Multidrug-resistant (MDR) gram-negative bacteria have increased the prevalence of fatal sepsis in modern times. Colistin is a cationic antimicrobial peptide (CAMP) antibiotic that permeabilizes the bacterial outer membrane (OM) and has been used to treat these infections. The OM outer leaflet is comprised of endotoxin containing lipid A, which can be modified to increase resistance to CAMPs and prevent clearance by the innate immune response. One type of lipid A modification involves the addition of phosphoethanolamine to the 1 and 4′ headgroup positions by phosphoethanolamine transferases. Previous structural work on a truncated form of this enzyme suggested that the full-length protein was required for correct lipid substrate binding and catalysis. We now report the crystal structure of a full-length lipid A phosphoethanolamine transferase from Neisseria meningitidis, determined to 2.75-Å resolution. The structure reveals a previously uncharacterized helical membrane domain and a periplasmic facing soluble domain. The domains are linked by a helix that runs along the membrane surface interacting with the phospholipid head groups. Two helices located in a periplasmic loop between two transmembrane helices contain conserved charged residues and are implicated in substrate binding. Intrinsic fluorescence, limited proteolysis, and molecular dynamics studies suggest the protein may sample different conformational states to enable the binding of two very different-sized lipid substrates. These results provide insights into the mechanism of endotoxin modification and will aid a structure-guided rational drug design approach to treating multidrug-resistant bacterial infections.lipid modification | multidrug resistance | molecular dynamics | Neisseria | membrane protein structure

Neisseria gonorrhoeae is a common sexually transmitted pathogen that significantly impacts female fertility, neonatal health, and transmission of HIV worldwide. N. gonorrhoeae usually causes localized inflammation of the urethra and cervix by inducing production of IL-1␤ and other inflammatory cytokines. Several NLR (nucleotide-binding domain, leucine-rich repeat) proteins are implicated in the formation of pro-IL-1␤-processing complexes called inflammasomes in response to pathogens. We demonstrate that NLRP3 (cryopyrin, NALP3) is the primary NLR required for IL-1␤/IL-18 secretion in response to N. gonorrhoeae in monocytes. We also show that N. gonorrhoeae infection promotes NLRP3-dependent monocytic cell death via pyronecrosis, a recently described pathway with morphological features of necrosis, including release of the strong inflammatory mediator HMBG1. Additionally, N. gonorrhoeae activates the cysteine protease cathepsin B as measured by the breakdown of a cathepsin B substrate. Inhibition of cathepsin B shows that this protease is an apical controlling step in the downstream activities of NLRP3 including IL-1␤ production, pyronecrosis, and HMGB1 release. Nonpathogenic Neisseria strains (Neisseria cinerea and Neisseria flavescens) do not activate NLRP3 as robustly as N. gonorrhoeae. Conditioned medium from N. gonorrhoeae contains factors capable of initiating the NLRP3-mediated signaling events. Isolated N. gonorrhoeae lipooligosaccharide, a known virulence factor from this bacterium that is elaborated from the bacterium in the form of outer membrane blebs, activates both NLRP3-induced IL-1␤ secretion and pyronecrosis. Our findings indicate that activation of NLRP3-mediated inflammatory response pathways is an important venue associated with host response and pathogenesis of N.

SummaryGspB and Hsa are homologous serine-rich surface glycoproteins of Streptococcus gordonii strains M99 and Challis, respectively, that mediate the binding of these organisms to platelet membrane glycoprotein (GP) Ib α α α α . Both GspB and Hsa consist of an N-terminal putative signal peptide, a short serine-rich region, a region (BR) that is rich in basic amino acids, a longer serine-rich region and a C-terminal cell wall anchoring domain. To further assess the mechanisms for GspB and Hsa binding, we investigated the binding of the BRs of GspB and Hsa (expressed as glutathione Stranferase fusion proteins) to sialylated glycoproteins in vitro . Both fusion proteins showed significant levels of binding to sialylated moieties on fetuin and GPIb α α α α . In contrast, the corresponding region of a GspB homologue of Streptococcus agalactiae , which is acidic rather than basic, showed no binding to either fetuin or GPIb α α α α . As measured by surface plasmon resonance kinetic analysis, GspB-and Hsaderived fusion proteins had high affinity for GPIb α α α α , but with somewhat different dissociation constants. Dot blot analysis using a panel of synthesized oligosaccharides revealed that the BR of Hsa can bind both α α α α (2-3) sialyllactosamine [NeuAc α α α α (2-3)Gal β β β β (1-4)GlcNAc] and sialyl-T antigen [NeuAc α α α α (2-3)Gal β β β β (1-3)GalNAc], whereas the BR of GspB only bound sialyl-T antigen. Moreover, far Western blotting using platelet membrane proteins revealed that GPIb α α α α is the principal receptor for GspB and Hsa on human platelets. The combined results indicate that the BRs of GspB and Hsa are the binding domains of these adhesins. However, the subsets of carbohydrate structures on GPIb α α α α recognized by the binding domains appear to be different between the two proteins.