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Necrotic enteritis is an economically important poultry disease caused by the bacterium Clostridium perfringens. There are currently no necrotic enteritis vaccines commercially available for use in broiler birds, the most important target population. Salmonella-vectored vaccines represent a convenient and effective option for controlling this disease. We used a single attenuated Salmonella vaccine strain, engineered to lyse within the host, to deliver up to three C. perfringens antigens. Two of the antigens were toxoids, based on C. perfringens α-toxin and NetB toxin. The third antigen was fructose-1,6-bisphosphate aldolase (Fba), a metabolic enzyme with an unknown role in virulence. Oral immunization with a single Salmonella vaccine strain producing either Fba, α-toxoid and NetB toxoid, or all three antigens, was immunogenic, inducing serum, cellular and mucosal responses against Salmonella and the vectored C. perfringens antigens. All three vaccine strains were partially protective against virulent C. perfringens challenge. The strains delivering Fba only or all three antigens provided the best protection. We also demonstrate that both toxins and Fba are present on the C. perfringens cell surface. The presence of Fba on the cell surface suggests that Fba may function as an adhesin.

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.

Necrotic enteritis (NE) is caused by type A strains of the bacterium . Total global economic losses to the poultry industry due to NE is estimated to be over two billion dollars annually. Traditionally, NE has been effectively controlled by inclusion of antibiotics in the diet of poultry. However, recent concerns regarding the impact of this practice on increasing antibiotic resistance in human pathogens have led us to consider alternative approaches, such as vaccination, for controlling this disease. NE strains of produce two major toxins, a-toxin and NetB. Immune responses against either toxin can provide partial protection against NE. We have developed a fusion protein combining a non-toxic carboxyl-terminal domain of a-toxin (PlcC) and an attenuated, mutant form of NetB (NetB-W262A) for use as a vaccine antigen to immunize poultry against NE. We utilized a DNA sequence that was codon-optimized for to enable high levels of expression. The 6-His tagged PlcC-NetB fusion protein was synthesized in using a geminiviral replicon transient expression system, purified by metal affinity chromatography, and used to immunize broiler birds. Immunized birds produced a strong serum IgY response against both the plant produced PlcC-NetB protein and against bacterially produced His-PlcC and His-NetB. Immunized birds were significantly protected against a subsequent in-feed challenge with virulent when treated with the fusion protein. These results indicate that a plant-produced PlcC-NetB toxoid is a promising vaccine candidate for controlling NE in poultry.

Gasdermins (GSDMs) are a family of pore-forming effectors that permeabilize the cell membrane during the cell death program pyroptosis 1 . GSDMs are activated by proteolytic removal of autoinhibitory carboxy-terminal domains, typically by caspase regulators 1 – 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 skin 10 , 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. Group A Streptococcus secretes a protease, SpeB, that directly cleaves and activates gasdermin A to induce pyroptosis of infected keratinocytes, demonstrating a role for gasdermin A in immune defence against invasive microorganisms.

Streptococcus pyogenes (group A Streptococcus; GAS) is an exclusively human pathogen that causes roughly 600 million infections annually. Infection can be relatively mild in the form of ‘strep throat’ or pharyngitis, or manifest as an invasive infection such as bacteremia, streptococcal toxic shock syndrome, or necrotizing fasciitis. GAS infection can also result in post-infectious immune sequelae like rheumatic fever and rheumatic heart disease. GAS causes an estimated 18 million invasive infections and 600,000 annual deaths due to these diseases.Invasive GAS in immunocompetent individuals is largely linked to hypervirulent strains, such as strain 5448, that have developed strategies to overcome host immune defenses. Congenital immunodeficiencies and those acquired from chronic disease or immunosuppressant drugs also increase risk of severe illness, specifically, drugs inhibiting Interleukin-1 or -6 (IL-1/6) signaling. We recovered a non-hypervirulent strain of GAS, M4C20, from the blood of a patient receiving a biologic inhibitor of IL-6. Survival of this strain and 5448 were markedly different in both in vitro and in vivo infection models. M4C20 was only virulent in the presence of IL-1 or IL-6 inhibitors, but 5448 was broadly virulent and resisted IL-6-mediated killing. These findings introduced IL-6 signaling deficiencies as a risk factor for invasive GAS infection, but the mechanism by which IL-6 contributes to GAS killing remained unclear.Our work was later able to demonstrate that IL-6 acts against GAS by inducing the production of antimicrobial reactive oxygen species (ROS). GAS lacks catalase, a virulence factor used by many diverse species for detoxifying ROS, yet some strains of GAS can withstand ROS and cause severe disease. Through analysis of clinical isolates, we found that the capsule of GAS, composed of hyaluronic acid, also confers protection against ROS. We also showed that hyaluronic acid can act as a direct antioxidant against ROS in vitro. Nonetheless, we find that in vivo ROS is not essential for killing of GAS in an intradermal infection model. However, lesion size was significantly impacted by both the absence of ROS in host cells and production of capsule by GAS, supporting a model in which ROS and hyaluronic acid regulate pathology during invasive GAS skin infections.

