Gary A. Jarvis

Neisseria gonorrhoeae Activates the Proteinase Cathepsin B to Mediate the Signaling Activities of the NLRP3 and ASC-Containing Inflammasome

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-1β and other inflammatory cytokines. Several NLR (nucleotide-binding domain, leucine-rich repeat) proteins are implicated in the formation of pro-IL-1β-processing complexes called inflammasomes in response to pathogens. We demonstrate that NLRP3 (cryopyrin, NALP3) is the primary NLR required for IL-1β/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-1β 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-1β 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. gonorrhoeae.

Binding of the Streptococcus gordonii surface glycoproteins GspB and Hsa to specific carbohydrate structures on platelet membrane glycoprotein Ibα

GspB 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) Ibα. 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 S‐tranferase fusion proteins) to sialylated glycoproteins in vitro. Both fusion proteins showed significant levels of binding to sialylated moieties on fetuin and GPIbα. 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 GPIbα. As measured by surface plasmon resonance kinetic analysis, GspB‐ and Hsa‐derived fusion proteins had high affinity for GPIbα, 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(2‐3) sialyllactosamine [NeuAcα(2‐3)Galβ(1‐4)GlcNAc] and sialyl‐T antigen [NeuAcα(2‐3)Galβ(1‐3)GalNAc], whereas the BR of GspB only bound sialyl‐T antigen. Moreover, far Western blotting using platelet membrane proteins revealed that GPIbα 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 GPIbα recognized by the binding domains appear to be different between the two proteins.

Candidate Sulfonated and Sulfated Topical Microbicides: Comparison of Anti-Human Immunodeficiency Virus Activities and Mechanisms of Action

ABSTRACT Poly(styrene 4-sulfonate), cellulose sulfate, polymethylenehydroquinone, and PRO 2000 are sulfated or sulfonated polymers (SPs) under development as topical microbicides. They are presumed to work through similar mechanisms of action, although to date there has been no extensive comparison of their anti-human immunodeficiency virus activities. To determine whether any of these candidate microbicides offers a potential advantage, their in vitro activities, mechanisms of action, stabilities in biological secretions, and toxicities were compared. All four compounds were found to be active against X4, R5, and dualtropic primary isolates and against X4 and R5 laboratory-adapted strains in CD4+ T cells, macrophages, and single-coreceptor cell lines. Our single-cycle experiments using pseudotyped virus suggest that all four SPs function at the binding and entry stages of the viral life cycle but differ in degree of postentry effect. Surface plasmon resonance analyses demonstrate that SPs bind to X4 and R5 monomeric glycoprotein 120 with similar high binding affinities. When mixed with cervicovaginal lavage fluid, SPs maintain inhibitory activity at concentrations achievable in formulations.

Candidate Topical Microbicides Bind Herpes Simplex Virus Glycoprotein B and Prevent Viral Entry and Cell-to-Cell Spread

ABSTRACT Topical microbicides designed to prevent acquisition of sexually transmitted infections are urgently needed. Nonoxynol-9, the only commercially available spermicide, damages epithelium and may enhance human immunodeficiency virus transmission. The observation that herpes simplex virus (HSV) and human immunodeficiency virus bind heparan sulfate provided the rationale for the development of sulfated or sulfonated polymers as topical agents. Although several of the polymers have advanced to clinical trials, the spectrum and mechanism of anti-HSV activity and the effects on soluble mediators of inflammation have not been evaluated. The present studies address these gaps. The results indicate that PRO 2000, polystyrene sulfonate, cellulose sulfate, and polymethylenehydroquinone sulfonate inhibit HSV infection 10,000-fold and are active against clinical isolates, including an acyclovir-resistant variant. The compounds formed stable complexes with glycoprotein B and inhibit viral binding, entry, and cell-to-cell spread. The effects may be long lasting due to the high affinity and stability of the sulfated compound-virus complex, as evidenced by surface plasmon resonance studies. The candidate microbicides retained their antiviral activities in the presence of cervical secretions and over a broad pH range. There was little reduction in cell viability following repeated exposure of human endocervical cells to these compounds, although a reduction in secretory leukocyte protease inhibitor levels was observed. These studies support further development and rigorous evaluation of these candidate microbicides.

