Group A Streptococcus induces GSDMA-dependent pyroptosis in keratinocytes.

Gasdermins (GSDMs) are a family of pore-forming effectors that permeabilize the cell membrane during the cell death program pyroptosis1. GSDMs are activated by proteolytic removal of autoinhibitory carboxy-terminal domains, typically by caspase regulators1-9. However, no activator is known for one member of this family, GSDMA. Here we show that the major human pathogen group A Streptococcus (GAS) secretes a protease virulence factor, SpeB, that induces GSDMA-dependent pyroptosis. SpeB cleavage of GSDMA releases an active amino-terminal fragment that can insert into membranes to form lytic pores. GSDMA is primarily expressed in the skin10, and keratinocytes infected with SpeB-expressing GAS die of GSDMA-dependent pyroptosis. Mice have three homologues of human GSDMA, and triple-knockout mice are more susceptible to invasive infection by a pandemic hypervirulent M1T1 clone of GAS. These results indicate that GSDMA is critical in the immune defence against invasive skin infections by GAS. Furthermore, they show that GSDMs can act independently of host regulators as direct sensors of exogenous proteases. As SpeB is essential for tissue invasion and survival within skin cells, these results suggest that GSDMA can act akin to a guard protein that directly detects concerning virulence activities of microorganisms that present a severe infectious threat.

Constitutive secretion of pro-IL-18 allows keratinocytes to initiate inflammation during bacterial infection.

Group A Streptococcus (GAS, Streptococcus pyogenes) is a professional human pathogen that commonly infects the skin. Keratinocytes are one of the first cells to contact GAS, and by inducing inflammation, they can initiate the earliest immune responses to pathogen invasion. Here, we characterized the proinflammatory cytokine repertoire produced by primary human keratinocytes and surrogate cell lines commonly used in vitro. Infection induces several cytokines and chemokines, but keratinocytes constitutively secrete IL-18 in a form that is inert (pro-IL-18) and lacks proinflammatory activity. Canonically, IL-18 activation and secretion are coupled through a single proteolytic event that is regulated intracellularly by the inflammasome protease caspase-1 in myeloid cells. The pool of extracellular pro-IL-18 generated by keratinocytes is poised to sense extracellular proteases. It is directly processed into a mature active form by SpeB, a secreted GAS protease that is a critical virulent factor during skin infection. This mechanism contributes to the proinflammatory response against GAS, resulting in T cell activation and the secretion of IFN-γ. Under these conditions, isolates of several other major bacterial pathogens and microbiota of the skin were found to not have significant IL-18-maturing ability. These results suggest keratinocyte-secreted IL-18 is a sentinel that sounds an early alarm that is highly sensitive to GAS, yet tolerant to non-invasive members of the microbiota.

Detoxification of reactive oxygen species by the hyaluronic acid capsule of group A Streptococcus.

The pro-inflammatory cytokine IL-6 regulates antimicrobial responses that are broadly crucial in the defense against infection. Our prior work shows that IL-6 promotes the killing of the M4 serotype group A Streptococcus (GAS) but does not impact the globally disseminated M1T1 serotype associated with invasive infections. Using in vitro and in vivo infection models, we show that IL-6 induces phagocyte reactive oxygen species (ROS) that are responsible for the differential susceptibility of M4 and M1T1 GAS to IL-6-mediated defenses. Clinical isolates naturally deficient in capsule, or M1T1 strains deficient in capsule production, are sensitive to this ROS killing. The GAS capsule is made of hyaluronic acid, an antioxidant that detoxifies ROS and can protect acapsular M4 GAS when added exogenously. During in vitro interactions with macrophages and neutrophils, acapsular GAS can also be rescued with the antioxidant N-acetylcysteine, suggesting this is a major virulence contribution of the capsule. In an intradermal infection model with gp91phox -/- (chronic granulomatous disease [CGD]) mice, phagocyte ROS production had a modest effect on bacterial proliferation and the cytokine response but significantly limited the size of the bacterial lesion in the skin. These data suggest that the capsule broadly provides enhanced resistance to phagocyte ROS but is not essential for invasive infection. Since capsule-deficient strains are observed across several GAS serotypes and are competent for transmission and both mild and invasive infections, additional host or microbe factors may contribute to ROS detoxification during GAS infections.

