiltration into GAS-infected fascia and reducing the pro-inflammatory response that causes tissue damage. Inhibitor treatment was also effective in mice when started at 12 hours after infection. We conclude that host metabolic alteration induced by PERK or ISR inhibitors is a promising therapeutic strategy to treat highly invasive GAS infections.

LL-37-mediated activation of host receptors is critical for defense against group A streptococcal infection.

Group A Streptococcus (GAS) causes diverse human diseases, including life-threatening soft-tissue infections. It is accepted that the human antimicrobial peptide LL-37 protects the host by killing GAS. Here, we show that GAS extracellular protease ScpC N-terminally cleaves LL-37 into two fragments of 8 and 29 amino acids, preserving its bactericidal activity. At sub-bactericidal concentrations, the cleavage inhibits LL-37-mediated neutrophil chemotaxis, shortens neutrophil lifespan, and eliminates P2X7 and EGF receptors' activation. Mutations at the LL-37 cleavage site protect the peptide from ScpC-mediated splitting, maintaining all its functions. The mouse LL-37 ortholog CRAMP is neither cleaved by ScpC nor does it activate P2X7 or EGF receptors. Treating wild-type or CRAMP-null mice with sub-bactericidal concentrations of the non-cleavable LL-37 analogs promotes GAS clearance that is abolished by the administration of either P2X7 or EGF receptor antagonists. We demonstrate that LL-37-mediated activation of host receptors is critical for defense against GAS soft-tissue infections.

Advanced strategies for development of vaccines against human bacterial pathogens.

Infectious diseases are one of the main grounds of death and disabilities in human beings globally. Lack of effective treatment and immunization for many deadly infectious diseases and emerging drug resistance in pathogens underlines the need to either develop new vaccines or sufficiently improve the effectiveness of currently available drugs and vaccines. In this review, we discuss the application of advanced tools like bioinformatics, genomics, proteomics and associated techniques for a rational vaccine design.

Cross-serotype protection against group A Streptococcal infections induced by immunization with SPy_2191.

Group A Streptococcus (GAS) infection causes a range of diseases, but vaccine development is hampered by the high number of serotypes. Here, using reverse vaccinology the authors identify SPy_2191 as a cross-protective vaccine candidate. From 18 initially identified surface proteins, only SPy_2191 is conserved, surface-exposed and inhibits both GAS adhesion and invasion. SPy_2191 immunization in mice generates bactericidal antibodies resulting in opsonophagocytic killing of prevalent and invasive GAS serotypes of different geographical regions, including M1 and M49 (India), M3.1 (Israel), M1 (UK) and M1 (USA). Resident splenocytes show higher interferon-γ and tumor necrosis factor-α secretion upon antigen re-stimulation, suggesting activation of cell-mediated immunity. SPy_2191 immunization significantly reduces streptococcal load in the organs and confers ~76-92% protection upon challenge with invasive GAS serotypes. Further, it significantly suppresses GAS pharyngeal colonization in mice mucosal infection model. Our findings suggest that SPy_2191 can act as a universal vaccine candidate against GAS infections.

Ribifolin, an orbitide from Jatropha ribifolia, and its potential antimalarial activity.

A new orbitide named ribifolin was isolated and characterized from Jatropha ribifolia using mass spectrometry, NMR spectroscopy, quantitative amino acid analysis, molecular dynamics/simulated annealing, and Raman optical activity measurements and calculations. Ribifolin (1) and its linear form (1a) were synthesized by solid-phase peptide synthesis, followed by evaluation of its antiplasmodial and cytotoxicity activities. Compound 1 was moderately effective (IC50 = 42 µM) against the Plasmodium falciparum strain 3D7.

Molecular epidemiology and virulence characteristics of prevalent group A streptococci recovered from patients in northern India.

