J. McLeod Griffiss

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.

Meningococcal molecular mimicry and the search for an ideal vaccine

The carbohydrates expressed on the surface of meningococcal strains of groups B and C mimic those commonly found on human cells and thus are not functionally antigenic in infancy. In order to develop an effective vaccine, it will be necessary to find ways of circumventing this molecular mimicry. Three possible ways of achieving this are discussed. (i) The surface polysaccharides can theoretically present conformationally different epitopes, some of which might be recognized as antigenic by the host. Experimental evidence is presented that such differences do indeed exist; what is needed is to determine which of these conformations are unique to the organism and hence potentially antigenic. (ii) Precursors of the surface lipooligosaccharides may be unable to mimic human antigens, and so may be potential candidates for vaccine development. (iii) Natural immunity to some strains of meningococci develops in young children who are colonized with strains of Neisseria lactamica, and it is possible that its development could be enhanced by widespread intentional colonization by N. lactamica strains that are particularly efficient inducers of broad immunity.

Experimental Gonococcal Urethritis and Reinfection with Homologous Gonococci in Male Volunteers

Background Reinfection, a common occurrence with gonorrhea, may result from a lack of protective immune response, or from the tremendous gonococcal strain variation. Goal A two-phase study in human volunteers tested whether experimental infection with Neisseria gonorrhoeae MS11mkC would protect against reinfection with the same organisms. Study Design In phase 1, an intraurethral inoculum of 57,000 piliated, transparent (opacity protein–negative [Opa−]) MS11mkC N gonorrhoeae infected 14 of 15 (93%) volunteers. The volunteers were encouraged to delay treatment for at least 5 days. In phase 2, which began 2 weeks after treatment for the initial infection, volunteers were inoculated with 7,100 piliated, Opa− MS11mkC. Results The phase 2 challenge infected 6 of 14 (43%) previously infected volunteers and 5 of 10 (50%) naïve control subjects. Phase 1 volunteers who resisted reinfection were significantly more likely to have had a fourfold or greater increase in lipooligosaccharide immunoglobulin G during phase 1 than those who did not resist reinfection (P = 0.026). Conclusions Although infection did not provide protection from reinfection under the conditions used, the results suggest that immunity to reinfection is more complex than anticipated by the experimental design.

Lack of Lipid A Pyrophosphorylation and Functional lptA Reduces Inflammation by Neisseria Commensals

ABSTRACT The interaction of the immune system with Neisseria commensals remains poorly understood. We have previously shown that phosphoethanolamine on the lipid A portion of lipooligosaccharide (LOS) plays an important role in Toll-like receptor 4 (TLR4) signaling. For pathogenic Neisseria, phosphoethanolamine is added to lipid A by the phosphoethanolamine transferase specific for lipid A, which is encoded by lptA. Here, we report that Southern hybridizations and bioinformatics analyses of genomic sequences from all eight commensal Neisseria species confirmed that lptA was absent in 15 of 17 strains examined but was present in N. lactamica. Mass spectrometry of lipid A and intact LOS revealed the lack of both pyrophosphorylation and phosphoethanolaminylation in lipid A of commensal species lacking lptA. Inflammatory signaling in human THP-1 monocytic cells was much greater with pathogenic than with commensal Neisseria strains that lacked lptA, and greater sensitivity to polymyxin B was consistent with the absence of phosphoethanolamine. Unlike the other commensals, whole bacteria of two N. lactamica commensal strains had low inflammatory potential, whereas their lipid A had high-level pyrophosphorylation and phosphoethanolaminylation and induced high-level inflammatory signaling, supporting previous studies indicating that this species uses mechanisms other than altering lipid A to support commensalism. A meningococcal lptA deletion mutant had reduced inflammatory potential, further illustrating the importance of lipid A pyrophosphorylation and phosphoethanolaminylation in the bioactivity of LOS. Overall, our results indicate that lack of pyrophosphorylation and phosphoethanolaminylation of lipid A contributes to the immune privilege of most commensal Neisseria strains by reducing the inflammatory potential of LOS.

