Factor H binding and function in sialylated pathogenic neisseriae is influenced by gonococcal, but not meningococcal, porin

Neisseria gonorrhoeae and Neisseria meningitidis both express the lacto-N-neotetraose (LNT) lipooligosaccharide (LOS) molecule that can be sialylated. Although gonococcal LNT LOS sialylation enhances binding of the alternative pathway complement inhibitor factor H and renders otherwise serum-sensitive bacteria resistant to complement-dependent killing, the role of LOS sialylation in meningococcal serum resistance is less clear. We show that only gonococcal, but not meningococcal, LNT LOS sialylation enhanced factor H binding. Replacing the porin (Por) B molecule of a meningococcal strain (LOS sialylated) that did not bind factor H with gonococcal Por1B augmented factor H binding. Capsule expression did not alter factor H binding to meningococci that express gonococcal Por. Conversely, replacing gonococcal Por1B with meningococcal PorB abrogated factor H binding despite LNT LOS sialylation. Gonococcal Por1B introduced in the background of an unsialylated meningococcus itself bound small amounts of factor H, suggesting a direct factor H-Por1B interaction. Factor H binding to unsialylated meningococci transfected with gonococcal Por1B was similar to the sialylated counterpart only in the presence of higher (20 microg/ml) concentrations of factor H and decreased in a dose-responsive manner by approximately 80% at 1.25 microg/ml. Factor H binding to the sialylated strain remained unchanged over this factor H concentration range however, suggesting that LOS sialylation facilitated optimal factor H-Por1B interactions. The functional counterpart of factor H binding showed that sialylated meningococcal mutants that possessed gonococcal Por1B were resistant to complement-mediated killing by normal human serum. Our data highlight the different mechanisms used by these two related species to evade complement.     

Arginase 2 deletion reduces neuro-glial injury and improves retinal function in a model of retinopathy of prematurity

Background

Retinopathy of prematurity (ROP) is a major cause of vision impairment in low birth weight infants. While previous work has focused on defining the mechanisms of vascular injury leading to retinal neovascularization, recent studies show that neurons are also affected. This study was undertaken to determine the role of the mitochondrial arginine/ornithine regulating enzyme arginase 2 (A2) in retinal neuro-glial cell injury in the mouse model of ROP.

Methods and Findings

Studies were performed using wild type (WT) and A2 knockout (A2−/−) mice exposed to Oxygen Induced Retinopathy (OIR). Neuronal injury and apoptosis were assessed using immunohistochemistry, TUNEL (terminal deoxynucleotidyl transferase dUTP nick end) labeling and Western blotting. Electroretinography (ERG) was used to assess retinal function. Neuro-glial injury in WT ROP mice was evident by TUNEL labeling, retinal thinning, decreases in number of rod bipolar cells and glial cell activation as compared with room air controls. Significant reduction in numbers of TUNEL positive cells, inhibition of retinal thinning, preservation of the rod bipolar cells and prevention of glial activation were observed in the A2−/− retinas. Retinal function was markedly impaired in the WT OIR mice as shown by decreases in amplitude of the b-wave of the ERG. This defect was significantly reduced in A2−/− mice. Levels of the pro-apoptotic proteins p53, cleaved caspase 9, cytochrome C and the mitochondrial protein Bim were markedly increased in WT OIR retinas compared to controls, whereas the pro-survival mitrochondrial protein BCL-xl was reduced. These alterations were largely blocked in the A2−/− OIR retina.

Conclusions

Our data implicate A2 in neurodegeneration during ROP. Deletion of A2 significantly improves neuronal survival and function, possibly through the regulation of mitochondrial membrane permeability mediated apoptosis during retinal ischemia. These molecular events are associated with decreased activation of glial cells, suggesting a rescue effect on macroglia as well.     

The meningococcal vaccine candidate GNA1870 binds the complement regulatory protein factor H and enhances serum resistance

Neisseria meningitidis binds factor H (fH), a key regulator of the alternative complement pathway. A approximately 29 kD fH-binding protein expressed in the meningococcal outer membrane was identified by mass spectrometry as GNA1870, a lipoprotein currently under evaluation as a broad-spectrum meningococcal vaccine candidate. GNA1870 was confirmed as the fH ligand on intact bacteria by 1) abrogation of fH binding upon deleting GNA1870, and 2) blocking fH binding by anti-GNA1870 mAbs. fH bound to whole bacteria and purified rGNA1870 representing each of the three variant GNA1870 families. We showed that the amount of fH binding correlated with the level of bacterial GNA1870 expression. High levels of variant 1 GNA1870 expression (either by allelic replacement of gna1870 or by plasmid-driven high-level expression) in strains that otherwise were low-level GNA1870 expressers (and bound low amounts of fH by flow cytometry) restored high levels of fH binding. Diminished fH binding to the GNA1870 deletion mutants was accompanied by enhanced C3 binding and increased killing of the mutants. Conversely, high levels of GNA1870 expression and fH binding enhanced serum resistance. Our findings support the hypothesis that inhibiting the binding of a complement down-regulator protein to the neisserial surface by specific Ab may enhance intrinsic bactericidal activity of the Ab, resulting in two distinct mechanisms of Ab-mediated vaccine efficacy. These data provide further support for inclusion of this molecule in a meningococcal vaccine. To reflect the critical function of this molecule, we suggest calling it fH-binding protein.     