Opportunistic Invasive Infection by Group A Streptococcus During Anti-Interleukin-6 Immunotherapy.

Invasive group A Streptococcus (GAS) in immunocompetent individuals is largely linked to hypervirulent strains. Congenital immunodeficiencies and those acquired from chronic disease or immunosuppressant drugs also increase risk of severe illness. We recovered GAS from the blood of a patient receiving a biologic inhibitor of interleukin 6 (IL-6). Growth of this serotype M4 isolate in human blood or a murine bacteremia model was promoted by interleukin 1 or IL-6 inhibition. Hyperinvasive M1T1 GAS was unaffected by IL-6 in both models. These findings based on a natural experiment introduce IL-6 signaling deficiencies as a risk factor for invasive GAS.

Restoring Endogenous Repair Mechanisms to Heal Chronic Wounds with a Multifunctional Wound Dressing.

Current treatment of chronic wounds has been critically limited by various factors, including bacterial infection, biofilm formation, impaired angiogenesis, and prolonged inflammation. Addressing these challenges, we developed a multifunctional wound dressing-based three-pronged approach for accelerating wound healing. The multifunctional wound dressing, composed of nanofibers, functional nanoparticles, natural biopolymers, and selected protein and peptide, can target multiple endogenous repair mechanisms and represents a promising alternative to current wound healing products.

Inflammasome inhibition blocks cardiac glycoside cell toxicity.

Chronic heart failure and cardiac arrhythmias have high morbidity and mortality, and drugs for the prevention and management of these diseases are a large part of the pharmaceutical market. Among these drugs are plant-derived cardiac glycosides, which have been used by various cultures over millennia as both medicines and poisons. We report that digoxin and related compounds activate the NLRP3 inflammasome in macrophages and cardiomyocytes at concentrations achievable during clinical use. Inflammasome activation initiates the maturation and release of the inflammatory cytokine IL-1ß and the programmed cell death pathway pyroptosis in a caspase-1-dependent manner. Notably, the same fluxes of potassium and calcium cations that affect heart contraction also induce inflammasome activation in human but not murine cells. Pharmaceuticals that antagonize these fluxes, including glyburide and verapamil, also inhibit inflammasome activation by cardiac glycosides. Cardiac glycoside-induced cellular cytotoxicity and IL-1ß signaling are likewise antagonized by inhibitors of the NLRP3 inflammasome or the IL-1 receptor-targeting biological agent anakinra. Our results inform on the molecular mechanism by which the inflammasome integrates the diverse signals that activate it through secondary signals like cation flux. Furthermore, this mechanism suggests a contribution of the inflammasome to the toxicity and adverse events associated with cardiac glycosides use in humans and that targeted anti-inflammatories could provide an additional adjunct therapeutic countermeasure.

Group A Streptococcus Infection of the Nasopharynx Requires Proinflammatory Signaling through the Interleukin-1 Receptor.

Group A Streptococcus (GAS) is the etiologic agent of numerous high-morbidity and high-mortality diseases. Infections are typically highly proinflammatory. During the invasive infection necrotizing fasciitis, this is in part due to the GAS protease SpeB directly activating interleukin-1ß (IL-1ß) independent of the canonical inflammasome pathway. The upper respiratory tract is the primary site for GAS colonization, infection, and transmission, but the host-pathogen interactions at this site are still largely unknown. We found that in the murine nasopharynx, SpeB enhanced IL-1ß-mediated inflammation and the chemotaxis of neutrophils. However, neutrophilic inflammation did not restrict infection and instead promoted GAS replication and disease. Inhibiting IL-1ß or depleting neutrophils, which both promote invasive infection, prevented GAS infection of the nasopharynx. Mice pretreated with penicillin became more susceptible to GAS challenge, and this reversed the attenuation from neutralization or depletion of IL-1ß, neutrophils, or SpeB. Collectively, our results suggest that SpeB is essential to activate an IL-1ß-driven neutrophil response. Unlike during invasive tissue infections, this is beneficial in the upper respiratory tract because it disrupts colonization resistance mediated by the microbiota. This provides experimental evidence that the notable inflammation of strep throat, which presents with significant swelling, pain, and neutrophil influx, is not an ineffectual immune response but rather is a GAS-directed remodeling of this niche for its pathogenic benefit.

Modeling neuro-immune interactions during Zika virus infection.