Background. Necrotic enteritis (NE) is caused by type A strains of the bacterium Clostridium perfringens. Total global economic losses to the poultry industry due to NE is estimated to be over 2 billion dollars annually. Traditionally, NE has been effectively controlled by inclusion of antibiotics in the diet of poultry. However, recent concerns regarding the impact of this practice on increasing antibiotic resistance in human pathogens have led us to consider alternative approaches, such as vaccination, for controlling this disease. NE strains of C. perfringensproduce two major toxins, a-toxin and NetB. Immune responses against either toxin can provide partial protection against NE. Methods. We have developed a fusion protein combining a non-toxic carboxyl-terminal domain of a-toxin (PlcC) and an attenuated, mutant form of NetB (NetB-W262A) for use as a vaccine antigen to immunize poultry against NE. We utilized a DNA sequence that was codon-optimized for Nicotiana benthamianato enable high levels of expression. The 6-His tagged PlcC-NetB fusion protein was synthesized in N. benthamianausing a geminiviral replicon transient expression system, purified by metal affinity chromatography, and used to immunize broiler birds. Results. Immunized birds produced a strong serum IgY response against both the plant produced PlcC-NetB protein and against bacterially produced His-PlcC and His-NetB. Immunized birds were significantly protected against a subsequent in-feed challenge with virulent C. perfringenswhen treated with the fusion protein. These results indicate that a plant-produced PlcC-NetB toxoid is a promising vaccine candidate for controlling NE in poultry.

Group A Streptococcus (GAS) is the etiologic agent of numerous high morbidity and high mortality diseases which commonly have a highly proinflammatory pathology. One factor contributing to this inflammation is the GAS protease SpeB, which directly activates the proinflammatory cytokine interleukin-1β (IL-1β), independent of the canonical inflammasome pathway. IL-1β drives neutrophil activation and recruitment that limits bacterial growth and invasion during invasive skin and soft tissue infections like necrotizing fasciitis. GAS also causes pharyngitis (strep throat), and the upper respiratory tract is its primary nidus for growth and transmission. Since the fitness selection for the species is likely primarily for this site, we examined the process of IL-1β activation in the murine nasopharynx. SpeB still activated IL-1β, which was required for neutrophil migration, but this inflammation instead increased GAS replication. Inhibiting IL-1β or depleting neutrophils, which both promote invasive infection, prevented GAS infection of the nasopharynx. Prior antibiotic exposure increased GAS growth in the murine nasopharynx, and antibiotics were sufficient to reverse the attenuation previously observed when IL-1β, neutrophils, or SpeB were not present to drive inflammation. Therefore, the same fundamental mechanism has opposing effects on virulence at different body sites. Invasive disease may be limited in part due to specific adaptations for inducing host inflammation that are beneficial for pharyngitis.

Necrotic enteritis is an economically important poultry disease caused by the bacterium Clostridium perfringens. There are currently no necrotic enteritis vaccines available for use in broiler birds, the most important target population. Salmonella-vectored vaccines represent a convenient and effective option for controlling this disease. We used a single attenuated Salmonella vaccine strain, engineered to lyse within the host, to deliver up to three C. perfringens antigens. Two of the antigens were toxoids, based on C. perfringens ?-toxin and NetB toxin. The third antigen was fructose-1,6-bisphosphate aldolase (Fba), an metabolic enzyme with an unknown role in virulence. Oral immunization with a single Salmonella vaccine strain producing either Fba, ?-toxoid and NetB toxoid, or all three antigens, was immunogenic, inducing serum, cellular and mucosal responses against Salmonella and the vectored C. perfringens antigens. All three vaccine strains were protective against virulent C. perfringens challenge. The strains delivering Fba only or all three antigens provided the best protection. We also demonstrate that both toxins and Fba are present on the C. perfringens cell surface. The presence of Fba on the cell surface suggests that Fba may function as an adhesin.