Synthetic analogues of durantoside I from Citharexylum spinosum L. and their cytotoxic activity

New iridoid glycoside derivatives from durantoside I, the latter from the dried flowers and leaves of Citharexylum spinosum, were synthesized by variously modifying a sugar moiety by silylation or acetylation and/or removal of cinnamate group at C-7 position and subsequent screening for comparative cytotoxicity against several cancer cell lines. Addition of alkylsilane to durantoside I and removal of cinnamate group were most effective in improving cytotoxicity.     

A meningococcal factor H binding protein mutant that eliminates factor H binding enhances protective antibody responses to vaccination

Certain pathogens recruit host complement inhibitors such as factor H (fH) to evade the immune system. Microbial complement inhibitor-binding molecules can be promising vaccine targets by eliciting Abs that neutralize this microbial defense mechanism. One such Ag, meningococcal factor H-binding protein (fHbp), was used in clinical trials before the protein was discovered to bind fH. The potential effect of fH binding on vaccine immunogenicity had not been assessed in experimental animals because fHbp binds human fH specifically. In this study, we developed a human fH transgenic mouse model. Transgenic mice immunized with fHbp vaccine had 4- to 8-fold lower serum bactericidal Ab responses than those of control mice whose native fH did not bind the vaccine. In contrast, Ab responses were unimpaired in transgenic mice immunized with a control meningococcal group C polysaccharide-protein conjugate vaccine. In transgenic mice, immunization with an fH nonbinding mutant of fHbp elicited Abs with higher bactericidal activity than that of fHbp vaccination itself. Abs elicited by the mutant fHbp more effectively blocked fH binding to wild-type fHbp than Abs elicited by fHbp that bound fH. Thus, a mutant fHbp vaccine that does not bind fH but that retains immunogenicity is predicted to be superior in humans to an fHbp vaccine that binds human fH. In the case of mutant fHbp vaccination, the resultant Ab responses may be directed more at epitopes in or near the fH binding site, which result in greater complement-mediated serum bactericidal activity; these epitopes may be obscured when human fH is bound to the wild-type fHbp vaccine.     

Molecular characterization of the interaction between sialylated Neisseria gonorrhoeae and factor H

Human factor H (HufH), a key inhibitor of the alternative pathway of complement, binds to Neisseria gonorrhoeae and constitutes an important mechanism of human-specific complement evasion. The C-terminal domain 20 of HufH contains the binding site for sialylated gonococci. We exploited differences in amino acid sequences between human and non-binding chimpanzee fH domain 20 to create cross-species mutations to define amino acids important for binding to sialylated gonococci. We used fH/Fc fusion constructs that contained contiguous fH domains 18-20 fused to Fc fragments of murine IgG2a. The Fc region was used both as a tag for detection of each fusion molecule on the bacterial surface and as an indicator for complement-dependent killing. Arg-1203 was critical for binding to both porin (Por) B.1A and PorB.1B strains. Modeling of the R1203N human-to-chimpanzee mutation using the crystal structure of HufH19-20 as a template showed a loss of positive charge that protrudes at the C terminus of domain 20. We tested the functional importance of Arg-1203 by incubating sialylated gonococci with normal human serum, in the presence of wild-type HufH18-20/Fc or its R1203A mutant. Gonococci bound and were killed by wild-type HufH18-20/Fc but not by the R1203A mutant. A recombinant fH/Fc molecule that contained chimpanzee domain 20, humanized only at amino acid 1203 (N1203R) also bound to sialylated gonococci and restored killing. These findings provide further insights into the species specificity of gonococcal infections and proof-of-concept of a novel therapeutic approach against gonorrhea, a disease rapidly becoming resistant to conventional antibiotics.     

Arginase 2 Deficiency Prevents Oxidative Stress and Limits Hyperoxia-Induced Retinal Vascular Degeneration

Background

Hyperoxia exposure of premature infants causes obliteration of the immature retinal microvessels, leading to a condition of proliferative vitreoretinal neovascularization termed retinopathy of prematurity (ROP). Previous work has demonstrated that the hyperoxia-induced vascular injury is mediated by dysfunction of endothelial nitric oxide synthase resulting in peroxynitrite formation. This study was undertaken to determine the involvement of the ureahydrolase enzyme arginase in this pathology.

