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Many bacterial surface glycans such as the peptidoglycan (PG) cell wall are built from monomeric units linked to a polyprenyl lipid carrier. How this limiting carrier is distributed among competing pathways has remained unclear. Here we describe the isolation of hyperactive variants of Pseudomonas aeruginosa MraY, the enzyme that forms the first lipid-linked PG precursor. These variants result in the elevated production of the final PG precursor lipid II in cells and are hyperactive in vitro. The activated MraY variants have substitutions that map to a cavity on the extracellular side of the dimer interface, far from the active site. Our structural and molecular dynamics results suggest that this cavity is a binding site for externalized lipid II. Overall, our results support a model in which excess externalized lipid II allosterically inhibits MraY, providing a feedback mechanism that prevents the sequestration of lipid carrier in the PG biogenesis pathway., (© 2024. The Author(s).)

A cell wall made of the heteropolymer peptidoglycan (PG) surrounds most bacterial cells. This essential surface layer is required to prevent lysis from internal osmotic pressure. The class A penicillin-binding proteins (aPBPs) play key roles in building the PG network. These bifunctional enzymes possess both PG glycosyltransferase (PGT) and transpeptidase (TP) activity to polymerize the wall glycans and cross-link them, respectively. In Escherichia coli and other gram-negative bacteria, aPBP function is dependent on outer membrane lipoproteins. The lipoprotein LpoA activates PBP1a and LpoB promotes PBP1b activity. In a purified system, the major effect of LpoA on PBP1a is TP stimulation. However, the relevance of this activation to the cellular function of LpoA has remained unclear. To better understand why PBP1a requires LpoA for its activity in cells, we identified variants of PBP1a from E. coli and Pseudomonas aeruginosa that function in the absence of the lipoprotein. The changes resulting in LpoA bypass map to the PGT domain and the linker region between the two catalytic domains. Purification of the E. coli variants showed that they are hyperactivated for PGT but not TP activity. Furthermore, in vivo analysis found that LpoA is necessary for the glycan synthesis activity of PBP1a in cells. Thus, our results reveal that LpoA exerts a much greater control over the cellular activity of PBP1a than previously appreciated. It not only modulates PG cross-linking but is also required for its cognate synthase to make PG glycans in the first place., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Competing Interests: Competing interests: Norm Campbell was a paid consultant to the Novartis Foundation (2016–2017) to support its program to improve hypertension control in low-to middle-income countries, which included travel support for site visits and a contract to develop a survey. Norm Campbell has provided paid consultative advice on accurate blood pressure assessment to Midmark Corporation (2017) and is an unpaid member of World Action on Salt and Health. As a senator, Nancy Greene Raine introduced Bill S-228, the Child Health Protection Act, and during its development met with professionals working in the health field, in particular those active in the Stop Marketing to Kids Coalition.

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 Ec LpoB was identified as a critical activator of Escherichia coli PBP1b ( Ec PBP1b), one of the major PG synthases of this organism. Activation of Ec PBP1b is mediated through the association of Ec LpoB with a regulatory domain on Ec PBP1b called UB2H. Notably, Pseudomonas aeruginosa also encodes PBP1b ( Pa PBP1b), which possesses a UB2H domain, but this bacterium lacks an identifiable LpoB homolog. We therefore searched for potential Pa PBP1b activators and identified a lipoprotein unrelated to LpoB that is required for the in vivo activity of Pa PBP1b. We named this protein LpoP and found that it interacts directly with Pa PBP1b in vitro and is conserved in many Gram-negative species. Importantly, we also demonstrated that Pa LpoP- Pa PBP1b as well as an equivalent protein pair from Acinetobacter baylyi can fully substitute for Ec LpoB- Ec PBP1b in E. coli for PG synthesis. Furthermore, we show that amino acid changes in Pa PBP1b that bypass the Pa LpoP requirement map to similar locations in the protein as changes promoting Ec LpoB bypass in Ec 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., Competing Interests: The authors declare no conflict of interest.

Although youth are increasingly going online to fulfill their needs for information, many youth struggle with information and digital literacy skills, such as the abilities to conduct a search and assess the credibility of online information. Ideally, these skills encompass an accurate and comprehensive understanding of the ways in which a system, such as a Web search engine, functions. In order to investigate youths' conceptions of the Google search engine, a drawing activity was conducted with 26 HackHealth after-school program participants to elicit their mental models of Google. The findings revealed that many participants personified Google and emphasized anthropomorphic elements, computing equipment, and/or connections (such as cables, satellites and antennas) in their drawings. Far fewer participants focused their drawings on the actual Google interface or on computer code. Overall, their drawings suggest a limited understanding of Google and the ways in which it actually works. However, an understanding of youths' conceptions of Google can enable educators to better tailor their digital literacy instruction efforts and can inform search engine developers and search engine interface designers in making the inner workings of the engine more transparent and their output more trustworthy to young users. With a better understanding of how Google works, young users will be better able to construct effective queries, assess search results, and ultimately find relevant and trustworthy information that will be of use to them., Competing Interests: Conflict of interestThe authors declare that they have no conflict of interest., (© The Author(s) 2017.)