Although Zika virus (ZIKV) infection is often asymptomatic, in some cases, it can lead to birth defects in newborns or serious neurologic complications in adults. However, little is known about the interplay between immune and neural cells that could contribute to the ZIKV pathology. To understand the mechanisms at play during infection and the antiviral immune response, we focused on neural precursor cells (NPCs)-microglia interactions. Our data indicate that human microglia infected with the current circulating Brazilian ZIKV induces a similar pro-inflammatory response found in ZIKV-infected human tissues. Importantly, using our model, we show that microglia interact with ZIKV-infected NPCs and further spread the virus. Finally, we show that Sofosbuvir, an FDA-approved drug for Hepatitis C, blocked viral infection in NPCs and therefore the transmission of the virus from microglia to NPCs. Thus, our model provides a new tool for studying neuro-immune interactions and a platform to test new therapeutic drugs.

Cationic antimicrobial peptide resistance mechanisms of streptococcal pathogens.

Cationic antimicrobial peptides (CAMPs) are critical front line contributors to host defense against invasive bacterial infection. These immune factors have direct killing activity toward microbes, but many pathogens are able to resist their effects. Group A Streptococcus, group B Streptococcus and Streptococcus pneumoniae are among the most common pathogens of humans and display a variety of phenotypic adaptations to resist CAMPs. Common themes of CAMP resistance mechanisms among the pathogenic streptococci are repulsion, sequestration, export, and destruction. Each pathogen has a different array of CAMP-resistant mechanisms, with invasive disease potential reflecting the utilization of several mechanisms that may act in synergy. Here we discuss recent progress in identifying the sources of CAMP resistance in the medically important Streptococcus genus. Further study of these mechanisms can contribute to our understanding of streptococcal pathogenesis, and may provide new therapeutic targets for therapy and disease prevention. This article is part of a Special Issue entitled: Bacterial Resistance to Antimicrobial Peptides.

Group A Streptococcus interactions with the host across time and space.

Group A Streptococcus (GAS) has a fantastically wide tissue tropism in humans, manifesting as different diseases depending on the strain's virulence factor repertoire and the tissue involved. Activation of immune cells and pro-inflammatory signaling has historically been considered an exclusively host-protective response that a pathogen would seek to avoid. However, recent advances in human and animal models suggest that in some tissues, GAS will activate and manipulate specific pro-inflammatory pathways to promote growth, nutrient acquisition, persistence, recurrent infection, competition with other microbial species, dissemination, and transmission. This review discusses molecular interactions between the host and pathogen to summarize how infection varies across tissue and stages of inflammation. A need for inflammation for GAS survival during common, mild infections may drive selection for mechanisms that cause pathological and excess inflammation severe diseases such as toxic shock syndrome, necrotizing fasciitis, and rheumatic heart disease.

Improved Methodology for Evaluating Nanomedicine Antibacterial Properties in Biological Fluids.

Accurate assessment of nanomedicines' antibacterial properties is pivotal for their effective use in both in vitro and in vivo settings. Conventional antibacterial activity assessment methods, involving bacterial coculture with compounds on agar plates, may not fully suit nanomedicines due to their susceptibility to alterations in physicochemical properties induced by biological fluids. Furthermore, these biological fluids might even enhance the bacterial growth. This study introduces a novel, rigorous, and reproducible methodology for evaluating nanomedicine antibacterial properties using cell culture media (i.e., DMEM-FBS10%). To assess the antibacterial activity of the nanoparticles in cell culture media, superparamagnetic iron oxide nanoparticles (SPIONs) were chosen as the model nanomedicine due to their clinical significance. A comparative analysis between the traditional and our proposed methods yielded contrasting outcomes, shedding light on the significant impact of biological fluids on nanoparticle antibacterial activities. While the conventional approach suggested the antibacterial effectiveness of SPIONs against Staphylococcus aureus, our innovative method unveiled a substantial increase in bacterial growth in the presence of biological fluids. More specifically, we found a significant increase in bacterial growth when exposed to bare SPIONs at various concentrations, while the formation of a protein corona on SPION surfaces could markedly reduce the observed bacterial growth compared to the control group. These findings underscore the necessity for more refined evaluation techniques that can better replicate the in vivo environment when studying the nanomedicine's antibacterial capabilities.