INTRODUCTION: In this study, the prevalence of M types of Group A Streptococcus (GAS) in North India, invasive behavior of prevalent M types, and inflammatory immune response by host cells were studied. METHODOLOGY: A total of 1,047 clinical samples were collected between 2004 and 2010. Confirmation of GAS was determined by serotyping and M types were identified by emm gene sequencing. The most prevalent serotypes were selected to study their invasive behavior and inflammatory immune response under different temperatures and salt concentrations in A549 and HEp-2 cells. RESULTS: Ninety-two isolates were identified as GAS of which 17 were M types with 18.5% heterogeneity. The most prevalent M types were M1 (21.73%) and M49 (8.7%), respectively. M1 and M49 were used to study virulence potential and inflammatory immune responses. The efficiency of cell infection decreased with increased temperature for both M types, increasing with lowering temperatures compared to the uninfected control (37°C). As salt concentration was increased, cell infection efficiency was lowered with some exceptions; the infection efficiency of M1 strain in A549 cells with 0.6 M NaCl was 50 fold higher (p ≤ 0.03). Significantly increased production of IL-6 and IL-8 was observed in both cell lines infected with GAS and when grown under different environmental conditions compared to uninfected cell lines. CONCLUSIONS: This study determined the prevalence of different M types in North India and showed that environmental conditions can regulate cell infection by GAS . This information may influence the selection of GAS serotypes used in vaccine development.

Identification of potential universal vaccine candidates against group A Streptococcus by using high throughput in silico and proteomics approach.

Streptococcus pyogenes or group A Streptococcus (GAS) causes ~700 million human infections each year, resulting in over 500,000 deaths. The development of a commercial GAS vaccine is hampered due to high strain and serotype diversity in different geographical regions, and the generation of cross-reactive antibodies that may induce autoimmune disease. There is an urgent need to search for alternative vaccine candidates. High throughput multigenome data mining coupled with proteomics seems to be a promising approach to identify the universal vaccine candidates. In the present study, in silico analysis led to prediction of 147 proteins as universal vaccine candidates. Distribution pattern of these predicted candidates was explored in nonsequenced Indian GAS strains (n = 20) by using DNA array hybridization validating in silico analysis. High throughput analyses of surface proteins using 1D-SDS-PAGE coupled with ESI-LC-MS/MS was applied on highly (M49) and less (M1) invasive GAS strains of Indian origin. Comparative proteomics analysis revealed that highly invasive GAS M49 had metabolically more active membrane associated protein machinery than less invasive M1. Further, by overlapping proteomics data with in silico predicted vaccine candidate genes, 52 proteins were identified as probable universal vaccine candidates, which were expressed in these GAS serotypes. These proteins can further be investigated as universal vaccine candidates against GAS. Moreover, this robust approach may serve as a model that can be applied to identify the universal vaccine candidates in case of other pathogenic bacteria with high strain and genetic diversity.

Addition of thiols to o-quinone methide: new 2-hydroxy-3-phenylsulfanylmethyl[1,4]naphthoquinones and their activity against the human malaria parasite Plasmodium falciparum (3D7).

A series of 36 new phenylsulfanylmethyl[1,4]naphthoquinones (7-42) were synthesized by a three-component reaction that involves lawsone, the appropriate aldehyde and thiols with variable substitution patterns. These reactions involve the in situ generation of o-quinone methides (o-QM) via Knoevenagel condensation and 1,4-nucleophilic addition under conventional heating or microwave irradiation. The new naphthoquinones obtained by this methodology were shown to have moderate to good in vitro antimalarial activity against Plasmodium falciparum (3D7).

Nano-technology for targeted drug delivery to combat antibiotic resistance.

Several microbes have evolved clinically significant resistance against almost every available antibiotic. Yet the development of new classes of antibiotics has lagged far behind our growing need. Frequent and suboptimal use of antibiotics particularly in developing countries aggravated the problem by increasing the rate of resistance. Therefore, developing new and multidimensional strategies to combat microbial infections is warranted. These include i) modification of existing antibiotics, ii) searching new and novel antibiotics, iii) development and improvement of antibiotics carrier system to reduce amount and frequency of antibiotic doses, iv) development of targeted antibiotic delivery systems. Here, the authors discuss trends and development of nano-materials and alternative antimicrobials to solve the problem of antibiotic resistance.