Streptococcus pneumoniae (the pneumococcus) colonizes the human nasopharynx and is a significant pathogen worldwide. Pneumolysin (Ply) is a multi-functional, extracellular virulence factor produced by this organism that is critical for pathogenesis. Despite the absence of any apparent secretion or cell surface attachment motifs, Ply localizes to the cell envelope of actively growing cells. We sought to characterize the consequences of this surface localization. Through functional assays with whole cells and subcellular fractions, we determined that Ply activity and its release into the extracellular environment are inhibited by peptidoglycan (PG) structure. The ability of PG to inhibit Ply release was dependent on the stem peptide composition of this macromolecule, which was manipulated by mutation of the murMN operon that encodes proteins responsible for branched stem peptide synthesis. Additionally, removal of choline-binding proteins from the cell surface significantly reduced Ply release to levels observed in a mutant with a high proportion of branched stem peptides suggesting a link between this structural feature and surface-associated choline-binding proteins involved in PG metabolism. Of clinical relevance, we also demonstrate that a hyperactive, mosaic murMN allele associated with penicillin resistance causes decreased Ply release with concomitant increases in the amount of branched stem peptides. Finally, using a murMN deletion mutant, we observed that increased Ply release is detrimental to virulence during a murine model of pneumonia. Taken together, our results reveal a novel role for branched stem peptides in pneumococcal pathogenesis and demonstrate the importance of controlled Ply release during infection. These results highlight the importance of PG composition in pathogenesis and may have broad implications for the diverse PG structures observed in other bacterial pathogens.

Background: Although a low health literacy level has been found to be among the most powerful predictors of poor health outcomes, there is very little research focused on assessing and improving the health literacy skills of adolescents, particularly those from socioeconomically disadvantaged backgrounds. The vast majority of existing research focuses solely on reading comprehension, despite the fact that health literacy is actually a multifaceted concept, which entails many different types of skills., Objective: The aim of this paper is to first mine existing literature to identify the many different skills that have been posited to constitute health literacy, and then, using this collection of skills as an overarching structure, to highlight the challenges that disadvantaged youth participating in our HackHealth after-school program encounter as they identify and articulate their health-related information needs, search for health-related information online, assess the relevance and credibility of this information, and manage and make use of it., Methods: We utilized the design-based research method to design, implement, and revise our HackHealth program. To collect data regarding HackHealth participants' health literacy skills and associated challenges, we used a variety of methods, including participant observation, surveys, interviews, focus groups, and logging of Web browser activities. We also collected data through specialized instructional activities and data collection forms that we developed for this purpose. Quantitative and qualitative techniques were used to analyze this data, as well as all of the artifacts that each student produced, including their final projects., Results: We identified the various challenges that the 30 HackHealth participants faced in completing various health-related information activities during the course of the program. Based on these findings, we describe important implications for working with youth from socioeconomically disadvantaged backgrounds, how to assess and improve their health literacy skills, and offer specific recommendations for health literacy instruction aimed at this population., Conclusions: With an increased societal focus on health and a shift from viewing patients as passive recipients of medical care to viewing them as active arbiters of their own health, today's youth need to possess an array of health literacy skills to ensure that they can live long and healthy lives. Working with adolescents to help them develop and practice these skills will also help to break the cycle between poor health literacy and poor health outcomes, thereby reducing health disparities and improving the long-term outlook for the health of our nation.

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 present 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 we showed that Nod-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (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 (YVAD-FLICA 660), and bone marrow neutrophils stimulated with heat-killed S. pneumoniae (signal 1) and pneumolysin (signal 2) exhibited multiple specks when stained for NLRP3, ASC, or Caspase-1. High-molecular mass 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. Taken together, these findings demonstrate an essential role for neutrophil-derived IL-1β in S. pneumoniae infection, and they elucidate the role of the NLRP3 inflammasome in cleavage and secretion of IL-1β in neutrophils. Given the ubiquitous presence of neutrophils in acute bacterial and fungal infections, these findings will have implications for other microbial diseases., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Herpes 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.

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.