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.

Utility of Human In Vitro Data in Risk Assessments of Influenza A Virus Using the Ferret Model.

As influenza A viruses (IAV) continue to cross species barriers and cause human infection, the establishment of risk assessment rubrics has improved pandemic preparedness efforts. In vivo pathogenicity and transmissibility evaluations in the ferret model represent a critical component of this work. As the relative contribution of in vitro experimentation to these rubrics has not been closely examined, we sought to evaluate to what extent viral titer measurements over the course of in vitro infections are predictive or correlates of nasal wash and tissue measurements for IAV infections in vivo. We compiled data from ferrets inoculated with an extensive panel of over 50 human and zoonotic IAV (inclusive of swine-origin and high- and low-pathogenicity avian influenza viruses associated with human infection) under a consistent protocol, with all viruses concurrently tested in a human bronchial epithelial cell line (Calu-3). Viral titers in ferret nasal wash specimens and nasal turbinate tissue correlated positively with peak titer in Calu-3 cells, whereas additional phenotypic and molecular determinants of influenza virus virulence and transmissibility in ferrets varied in their association with in vitro viral titer measurements. Mathematical modeling was used to estimate more generalizable key replication kinetic parameters from raw in vitro viral titers, revealing commonalities between viral infection progression in vivo and in vitro. Meta-analyses inclusive of IAV that display a diverse range of phenotypes in ferrets, interpreted with mathematical modeling of viral kinetic parameters, can provide critical information supporting a more rigorous and appropriate contextualization of in vitro experiments toward pandemic preparedness. IMPORTANCE Both in vitro and in vivo models are employed for assessing the pandemic potential of novel and emerging influenza A viruses in laboratory settings, but systematic examinations of how well viral titer measurements obtained in vitro align with results from in vivo experimentation are not frequently performed. We show that certain viral titer measurements following infection of a human bronchial epithelial cell line are positively correlated with viral titers in specimens collected from virus-inoculated ferrets and employ mathematical modeling to identify commonalities between viral infection progression between both models. These analyses provide a necessary first step in enhanced interpretation and incorporation of in vitro-derived data in risk assessment activities and highlight the utility of employing mathematical modeling approaches to more closely examine features of virus replication not identifiable by experimental studies alone.

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.

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.

Antibiotic-Selected Gene Amplification Heightens Metal Resistance.

The increasing frequency of antibiotic resistance poses myriad challenges to modern medicine. Environmental survival of multidrug-resistant bacteria in health care facilities, including hospitals, creates reservoirs for transmission of these difficult to treat pathogens. To prevent bacterial colonization, these facilities deploy an array of infection control measures, including bactericidal metals on surfaces, as well as implanted devices. Although antibiotics are routinely used in these health care environments, it is unknown whether and how antibiotic exposure affects metal resistance. We identified a multidrug-resistant Enterobacter clinical isolate that displayed heteroresistance to the antibiotic colistin, where only a minor fraction of cells within the population resist the drug. When this isolate was grown in the presence of colistin, a 9-kb DNA region was duplicated in the surviving resistant subpopulation, but surprisingly, was not required for colistin heteroresistance. Instead, the amplified region included a three-gene locus (ncrABC) that conferred resistance to the bactericidal metal, nickel. ncrABC expression alone was sufficient to confer nickel resistance to E. coli K-12. Due to its selection for the colistin-resistant subpopulation harboring the duplicated 9-kb region that includes ncrABC, colistin treatment led to enhanced nickel resistance. Taken together, these data suggest that the use of antibiotics may inadvertently promote enhanced resistance to antimicrobial metals, with potentially profound implications for bacterial colonization and transmission in the health care environment.IMPORTANCE To inhibit bacterial transmission and infection, health care facilities use bactericidal metal coatings to prevent colonization of surfaces and implanted devices. In these environments, antibiotics are commonly used, but their effect on metal resistance is unclear. The data described here reveal that exposure of a human isolate of Enterobacter cloacae to a last-line antibiotic, colistin, resulted in a DNA amplification that does not confer antibiotic resistance but instead facilitates resistance to the toxic metal nickel. This highlights a novel aspect of antibiotic and metal interplay. Concerningly, these data suggest the use of antibiotics could in some cases promote bacterial survival and colonization in the health care environment and ultimately increase transmission and infection of patients.