Expression and function of the complement membrane attack complex inhibitor protectin (CD59) in human prostate cancer

Protectin (CD59) inhibits homologous complement‐mediated cytolysis by preventing formation of the membrane attack complex at the point of insertion and polymerization of C9 into cell membranes. The present study investigated the expression and function of CD59 on human prostatic tumor cells in situ and on 5 human prostate cell lines in vitro originating from either metastatic tumors or benign prostate hypertrophy epithelial cells. Immunohistochemical staining of prostate carcinoma tissue with monoclonal antibody (MAb) MEM43 revealed weak to moderately strong expression of CD59 by prostate glandular epithelial cells. Flow cytometry with MEM43 demonstrated that the 5 prostate cell lines expressed different relative quantities of CD59. Indirect immunofluorescence analysis revealed uniform membrane staining of DU145 and PC3 cell lines with no membranous granularity in the staining pattern. Western immunoblots with MAb BRIC 229 showed that PC3 and DU145 cells express CD59 with a m.w. of 18‐25 kDa. Treatment of DU145 and PC3 cells with phosphatidylinositol‐specific phospholipase C caused a significant decrease of CD59 expression indicating that the CD59 expressed by prostate cancer cells is anchored to the cell membrane via a glycosylphosphatidylinositol (GPI) linkage. PC3 and DU145 cells were completely resistant to human complement‐mediated cytolysis but became sensitive to killing in the presence of the CD59‐neutralizing MAb YTH53.1. We conclude that malignant and benign human prostate cells express CD59 that is GPI‐linked to the cell surface and that CD59 may regulate the immunological response to cancerous prostate cells by protecting the cells from the cytolytic activity of complement. Int. J. Cancer 71: 1049‐1055, 1997.© 1997 Wiley‐Liss Inc.

Neisseria gonorrhoeae-Induced Human Defensins 5 and 6 Increase HIV Infectivity: Role in Enhanced Transmission

Sexually transmitted infections (STIs) increase the likelihood of HIV transmission. Defensins are part of the innate mucosal immune response to STIs and therefore we investigated their role in HIV infection. We found that human defensins 5 and 6 (HD5 and HD6) promoted HIV infection, and this effect was primarily during viral entry. Enhancement was seen with primary viral isolates in primary CD4+ T cells and the effect was more pronounced with R5 virus compared with X4 virus. HD5 and HD6 promoted HIV reporter viruses pseudotyped with vesicular stomatitis virus and murine leukemia virus envelopes, indicating that defensin-mediated enhancement was not dependent on CD4 and coreceptors. Enhancement of HIV by HD5 and HD6 was influenced by the structure of the peptides, as loss of the intramolecular cysteine bonds was associated with loss of the HIV-enhancing effect. Pro-HD5, the precursor and intracellular form of HD5, also exhibited HIV-enhancing effect. Using a cervicovaginal tissue culture system, we found that expression of HD5 and HD6 was induced in response to Neisseria gonorrhoeae (GC, for gonococcus) infection and that conditioned medium from GC-exposed cervicovaginal epithelial cells with elevated levels of HD5 also enhanced HIV infection. Introduction of small interfering RNAs for HD5 or HD6 abolished the HIV-enhancing effect mediated by GC. Thus, the induction of these defensins in the mucosa in the setting of GC infection could facilitate HIV infection. Furthermore, this study demonstrates the complexity of defensins as innate immune mediators in HIV transmission and warrants further investigation of the mechanism by which defensins modulate HIV infection.

Mannose-Binding Lectin Binds to Two Major Outer Membrane Proteins, Opacity Protein and Porin, of Neisseria meningitidis

Human mannose-binding lectin (MBL) provides a first line of defense against microorganisms by complement activation and/or opsonization in the absence of specific Ab. This serum collectin has been shown to activate complement when bound to repeating sugar moieties on several microorganisms, including encapsulated serogroup B and C meningococci, which leads to increased bacterial killing. In the present study, we sought to identify the meningococcal cell surface components to which MBL bound and to characterize such binding. Outer membrane complex containing both lipooligosaccharide (LOS) and proteins and LOS from Neisseria meningitidis were examined for MBL binding by dot blot and ELISA. MBL bound outer membrane complex but not LOS. The binding to bacteria by whole-cell ELISA did not require calcium and was not inhibited by N-acetyl-glucosamine or mannose. With the use of SDS-PAGE, immunoblot analysis, and mAbs specific for meningococcal opacity (Opa) proteins and porin proteins, we determined that MBL bound to Opa and porin protein B (porB). The N-terminal amino acid sequences of the two MBL binding proteins confirmed Opa and PorB. Purified PorB inhibited the binding of MBL to meningococci. Escherichia coli with surface-expressed gonococcal Opa bound significantly more MBL than did the control strain. The binding of human factor H to purified PorB was markedly inhibited by MBL in a dose-dependent manner. Meningococci incubated with human serum bound MBL as detected by ELISA. We conclude that MBL binds to meningococci by a novel target recognition of two nonglycosylated outer membrane proteins, Opa and PorB.