Human microglial cells as a therapeutic target in a neurodevelopmental disease model.

Although microglia are macrophages of the central nervous system, their involvement is not limited to immune functions. The roles of microglia during development in humans remain poorly understood due to limited access to fetal tissue. To understand how microglia can impact human neurodevelopment, the methyl-CpG binding protein 2 (MECP2) gene was knocked out in human microglia-like cells (MGLs). Disruption of the MECP2 in MGLs led to transcriptional and functional perturbations, including impaired phagocytosis. The co-culture of healthy MGLs with MECP2-knockout (KO) neurons rescued synaptogenesis defects, suggesting a microglial role in synapse formation. A targeted drug screening identified ADH-503, a CD11b agonist, restored phagocytosis and synapse formation in spheroid-MGL co-cultures, significantly improved disease progression, and increased survival in MeCP2-null mice. These results unveil a MECP2-specific regulation of human microglial phagocytosis and identify a novel therapeutic treatment for MECP2-related conditions.

Engineered probiotic overcomes pathogen defences using signal interference and antibiotic production to treat infection in mice.

Probiotic supplements are suggested to promote human health by preventing pathogen colonization. However, the mechanistic bases for their efficacy in vivo are largely uncharacterized. Here using metabolomics and bacterial genetics, we show that the human oral probiotic Streptococcus salivarius K12 (SAL) produces salivabactin, an antibiotic that effectively inhibits pathogenic Streptococcus pyogenes (GAS) in vitro and in mice. However, prophylactic dosing with SAL enhanced GAS colonization in mice and ex vivo in human saliva. We showed that, on co-colonization, GAS responds to a SAL intercellular peptide signal that controls SAL salivabactin production. GAS produces a secreted protease, SpeB, that targets SAL-derived salivaricins and enhances GAS survival. Using this knowledge, we re-engineered probiotic SAL to prevent signal eavesdropping by GAS and potentiate SAL antimicrobials. This engineered probiotic demonstrated superior efficacy in preventing GAS colonization in vivo. Our findings show that knowledge of interspecies interactions can identify antibiotic- and probiotic-based strategies to combat infection.

Assessing Interleukin-1ß Activation During Pyroptosis.

The cytokine interleukin-1ß (IL-1ß) is a major mediator of inflammation in autoinflammatory disease and the host response to infection. IL-1ß is stored within cells in an inert form, which requires proteolytic removal of an amino-terminal fragment in order to bind the IL-1 receptor complex and have pro-inflammatory activity. This cleavage event is canonically carried out by inflammasome-activated caspase proteases, but microbe and host proteases can also generate unique active forms. Post-translational regulation of IL-1ß and the diversity in resulting products can present challenges to the evaluation of IL-1ß activation. This chapter details methods and important controls for the accurate and sensitive measurement of IL-1ß activation within biological samples.

Chemical Optimization of Selective Pseudomonas aeruginosa LasB Elastase Inhibitors and Their Impact on LasB-Mediated Activation of IL-1ß in Cellular and Animal Infection Models.

LasB elastase is a broad-spectrum exoprotease and a key virulence factor of Pseudomonas aeruginosa, a major pathogen causing lung damage and inflammation in acute and chronic respiratory infections. Here, we describe the chemical optimization of specific LasB inhibitors with druglike properties and investigate their impact in cellular and animal models of P. aeruginosa infection. Competitive inhibition of LasB was demonstrated through structural and kinetic studies. In vitro LasB inhibition was confirmed with respect to several host target proteins, namely, elastin, IgG, and pro-IL-1ß. Furthermore, inhibition of LasB-mediated IL-1ß activation was demonstrated in macrophage and mouse lung infection models. In mice, intravenous administration of inhibitors also resulted in reduced bacterial numbers at 24 h. These highly potent, selective, and soluble LasB inhibitors constitute valuable tools to study the proinflammatory impact of LasB in P. aeruginosa infections and, most importantly, show clear potential for the clinical development of a novel therapy for life-threatening respiratory infections caused by this opportunistic pathogen.

3D bioprinting of nanoparticle-laden hydrogel scaffolds with enhanced antibacterial and imaging properties.

