Although neutrophils are the most abundant cells in acute infection and inflammation, relatively little attention has been paid to their role in inflammasome formation and IL-1β processing. In the current study, we investigated the mechanism by which neutrophils process IL-1β in response to Streptococcus pneumoniae. Using a murine model of S. pneumoniae corneal infection, we demonstrated a requirement for IL-1β in bacterial clearance, and showed that NLRP3, ASC and caspase-1 are essential for IL-1β production and bacterial killing in the cornea. Neutrophils in infected corneas had multiple specks with enzymatically active caspase-1 (FLICA-660+), and bone marrow neutrophils stimulated with heat killed S. pneumoniae (signal 1) and pneumolysin (signal 2) exhibited multiple specks after staining with FLICA-660, NLRP3 or ASC. High molecular weight ASC complexes were also detected, consistent with oligomer formation. Pneumolysin induced K+ efflux in neutrophils, and blocking K+ efflux inhibited caspase-1 activation and IL-1β processing; however, neutrophils did not undergo pyroptosis, indicating that K+ efflux and IL-1β processing is not a consequence of cell death. There was also no role for lysosomal destabilization or neutrophil elastase in pneumolysin mediated IL-1β processing in neutrophils. Together, these findings demonstrate an essential role for neutrophil derived IL-1β in S. pneumoniae infection, and elucidate the role of the NLRP3 inflammasome in neutrophil cleavage and secretion of mature IL-1β. Given the ubiquitous presence of neutrophils in acute bacterial and fungal infections, these findings will have implications for other microbial diseases.

bHerpes simplex virus type 1 (HSV-1) enters cells by fusion of its envelope with a host cell membrane, which requires four viral glycoproteins and a cellular receptor. Viral fusion glycoprotein B (gB) mediates membrane fusion through the action of its ectodomain, while its cytoplasmic domain (cytodomain) regulates fusion from the opposite face of the membrane by an unknown mechanism. The gB cytodomain appears to restrict fusion, because point or truncation mutations within it increase the extent of fusion (syn mutations). Previously, we showed that the hyperfusion phenotype correlated with reduced membrane binding in gB syn truncation mutants and proposed that membrane binding was important in regulating fusion. Here, we extended our analysis to three syn point mutants: A855V, R858H, and A874P. These mutations produce local conformational changes, with some affecting membrane interaction, which suggests that while syn mutants may deregulate fusion by somewhat different mechanisms, maintaining the wild-type (WT) conformation is critical for fusion regulation. We further show that the presence of a membrane is necessary for the cytodomain to achieve its fully folded conformation and propose that the membrane-bound form of the cytodomain represents its native conformation. Taken together, our data suggest that the cytodomain of gB regulates fusion by a novel mechanism in which membrane interaction plays a key role. Herpes simplex virus type 1 (HSV-1) causes diseases ranging from mild skin lesions to fatal encephalitis and neonatal herpes (28). Like all herpesviruses, HSV-1 enters host cells by fusion of the viral envelope with the plasma membrane or an endocytic vesicle. While the specific viral glycoproteins required for entry differ among herpesviruses, glycoprotein B (gB) and the heterodimer gH/gL are conserved. Entry of HSV-1 requires gD, gB, and gH/gL, in addition to a host cell receptor (Fig. 1A). These five proteins are also sufficient to mediate fusion of transiently transfected cells in the absence of any other viral proteins (47).gD initiates entry by binding one of its three cellular receptors (44). The exact role of gH/gL in entry remains unclear; however, recent evidence suggests that binding of a receptor by gD activates gH/gL, which in turn activates gB for fusion (3). gB is a class III viral fusion protein that enables membrane fusion, presumably by undergoing dramatic conformational changes that provide the energy to drive fusion (25, 29). However, unlike fusion proteins of other enveloped viruses, e.g., influenza virus hemagglutinin (HA) or HIV gp160, which accomplish fusion independently (15), HSV-1 gB mediates fusion only in the presence of gD, a gD receptor, and gH/gL (39, 47). gB is a trimeric, type I transmembrane protein with an N-terminal ectodomain displayed on the virion surface, a single-pass transmembrane helix, and a C-terminal cytoplasmic domain (cytodomain) situated on the opposite face of the viral envelope (29) (Fig. 1A). The ectodomain is presumably directly involved in the membra...

Penicillin-binding proteins (PBPs) are synthases required to build the essential peptidoglycan (PG) cell wall surrounding most bacterial cells. The mechanisms regulating the activity of these enzymes to control PG synthesis remain surprisingly poorly defined given their status as key antibiotic targets. Several years ago, the outer-membrane lipoprotein LpoB was identified as a critical activator of PBP1b (PBP1b), one of the major PG synthases of this organism. Activation of PBP1b is mediated through the association ofLpoB with a regulatory domain on PBP1b called UB2H. Notably, also encodes PBP1b (PBP1b), which possesses a UB2H domain, but this bacterium lacks an identifiable LpoB homolog. We therefore searched for potential PBP1b activators and identified a lipoprotein unrelated to LpoB that is required for the in vivo activity ofPBP1b. We named this protein LpoP and found that it interacts directly with PBP1b in vitro and is conserved in many Gram-negative species. Importantly, we also demonstrated thatLpoP-PBP1b as well as an equivalent protein pair from can fully substitute forLpoB-PBP1b in for PG synthesis. Furthermore, we show that amino acid changes inPBP1b that bypass the LpoP requirement map to similar locations in the protein as changes promotingLpoB bypass in PBP1b. Overall, our results indicate that, although different Gram-negative bacteria activate their PBP1b synthases with distinct lipoproteins, they stimulate the activity of these important drug targets using a conserved mechanism.

Streptococcus pneumoniae is a major causative agent of otitis media, pneumonia, bacteremia, and meningitis. Pneumolysin (Ply), a member of the cholesterol-dependent cytolysins (CDCs), is produced by virtually all clinical isolates of S. pneumoniae, and ply mutant strains are severely attenuated in mouse models of colonization and infection. In contrast to all other known members of the CDC family, Ply lacks a signal peptide for export outside the cell. Instead, Ply has been hypothesized to be released upon autolysis or, alternatively, via a nonautolytic mechanism that remains undefined. We show that an exogenously added signal sequence is not sufficient for Sec-dependent Ply secretion in S. pneumoniae but is sufficient in the surrogate host Bacillus subtilis. Previously, we showed that Ply is localized primarily to the cell wall compartment in the absence of detectable cell lysis. Here we show that Ply released by autolysis cannot reassociate with intact cells, suggesting that there is a Ply export mechanism that is coupled to cell wall localization of the protein. This putative export mechanism is capable of secreting a related CDC without its signal sequence. We show that B. subtilis can export Ply, suggesting that the export pathway is conserved. Finally, through truncation and domain swapping analyses, we show that export is dependent on domain 2 of Ply.