Activation of Toll-Like Receptor 2 (TLR2) and TLR4/MD2 by Neisseria Is Independent of Capsule and Lipooligosaccharide (LOS) Sialylation but Varies Widely among LOS from Different Strains

ABSTRACT Lipooligosaccharide (LOS) structure and capsular polysaccharide of Neisseria meningitidis each greatly influence the virulence of the organism and the quality of host innate immune responses. In this study, we found that production of the proinflammatory cytokine tumor necrosis factor (TNF) by a human monocyte-derived cell line (THP-1) exposed to strains of N. meningitidis lacking capsule and/or with truncated LOS was similar to that elicited by the isogenic wild-type strain. These mutants also exhibited no difference in induction of the interleukin-8 (IL-8) promoter in a transfected HeLa cell system of Toll-like receptor 2 (TLR2) and TLR4/MD2 signaling. However, purified LOS from diverse strains of Neisseria (both N. meningitidis and N. gonorrhoeae) caused widely variant levels of IL-8 promoter induction in cells expressing MD2 that correlated with the production of TNF from THP-1 cells. These data suggest that although modification of the oligosaccharide chain of LOS and/or absence of capsule do not affect cell signaling mediated by TLR4/MD2, fine-structural differences in the LOS do influence signaling through TLR4/MD2 and, through this pathway, influence some of the proinflammatory responses elicited by Neisseria.

Phosphoryl Moieties of Lipid A from Neisseria meningitidis and N. gonorrhoeae Lipooligosaccharides Play an Important Role in Activation of both MyD88- and TRIF-Dependent TLR4/MD-2 Signaling Pathways

We have previously shown that the lipooligosaccharide (LOS) from Neisseria meningitidis and N. gonorrhoeae engages the TLR4–MD-2 complex. In this study, we report that LOS from different meningococcal and gonococcal strains have different potencies to activate NF-κB through TLR4–MD-2 and that the relative activation can be correlated with ion abundances in MALDI-TOF mass spectrometry that are indicative of the number of phosphoryl substituents on the lipid A (LA) component of the LOS. The LOSs from three of the strains, meningococcal strain 89I and gonococcal strains 1291 and GC56, representing high, intermediate, and low potency on NF-κB activation, respectively, differently activated cytokine expression through the TLR4–MD-2 pathway in monocytes. In addition to induction of typical inflammatory cytokines such as TNF-α, IL-1β, and IL-6, MIP-1α and MIP-1β also were significantly higher in cells treated with 89I LOS, which had the most phosphoryl substitutions on the LA compared with 1291 LOS and GC56 LOS. We found that LOS activated both the MyD88- and TRIF-dependent pathways through NF-κB and IFN regulatory factor 3 transcription factors, respectively. Moreover, LOS induced the expression of costimulatory molecule CD80 on the surfaces of monocytes via upregulation of IFN regulatory factor 1. These results suggest that phosphoryl moieties of LA from N. meningitidis and N. gonorrhoeae LOSs play an important role in activation of both the MyD88- and TRIF-dependent pathways. Our findings are consistent with the concept that bacteria modulate pathogen-associated molecular patterns by expression of phosphoryl moieties on the LA to optimize interactions with the host.

Neisseria gonorrhoeae selectively suppresses the development of Th1 and Th2 cells, and enhances Th17 cell responses, through TGF-β-dependent mechanisms.

Infection with Neisseria gonorrhoeae does not induce specific immunity or immune memory. Our previous studies in a murine model of vaginal gonococcal infection showed that innate immunity governed by Th17 cells was a critical aspect of the immune response elicited by this pathogen. Herein we show that N. gonorrhoeae selectively inhibited Th1 and Th2 cells and enhanced Th17 cell development through the induction of TGF-β. Whereas Th17 responses depended on gonococcal lipooligosaccharide acting through TLR4, the inhibitory effect of N. gonorrhoeae on Th1/Th2 responses involved gonococcal Opa proteins. In vitro Th17 responses to N. gonorrhoeae could be diverted to Th1/Th2 by blockade of TGF-β, but not by blockade of IL-17. The results reveal that N. gonorrhoeae suppresses Th1/Th2-mediated adaptive immune response through mechanisms dependent on TGF-β, and that this effect can be manipulated to promote the development of adaptive immunity.