Biomaterial-associated microbial contaminations in biologically conducive three-dimensional (3D) tissue-engineered constructs have significantly limited the clinical applications of scaffold systems. To prevent such infections, antimicrobial biomaterials are rapidly evolving. Yet, the use of such materials in bioprinting-based approaches of scaffold fabrication has not been examined. This study introduces a new generation of bacteriostatic gelatin methacryloyl (GelMA)-based bioinks, incorporated with varying doses of antibacterial superparamagnetic iron oxide nanoparticles (SPIONs). The SPION-laden GelMA scaffolds showed significant resistance against the Staphylococcus aureus growth, while providing a contrast in magnetic resonance imaging. We simulated the bacterial contamination of cellular 3D GelMA scaffolds in vitro and demonstrated the significant effect of functionalized scaffolds in inhibiting bacterial growth, while maintaining cell viability and growth. Together, these results present a new promising class of functionalized bioinks to 3D bioprint tissue-engineered scaffold with markedly enhanced properties for the use in a variety of in vitro and clinical applications.

Playing With Fire: Proinflammatory Virulence Mechanisms of Group A Streptococcus.

Group A Streptococcus is an obligate human pathogen that is a major cause of infectious morbidity and mortality. It has a natural tropism for the oropharynx and skin, where it causes infections with excessive inflammation due to its expression of proinflammatory toxins and other virulence factors. Inflammation directly contributes to the severity of invasive infections, toxic shock syndrome, and the induction of severe post-infection autoimmune disease caused by autoreactive antibodies. This review discusses what is known about how the virulence factors of Group A Streptococcus induce inflammation and how this inflammation can promote disease. Understanding of streptococcal pathogenesis and the role of hyper-immune activation during infection may provide new therapeutic targets to treat the often-fatal outcome of severe disease.

Antibiotic Treatment, Mechanisms for Failure, and Adjunctive Therapies for Infections by Group A Streptococcus.

Group A Streptococcus (GAS; Streptococcus pyogenes) is a nearly ubiquitous human pathogen responsible for a significant global disease burden. No vaccine exists, so antibiotics are essential for effective treatment. Despite a lower incidence of antimicrobial resistance than many pathogens, GAS is still a top 10 cause of death due to infections worldwide. The morbidity and mortality are primarily a consequence of the immune sequelae and invasive infections that are difficult to treat with antibiotics. GAS has remained susceptible to penicillin and other ß-lactams, despite their widespread use for 80 years. However, the failure of treatment for invasive infections with penicillin has been consistently reported since the introduction of antibiotics, and strains with reduced susceptibility to ß-lactams have emerged. Furthermore, isolates responsible for outbreaks of severe infections are increasingly resistant to other antibiotics of choice, such as clindamycin and macrolides. This review focuses on the challenges in the treatment of GAS infection, the mechanisms that contribute to antibiotic failure, and adjunctive therapeutics. Further understanding of these processes will be necessary for improving the treatment of high-risk GAS infections and surveillance for non-susceptible or resistant isolates. These insights will also help guide treatments against other leading pathogens for which conventional antibiotic strategies are increasingly failing.

3D Bioprinted Bacteriostatic Hyperelastic Bone Scaffold for Damage-Specific Bone Regeneration.

Current strategies for regeneration of large bone fractures yield limited clinical success mainly due to poor integration and healing. Multidisciplinary approaches in design and development of functional tissue engineered scaffolds are required to overcome these translational challenges. Here, a new generation of hyperelastic bone (HB) implants, loaded with superparamagnetic iron oxide nanoparticles (SPIONs), are 3D bioprinted and their regenerative effect on large non-healing bone fractures is studied. Scaffolds are bioprinted with the geometry that closely correspond to that of the bone defect, using an osteoconductive, highly elastic, surgically friendly bioink mainly composed of hydroxyapatite. Incorporation of SPIONs into HB bioink results in enhanced bacteriostatic properties of bone grafts while exhibiting no cytotoxicity. In vitro culture of mouse embryonic cells and human osteoblast-like cells remain viable and functional up to 14 days on printed HB scaffolds. Implantation of damage-specific bioprinted constructs into a rat model of femoral bone defect demonstrates significant regenerative effect over the 2-week time course. While no infection, immune rejection, or fibrotic encapsulation is observed, HB grafts show rapid integration with host tissue, ossification, and growth of new bone. These results suggest a great translational potential for 3D bioprinted HB scaffolds, laden with functional nanoparticles, for hard tissue engineering applications.