Pigment gallstone pathogenesis: slime production by biliary bacteria is more important than beta-glucuronidase production☆☆☆

Pigment stones are thought to form as a result of deconjugation of bilirubin by bacterial β-glucuronidase, which results in precipitation of calcium bilirubinate. Calcium bilirubinate is then aggregated into stones by an anionic glycoprotein. Slime (glycocalyx), an anionic glycoprotein produced by bacteria causing foreign body infections, has been implicated in the formation of the precipitate that blocks biliary stems. We previously showed that bacteria are present within the pigment portions of gallstones and postulated a bacterial role in pigment stone formation through β-glucuronidase or slime production. Ninety-one biliary bacterial isolates from 61 patients and 12 control stool organisms were tested for their production of β-glucuronidase and slime. The average slime production was 42 for biliary bacteria and 2.5 for stool bacteria (P <0.001). Overall, 73% of biliary bacteria and 8% of stool bacteria produced slime (optical density >3). In contrast, only 38% of biliary bacteria produced β-glucuronidase. Eighty-two percent of all patients, 90% of patients with common bile duct (CBD) stones, 100% of patients with primary CBD stones, and 93% of patients with biliary tubes had one or more bacterial species in their stones that produced slime. By comparison, only 47% of all patients, 60% of patients with CBD stones, 62% of patients with primary CBD stones, and 50% of patients with biliary tubes had one or more bacteria that produced β-glucuronidase. Most biliary bacteria produced slime, and slime production correlated better than β-glucuronidase production did with stone formation and the presence of biliary tubes or stents. Patients with primary CBD stones and biliary tubes had the highest incidence of slime production. These findings suggest that bacterial slime is important in gallstone formation and the blockage of biliary tubes.

CEACAM is not necessary for Neisseria gonorrhoeae to adhere to and invade female genital epithelial cells.

Neisseria gonorrhoeae has a repertoire of up to 11 opacity‐associated (Opa) proteins that are adhesins. Most Opa proteins adhere to CEACAM antigens and when CEACAM molecules are present on the surface of transfected epithelial cells their binding by Opa is thought to induce invasion of these cells by gonococci. In this study, we investigated whether several malignant epithelial cell lines, normal cervical and fallopian tube epithelial cell cultures, as well as normal fallopian tube tissue express several of the CEACAM molecules, and whether gonococci use these molecules for adherence and invasion of these female genital epithelial cells. A primary cervical cell culture and metastatic cervical cell line ME180 both expressed CEACAM as shown by whole cell ELISA and flow cytometry, and increased the surface expression of total CEACAM during incubation with Opa+ gonococci. Opa+ gonococci both adhered to and invaded these cells; CEACAM‐specific monoclonal antibody (MAb) partially abolished this interaction. Two primary fallopian epithelial tube cell cultures, a primary cervical cell culture and two malignant cell lines, HEC‐1‐B and HeLa, did not express CEACAM nor was CEACAM mRNA present. No evidence of either intracellular or secreted extracellular CEACAM was found with HEC‐1‐B and HeLa cells. Opa+ gonococci both adhered to and invaded CEACAM non‐expressing cells; however, Opa+ gonococcal association with these non‐expressing cell lines could not be inhibited with CEACAM‐specific MAb. These data show that CEACAM is not always expressed on female genital epithelial cells and is not essential for gonococcal adherence and invasion. However, when CEACAM is expressed, Opa+ gonococci exploit it for the adherence to and invasion of these cells.

Affinity-Purified Human Immunoglobulin G That Binds a Lacto-N-Neotetraose-Dependent Lipooligosaccharide Structure Is Bactericidal for Serogroup B Neisseria meningitidis

ABSTRACT Despite technological advances, no vaccine to prevent serogroup B meningococcal disease is available. The failure to develop a vaccine has shifted the focus to an alternative outer membrane structure, lipooligosaccharide (LOS), because disseminated disease induces bactericidal immunoglobulin G (IgG) that binds LOS. The purpose of this study was to identify the LOS structure(s) that induces human bactericidal IgG by purification and characterization of these antibodies. Human LOS IgG antibodies were affinity purified by passage of intravenous immunoglobulin through purified, type-specific LOS having a known structure coupled to epoxy-activated Sepharose 6B. Pathogenic group B strains representing the major LOS serotypes were used to examine the binding and bactericidal activities of four LOS-specific IgG preparations. All four LOS-specific IgG preparations bound to strains expressing homologous, as well as heterologous, LOS serotypes as determined by flow cytometry and an enzyme-linked immunosorbent assay. With human complement, IgG that was purified with L7 LOS was bactericidal for strains expressing L3,7 and L2,4 LOS, serotypes expressed by the majority of disease-associated group B and C meningococci. In conclusion, we purified human LOS-specific IgG that binds meningococci across LOS glycose-specific serotypes. An antigen that is dependent on the glycose lacto-N-neotetraose induces IgG in humans that is bactericidal for L2, L3, L4, and L7 strains. A vaccine containing this antigen would have the potential to protect against the vast majority of group B meningococcal strains.