Methods and Findings

Studies were performed using hyperoxia-treated bovine retinal endothelial cells (BRE) and mice with oxygen-induced retinopathy (OIR) as experimental models of ROP. Treatment with the specific arginase inhibitor 2(S)-amino-6-boronohexanoic acid (ABH) prevented hyperoxia-induced apoptosis of BRE cells and reduced vaso-obliteration in the OIR model. Furthermore, deletion of the arginase 2 gene protected against hyperoxia-induced vaso-obliteration, enhanced physiological vascular repair, and reduced retinal neovascularization in the OIR model. Additional deletion of one copy of arginase 1 did not improve the vascular pathology. Analyses of peroxynitrite by quantitation of its biomarker nitrotyrosine, superoxide by dihydroethidium imaging and NO formation by diaminofluoroscein imaging showed that the protective actions of arginase 2 deletion were associated with blockade of superoxide and peroxynitrite formation and normalization of NOS activity.

Conclusions

Our data demonstrate the involvement of arginase activity and arginase 2 expression in hyperoxia-induced vascular injury. Arginase 2 deletion prevents hyperoxia-induced retinal vascular injury by preventing NOS uncoupling resulting in decreased reactive oxygen species formation and increased nitric oxide bioavailability.     

Virulence of Group A Streptococci Is Enhanced by Human Complement Inhibitors

Streptococcus pyogenes, also known as Group A Streptococcus (GAS), is an important human bacterial pathogen that can cause invasive infections. Once it colonizes its exclusively human host, GAS needs to surmount numerous innate immune defense mechanisms, including opsonization by complement and consequent phagocytosis. Several strains of GAS bind to human-specific complement inhibitors, C4b-binding protein (C4BP) and/or Factor H (FH), to curtail complement C3 (a critical opsonin) deposition. This results in diminished activation of phagocytes and clearance of GAS that may lead to the host being unable to limit the infection. Herein we describe the course of GAS infection in three human complement inhibitor transgenic (tg) mouse models that examined each inhibitor (human C4BP or FH) alone, or the two inhibitors together (C4BPxFH or ‘double’ tg). GAS infection with strains that bound C4BP and FH resulted in enhanced mortality in each of the three transgenic mouse models compared to infection in wild type mice. In addition, GAS manifested increased virulence in C4BPxFH mice: higher organism burdens and greater elevations of pro-inflammatory cytokines and they died earlier than single transgenic or wt controls. The effects of hu-C4BP and hu-FH were specific for GAS strains that bound these inhibitors because strains that did not bind the inhibitors showed reduced virulence in the ‘double’ tg mice compared to strains that did bind; mortality was also similar in wild-type and C4BPxFH mice infected by non-binding GAS. Our findings emphasize the importance of binding of complement inhibitors to GAS that results in impaired opsonization and phagocytic killing, which translates to enhanced virulence in a humanized whole animal model. This novel hu-C4BPxFH tg model may prove invaluable in studies of GAS pathogenesis and for developing vaccines and therapeutics that rely on human complement activation for efficacy.     

Molecular characterization of the interaction between porins of Neisseria gonorrhoeae and C4b-binding protein

Neisseria gonorrhoeae, the causative agent of gonorrhea, is a natural infection only in humans. The resistance of N. gonorrhoeae to normal human serum killing correlates with porin (Por)-mediated binding to the complement inhibitor, C4b-binding protein (C4BP). The entire binding site for both porin molecules resides within complement control protein domain 1 (CCP1) of C4BP. Only human and chimpanzee C4BPs bind to Por1B-bearing gonococci, whereas only human C4BP binds to Por1A strains. We have now used these species-specific differences in C4BP binding to gonococci to map the porin binding sites on CCP1 of C4BP. A comparison between human and chimpanzee or rhesus C4BP CCP1 revealed differences at 4 and 12 amino acid positions, respectively. These amino acids were targeted in the construction of 13 recombinant human mutant C4BPs. Overall, amino acids T43, T45, and K24 individually and A12, M14, R22, and L34 together were important for binding to Por1A strains. Altering D15 (found in man) to N15 (found in rhesus) introduced a glycosylation site that blocked binding to Por1A gonococci. C4BP binding to Por1B strains required K24 and was partially shielded by additional glycosylation in the D15N mutant. Only those recombinant mutant C4BPs that bound to bacteria rescued them from 100% killing by rhesus serum, thereby providing a functional correlate for the binding studies and highlighting C4BP function in gonococcal serum resistance.     

Triazolyl tryptoline derivatives as β-secretase inhibitors

Tryptoline, a core structure of ochrolifuanine E, which is a hit compound from virtual screening of the Thai herbal database against BACE1 was used as a scaffold for the design of BACE1 inhibitors. The tryptoline was linked with different side chains by 1,2,3-triazole ring readily synthesized by catalytic azide-alkyne cycloaddition reactions. Twenty two triazolyl tryptoline derivatives were synthesized and screened for the inhibitory action against BACE1. JJCA-140 was the most potent inhibitor (IC(50)=1.49 μM) and was 100 times more selective for BACE1 than for Cat-D.