Neisseria gonorrhoeae-induced transactivation of EGFR enhances gonococcal invasion

Neisseria gonorrhoeae, the causative agent of the sexually transmitted infection gonorrhoea, adheres to and invades into genital epithelial cells. Here, we investigate host components that are used by the bacteria for their entry into epithelial cells. We found that gonococcal microcolony formation on the surface of HEC‐1‐B cells disrupted the polarized, basolateral distribution of both epidermal growth factor receptor (EGFR) and ErbB2, a related family member, and induced their accumulation under the microcolonies at the apical membrane. Gonococcal infection increased EGFR and ErbB2 phosphorylation. The EGFR kinase inhibitor, AG1478, reduced gonococcal invasion by 80%, but had no effect on adherence or the recruitment of EGFR and ErbB2 to the microcolonies. Gonococcal inoculation upregulated the mRNA levels of several ligands of EGFR. Prevention of EGFR ligand shedding by blocking matrix metalloproteinase activation reduced gonococcal invasion without altering their adherence, while the addition of the EGFR ligand, HB‐EGF, was able to restore invasion to 66% of control levels. These data indicate that N. gonorrhoeae modulates the activity and cellular distribution of host EGFR, facilitating their invasion. EGFR activation does not appear to be due to direct gonococcal binding to EGFR, but instead by its transactivation by gonococcal induced increases in EGFR ligands.

Gallstones Containing Bacteria are Biofilms: Bacterial Slime Production and Ability to Form Pigment Solids Determines Infection Severity and Bacteremia

ObjectiveGallstone bacteria provide a reservoir for biliary infections. Slime production facilitates adherence, whereas β-glucuronidase and phospholipase generate colonization surface. These factors facilitate gallstone formation, but their influence on infection severity is unknown.MethodsTwo hundred ninety-two patients were studied. Gallstones, bile, and blood (as applicable) were cultured. Bacteria were tested for β-glucuronidase/phospholipase production and quantitative slime production. Infection severity was correlated with bacterial factors.ResultsBacteria were present in 43% of cases, 13% with bacteremia. Severe infections correlated directly with β-glucuronidase/phospholipase (55% with vs 13% without, P < 0.0001), but inversely with slime production (55 vs 8%, slime <75 or >75, P = 0.008). Low slime production and β-glucuronidase/phospholipase production were additive: Severe infections were present in 76% with both, but 10% with either or none (P < 0.0001). β-Glucuronidase/phospholipase production facilitated bactibilia (86% with vs 62% without, P = 0.03). Slime production was 19 (±8) vs 50 (±10) for bacteria that did or did not cause bacteremia (P = 0.004). No bacteria with slime >75 demonstrated bacteremia.ConclusionsBacteria-laden gallstones are biofilms whose characteristics influence illness severity. Factors creating colonization surface (β-glucuronidase/phospholipase) facilitated bacteremia and severe infections; but abundant slime production, while facilitating colonization, inhibited detachment and cholangiovenous reflux. This shows how properties of the gallstone biofilm determine the severity of the associated illness.

HLA Expression by Benign and Malignant Prostatic Epithelium: Augmentation by Interferon-Gamma

Chronic prostatitis is a poorly understood entity that is characterized by lymphocytic infiltration of benign prostatic epithelium. Previously, we and others have shown that prostatic epithelium involved by prostatitis is phenotypically different from uninvolved epithelium. In addition, we have shown that malignant prostatic epithelium is rarely, if ever, infiltrated by lymphocytes. We now report that benign prostatic epithelium expresses HLA-DR only in the presence of lymphocytic inflammation, and that benign epithelium without chronic prostatitis and malignant prostatic epithelium do not express HLA-DR. In order to determine whether HLA-DR expression is inducible on malignant prostatic epithelium and therefore, at least theoretically, susceptible to immune regulation, we studied the DU-145 cell line in culture under various conditions. DU-145 cells did not express HLA-DR under routine culture conditions. However, the addition of interferon-gamma (100 to 6000 U/ml.) resulted in HLA-DR expression by DU-145 cells at 24 hours with maximal expression by 72 hours. In contrast, other cytokines (tumor necrosis factor, interleukin-1, interleukin-2) had no effect on HLA-DR expression. These investigations show that interferon-gamma induces HLA-DR expression on the DU-145 prostatic adenocarcinoma cell line, raising the theoretical possibility that malignant prostatic cells may be induced in vivo to express HLA-DR and thus become susceptible to immune regulation.